Handbook of Neurochemistry and Molecular Neurobiology: Development and Aging Changes in the Nervous System [3 ed.] 9780387326702, 9780387326719

Table of contents :
Front Matter....Pages i-xvi
Back Matter....Pages 1-19
....Pages 21-59

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Neurotrophins and Central Nervous System Development

D. B. Pereira . M. V. Chao

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2 2.1 2.2 2.2.1 2.2.2 2.2.3

Neurotrophins and CNS Neuronal Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . NGF and the Survival of Central Cholinergic Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of BDNF, NT-3 and NT-4 in Central Neuron Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mesencephalic Trigeminal Nucleus Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurons of Other Regions of the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3 3.1 3.1.1 3.1.2 3.1.3 3.2

Neurotrophins and CNS Neuronal Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Neuronal Migration in the Cortex by BDNF and NT-4 . . . . . . . . . . . . . . . . . . . . . . . . . . Radial Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tangential Migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurotrophin Signaling Pathways Involved . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modulation of Neuronal Migration in the Cerebellum by BDNF and NT-3 . . . . . . . . . . . . . . . . . . . . .

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4 4.1 4.2

Neurotrophins and CNS Dendritic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Exogenous Neurotrophins on Pyramidal Neuron Dendrites . . . . . . . . . . . . . . . . . . . . . . . . . . Effects of Exogenous Neurotrophins on the Dendrites of Non-Pyramidal Interneurons and Other Neuronal Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of Endogenous Neurotrophins in Dendritic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurotrophin Receptors and Downstream Signaling Pathways Involved in Dendritic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Interplay Between Neurotrophins and Neuronal Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3 4.4 4.5

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2008 Springer ScienceþBusiness Media, LLC.

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Neurotrophins and central nervous system development

Abstract: The formation of the vertebrate nervous system is characterized by widespread programed cell death, which determines cell number and appropriate target innervation during development. The neurotrophins, which include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and NT-4, represent an important family of trophic factors that are essential for survival of selective populations of neurons during different developmental periods. Neurotrophins exert their cellular effects through the actions of two different receptors, the tropomyosin-related kinase (Trk) receptor tyrosine kinase and the p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily. Much attention has been given to the consequences of neurotrophin action in the peripheral nervous system (PNS); however, neurotrophins are widely expressed in the brain and spinal cord. This chapter focuses on new views concerning effects of neurotrophins in central nervous system (CNS) development. List of Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brainderived neurotrophic factor; CA1, cornu ammonis 1; CA3, cornu ammonis 3; CNS, central nervous system; CP, cortical plate; EGL, external germinal layer; ERK, extracellular signal-regulated kinase; GEF, guanine nucleotide-exchange factor; IgG, immunoglobulin G; IGL, internal germinal layer; MEK, mitogen activated protein kinase/ERK kinase; MGE, medial ganglionic eminence; ML, molecular layer; MTN, mesencephalic trigeminal nucleus; MZ, marginal zone; NGF, nerve growth factor; NMDA, N-methyl-D-aspartate; NPY, neuropeptide Y; NT-3, neurotrophin-3; NT-4, neurotrophin-4; PI3K, phosphatidylinositol 3-kinase; PLCg, phospholipase Cg; PNS, peripheral nervous system; RGC, retinal ganglion cell; SNP, single nucleotide polymorphism; Tiam1, T lymphoma invasion and metastasis; Trk, tropomyosin-related kinase; VTA, ventral tegmental area

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Introduction

The neurotrophic hypothesis postulates that during nervous system development, neurons approaching the same final target vie for limited amounts of target-derived trophic factor (Levi-Montalcini and Angeletti, 1968; Thoenen and Barde, 1980). In this way, the nervous system moulds itself to maintain only the most competitive and appropriate connections. Competition among neurons for limited amounts of neurotrophin molecules produced by target cells accounts for selective cell survival. Several predictions can be gleaned from this hypothesis. First, the efficacy of neuronal survival depends on the amount of trophic factors produced during development. Second, specific receptor expression in responsive cell population dictates neuronal responsiveness. The neurotrophins are initially synthesized as precursors or pro-neurotrophins that are cleaved to release the mature, active proteins. The mature proteins form stable, noncovalent dimers and are normally expressed at very low levels during development. Pro-neurotrophins are cleaved intracellularly by furin or pro-convertases utilizing a highly conserved dibasic amino acid cleavage site to release carboxy-terminal mature proteins of approximately 13 kD (Chao and Bothwell, 2002). The mature proteins mediate neurotrophin actions by selectively binding to members of the Trk family of receptor tyrosine kinases to regulate neuronal survival, differentiation, and synaptic plasticity. NGF binds most specifically to TrkA; BDNF and NT-4 to TrkB; and NT-3 to TrkC receptors (> Figure 1‐1). Neurotrophin binding to Trk receptors results in the activation of the phospholipase Cg (PLCg) pathway, involved in synaptic transmission and plasticity (Matsumoto et al., 2001; Minichiello et al., 2002), and in the recruitment of a number of proteins that are involved in the activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K) and its downstream target Akt. While ERK1/2 mediate neurotrophin-induced neuronal survival and differentiation (Huang and Reichardt, 2003), PI3K activity is responsible for retrograde survival signaling (Kuruvilla et al., 2000). The p75 receptor can bind to each neurotrophin but has the additional capability of regulating a Trk’s affinity for its cognate ligand. In addition, pro-neurotrophins can also interact with p75 independently (Lee et al., 2001). Neurotrophin factors fit well with the neurotrophic hypothesis, as many peripheral neuronal subpopulations exhibit a predominant dependence on a specific neurotrophin during the period of naturally

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. Figure 1‐1 Selectivity of neurotrophin binding to Trk receptors and p75. NGF binds most specifically to TrkA, BDNF and NT4 to TrkB and NT-3 to TrkC. NT-3 can also potentially interact with TrkB and TrkA receptors. In addition, all neurotrophins are capable of binding to the p75 receptor with equal affinity

occurring cell death. However, in the CNS, there appears to be a much more complex situation. In the brain and spinal cord, there is overlapping expression of multiple neurotrophin receptors and their cognate ligands. This allows for the generation of diverse connectivity that extends well into adulthood. In addition to promoting axonal and dendritic branching, neurotrophins also possess acute regulatory effects on neurotransmitter release, synaptic strength and connectivity (Thoenen, 1995; Bonhoeffer, 1996; McAllister et al., 1999). Many of these effects reflect activity-dependent events based upon competitive remodeling of axon and dendritic terminals. Other complexities remain, such as the molecular mechanisms underlying the retrograde signal, a pathway that must efficiently transmit information over long distances, at times over a meter, as well as anterograde transport of neurotrophins. The NGF family of trophic factors has provided a remarkable mechanism for controlling a wide gamut of cellular activities, including target innervation, apoptosis, differentiation, synaptic plasticity, and axon guidance. Defining the populations in the central nervous system (CNS) that are affected bidirectionally by neurotrophins provides insights into the mechanisms responsible for regulating cell growth, survival, and changes in complex circuits. This chapter considers some of the key decision-making events in different populations of central neurons that lead to these diverse biological outcomes.

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Neurotrophins and CNS Neuronal Survival

The broad distribution of TrkB, TrkC and their ligands throughout the CNS is suggestive of a putative role for neurotrophins in CNS neuronal survival during development, in similarity to their action in the PNS (peripheral nervous system). The result of initial studies showing that exogenously applied neurotrophins can rescue specific populations of central neurons from naturally occurring and induced cell death, seemed to support this idea. Among the neuronal populations that show increased survival when cultured in the presence of neurotrophins are embryonic cerebellar granule cells (reviewed in Segal et al., 1992) and substantia nigra dopaminergic neurons (Hyman et al., 1991), which are responsive to brain-derived

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neurotrophic factor (BDNF), cerebellar Purkinje cells (Cohen-Cory et al., 1991), which are sensitive to NGF, and septal cholinergic neurons, which are responsive to both (Hartikka and Hefti, 1988; Alderson et al., 1990). In vivo studies have also shown that exogenously applied NGF and BDNF or NT-3, can rescue axotomized basal forebrain neurons (Williams et al., 1986) and motor neurons (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992; Koliatsos et al., 1993), respectively. Despite what these pharmacological studies have initially suggested, mouse mutants lacking neurotrophins or their receptors, which were generated in the early-to-mid 1990s, have failed to confirm a significant neuroprotective role for neurotrophins in the CNS during embryonic development (reviewed in Snider, 1994).

2.1 NGF and the Survival of Central Cholinergic Neurons The TrkA
/
and the NGF
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mice, while exhibiting the expected loss of neurons in peripheral ganglia, do not show reduced numbers of basal forebrain cholinergic neurons (Crowley et al., 1994; Smeyne et al., 1994). These are the prototypical NGF-responsive neurons in the brain, which were thought to depend on NGF secreted by their hippocampal and cortical target fields for survival and function (Dreyfus, 1989). Striatal cholinergic neurons also do not seem to be affected in NGF-deficient mice (Crowley et al., 1994). Although cholinergic cell numbers are normal, a substantial decrease in acetylcholinesterase staining in fibers extending to the hippocampus and cerebral cortex was observed in the TrkA mutant mice (Smeyne et al., 1994). A reduction in the size of basal forebrain cholinergic neurons is also apparent in both NGF mutant homozygous and heterozygous animals (Crowley et al., 1994; Chen et al., 1997), arguing in favor of a physiological role for endogenous NGF on the differentiation of these neurons. We should also bear in mind, though, that both the TrkA and the NGF deficient mice die very early in their postnatal life, while the development of their CNS is still ongoing. This precludes a definitive answer on the NGF-dependence of brain cholinergic neurons. Understanding the role of NGF in the survival of these neurons is further complicated by the controversy surrounding the function of the p75 neurotrophin receptor in neuronal apoptosis in the CNS. This issue is aggravated by another controversy regarding the nature of the two existing p75-deficient mouse strains. The first p75 mutant mouse was generated by a targeted deletion of exon III and is commonly referred to as the p75exonIII mutant (Lee et al., 1992). This mouse was later suggested to express a shorter form of the p75 receptor, arising from alternative splicing of exon III, which lacks three of the four cysteine-rich domains responsible for neurotrophin binding (von Schack et al., 2001). A second p75-deficient mouse, the p75exonIV mutant, was then generated by deleting exon IV of the p75 locus, in an attempt to create animals lacking any form of the p75 receptor (von Schack et al., 2001). However, these mice were described to express a 26 kDa p75 gene product, containing the extracellular stalk region and the entire transmembrane and intracellular domains of p75 (Paul et al., 2004). This truncated protein, which was not observed in the wildtype or in the p75exonIII mutant animals, was able to activate p75 signaling cascades when overexpressed in heterologous cells (Paul et al., 2004). While this controversy remains unresolved, the study of the putative role of p75 in basal forebrain cholinergic neurons has originated discrepant results even when the same p75 mutant mouse strain was used. An initial report described an increase in the number of septum cholinergic neurons in the p75exonIII mutant in agreement with the previously purposed pro-apoptotic function of p75 (Van der Zee et al., 1996). Although subsequent studies reported a similar increase in basal forebrain cholinergic neuron numbers (Yeo et al., 1997), others have found exactly the opposite. Adult p75exonIII mice were described to have reduced septal region volume and cholinergic neuron numbers (Peterson et al., 1997, 1999; Greferath et al., 2000), which suggests a pro-survival function for p75, possibly through a positive regulation of neurotrophin signaling through Trk receptors. A lack of any significant change in cholinergic neuron numbers was also described in a reexamination of the p75exonIII mutant mice by the same group that first reported a decrease in neuronal numbers in these mice (Ward and Hagg, 1999). Differences in genetic background and age of the mice used in these studies and quantitative stereology methods may account for some of the discrepancies observed. In fact, a more recent study shows that a moderate increase in basal forebrain cholinergic neurons was only apparent in p75exonIII mutant mice after backcrossing in a C57BL/6

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background (Naumann et al., 2002). On the other hand, the same study reports a significant increase in the cholinergic neuron number in the p75exonIV mutant mouse (Naumann et al., 2002). The role of p75 in basal forebrain cholinergic neuron survival remains, therefore, unresolved. Moreover, the involvement of neurotrophins and Trk signaling in the putative role of p75 in the survival of the aforementioned neurons was not yet investigated. Clarifying the controversy regarding the two different strains of p75 mutant mice will require conditional mice, together with crossing these animals with other neurotrophin mutants. This will be crucial to the understanding of neurotrophin and p75 regulation of central cholinergic neuron survival.

2.2 Role of BDNF, NT-3 and NT-4 in Central Neuron Survival Studying the role of TrkB and TrkC in CNS development by genetic ablation of either one of these receptors or their specific ligands was complicated, as in the case of TrkA and NGF, by the limited life span of the animals. With the exception of the NT-4
/
mice that survive well into adulthood (Conover et al., 1995; Liu et al., 1995), these mutant mice die either shortly after birth, as it is the case for the NT-3 and the TrkB mutants (Klein et al., 1993; Farinas et al., 1994; Ernfors et al., 1994b) or within the second or third postnatal weeks, as for the TrkC and BNDF-deficient animals (Ernfors et al., 1994a; Jones et al., 1994; Klein et al., 1994). Analyzing the CNS of these animals did indicate, though, that neither TrkB nor TrkC activity is essential on its own for CNS neuronal survival, at least during embryonic development. Nevertheless, there are some exceptions and controversies that are worth mentioning.

2.2.1 Motor Neurons The TrkB
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mouse, which was the first neurotrophin receptor mutant to be reported, was initially described to have a decrease of 35% in the number of spinal cord motor neurons (Klein et al., 1993). This would be in agreement with the rescue of developmentally regulated or axotomy-induced motor neuron death by BDNF (Oppenheim et al., 1992; Sendtner et al., 1992; Yan et al., 1992; Koliatsos et al., 1993). However, two different strains of mice lacking BDNF failed to show any reduction in the survival of several motor neuron populations (Ernfors et al., 1994a; Jones et al., 1994), including spinal cord neurons. Mice deficient for the other two neurotrophins that can activate TrkB, NT-4 and NT-3, did not exhibit any reduction in facial motor neurons numbers (Ernfors et al., 1994b; Farinas et al., 1994; Conover et al., 1995; Liu et al., 1995). BDNF and NT-4 double mutants also have normal facial and lumbar cord motor neurons pools (Conover et al., 1995; Liu et al., 1995). A reduction in motor neuron survival was only observed in triple BDNF, NT-4 and NT-3 deficient mice, which show a modest 20% decrease in neuronal numbers in several nuclei (Liu and Jaenisch, 2000). Finally, reexamination of the TrkB mouse mutants and analysis of mice lacking both TrkB and TrkC showed no significant difference in the number of facial or spinal motor neurons between these mutant mice and their wildtype littermates (Silos-Santiago et al., 1997). Therefore, neurotrophins appear to play a redundant and modest role in the survival of motor neurons during embryonic development. It is still possible, since not all motor neuron pools were examined in each animal, that particular sets of motor neurons are dependent on specific neurotrophins for survival, as it is the case for other trophic factors (Huang and Reichardt, 2001). As an example, the survival of g-motor neurons, whose nerve fibers are present at the L4 ventral spinal root, is greatly reduced in the NT-3
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mouse (Kucera et al., 1995).

2.2.2 Mesencephalic Trigeminal Nucleus Neurons The most dramatic reduction in neuronal survival in the CNS of neurotrophin or neurotrophin-receptor mutant mice was observed in the mesencephalic trigeminal nucleus (MTN). The MTN, which is located in the brainstem, contains the proprioceptive neurons of the trigeminal system that convey information from

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the head region. Both the BDNF
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and the NT-3
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mice show an approximate 50% reduction in the number of neurons in this area (Ernfors et al., 1994a, b; Farinas et al., 1994; Jones et al., 1994). Double BDNF/NT-3-deficient mice show a loss of 88% of these neurons and triple BDNF/NT-3/NT-4 mutants are practically devoid of MTN neurons, with only about 5% of them surviving (Fan et al., 2000). Therefore, unlike their PNS counterparts, which are completely dependent on NT-3 (reviewed in Snider, 1994; Huang and Reichardt, 2001), different subsets of these CNS proprioceptive neurons seem to depend on different neurotrophins for survival.

2.2.3 Neurons of Other Regions of the Brain Most of the other regions of the brain were surprisingly normal in these mutant mice. No gross abnormality was detected in the cytoarchitecture of the brain areas examined, including the cortex, hippocampus, and thalamus, in TrkB and TrkC single mutants (Klein et al., 1993, 1994). TrkB/TrkC double mutants also appeared to have normal brain anatomy (Silos-Santiago et al., 1997), ruling out redundancy as a possibility for the lack of a phenotype. A more comprehensive examination was performed on the BDNF mutants. With the exception of the granule cerebellar neurons, which exhibit increased cell death in these animals (Schwartz et al., 1997), most of the neuronal populations known to be sensitive to this neurotrophin showed no decrease in survival. This included substantia nigra and VTA dopaminergic neurons, basal forebrain cholinergic neurons, cerebellar Purkinje neurons, and GABAergic and cholinergic neurons of the hippocampus and striatum (Jones et al., 1994; Ernfors et al., 1994a). There was, however, a marked decrease in the expression of certain neuronal markers, such as calbindin, parvalbumin, and neuropeptide Y (NPY; Jones et al., 1994). This was interpreted as a problem in neuronal differentiation since GABAergic neurons, where calbindin, parvalbumin, and NPY are expressed, are present in normal numbers in the BDNFdeficient mice. Importantly, although their brain structure seems normal, BDNF
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brains are smaller and have less neuropile (Conover et al., 1995). A reduction in the thickness of all cortical layers is also evident (Jones et al., 1994). Subsequent reports looking specifically for neuronal apoptosis in the postnatal brain of TrkB and double TrkB/TrkC mutants uncovered a need for these receptors in the survival of several populations of CNS postmitotic neurons. TrkB deficient mice show a progressive increase in the number of apoptotic cells from P10 to P18, in all cortical layers and hippocampal subfields (Alcantara et al., 1997). The dentate gyrus is the most affected area. In other brain regions, such as the striatum and the reticular thalamic nucleus, the increase in apoptotic cells in the TrkB mutant is apparent only during the first postnatal weeks (Alcantara et al., 1997). The majority of dying cells were confirmed to be neurons by the use of specific neuronal markers. Mice deficient in both TrkB and TrkC also exhibit increased cell death in the hippocampus, after the first postnatal week (Minichiello and Klein, 1996). Cerebellar granule neurons, but not Purkinje cells, are also reduced in these animals, in similarity to what was observed in the BDNF mouse mutants (Minichiello and Klein, 1996; Schwartz et al., 1997). Conditional mouse mutants lacking TrkB at specific developmental time points and in specific cell types have been generated using the Cre/loxP recombination system (Minichiello et al., 1999; Xu et al., 2000; Medina et al., 2004). These mice have been a valuable tool to study the role of TrkB in the CNS of adult mice. Two of these strains, where depletion of TrkB occurs either embryonically or during the second to third postnatal weeks, show a severe reduction in the thickness of their visual and somatosensory cortex (but Medina et al., 2004; see also Minichiello et al., 1999; Xu et al., 2000). Although it is likely that this compression of the cortical layers is associated with migration and differentiation problems (Xu et al., 2000; Medina et al., 2004), a decrease in neuronal survival was also observed in 10-week-old animals (Xu et al., 2000). Interestingly, the survival of different neuronal populations, identified by the expression of specific transcription factors, were differentially affected by the lack of TrkB (Xu et al., 2000). Taken together, all the work involving conventional and conditional neurotrophin and Trk receptor mouse mutants has revealed a disappointing small role for these molecules in the survival of CNS neurons during embryonic development, as compared with their PNS counterparts. With the exception of the MTN,

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other brain regions seem to be dependent on neurotrophins for neuronal survival only throughout postnatal development. As we will describe in the following sections, neurotrophins appear to have a bigger contribution to CNS development by modulating migration and differentiation of CNS neurons.

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Neurotrophins and CNS Neuronal Migration

Neuronal migration is an essential step in brain tissue formation during development, allowing the proper positioning of neurons before the onset of neuritogenesis and synaptogenesis. To reach their appropriate position, neurons undergo one or two different types of migration. In radial migration, neurons migrate from their progenitor regions following the radial disposition of the neural tube (reviewed in Marin and Rubenstein, 2003). This usually involves the use of radial glia processes, which extend from the ventricular zone to the pial surface of the brain, as a guide on which neurons migrate until they reach their destination. Radial glia-independent radial migration can also occur by a process called somal translocation, where neurons themselves extend a process to the pial surface and then translocate their soma to the proper position. The other type of migration, termed tangential migration, follows a path that is orthogonal to the direction of radial migration (reviewed in Marin and Rubenstein, 2003). The physical substrates used by cells migrating tangentially are other migrating neurons or growing axons. Alternatively, cells can just disperse in an individual manner not following any cellular substrates. Neurotrophins have been described to influence both types of neuronal migration by acting as motogenic factors that stimulate the motility of migrating cells. In the following sections, we will focus on studies performed on the cerebral cortex and the cerebellum, where the role of neurotrophins in neuronal migration has been most intensively investigated.

3.1 Modulation of Neuronal Migration in the Cortex by BDNF and NT-4 The formation of the layered architecture of the cerebral cortex relies on the radial migration of postmitotic neurons, originating from the ventricular zone that lines the wall of the cerebral ventricle, toward the surface of the brain (Rakic, 1975). The first migrating neurons form a subpial layer known as the preplate. This layer is then split into the marginal zone (MZ; Layer I, the outermost layer) and the subplate by successive migrating neurons that form the cortical plate in between (Marin-Padilla, 1971). The latter is generated by an inside-out migratory sequence that leads to the formation of layers II–VI (Angevine and Sidman, 1961; Rakic et al., 1974; Caviness, 1982; Bayer et al., 1991) (> Figure 1-2). In addition, GABAergic neurons generated mostly in the medial ganglionic eminence (MGE), with a smaller contribution of the lateral and caudal eminences, migrate tangentially into the cortex where they reach the appropriate layer by a final step of radial migration (Anderson et al., 2001; Nery et al., 2002; Ang et al., 2003).

3.1.1 Radial Migration Messenger RNA for the TrkB and TrkC receptors is present in high levels during cortical development, when neuronal migration occurs (Klein et al., 1990; Lamballe et al., 1994). On the other hand, their ligands, BDNF and NT-3, have opposite patterns of expression during corticogenesis: NT-3 expression is high during embryogenesis and decreases with maturation while BDNF expression is low during embryonic development increasing during adulthood (Maisonpierre et al., 1990; Timmusk et al., 1994). Surprisingly, in spite of these expression patterns, it is BDNF and not NT-3 that seems to play a role in neuronal migration in the neocortex. Both BDNF and NT-4 can induce chemotaxis (directed migration) of embryonic cortical neurons in vitro in a manner dependent on Ca2+ and Trk activation (Behar et al., 1997). In the same assay, NT-3 can only elicit migration when used at high concentrations (100 ng/ml). In vivo studies have shown that increasing the concentration of BDNF in the developing embryonic brain,

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. Figure 1-2 Cerebral cortex organization during development and adulthood. Postmitotic neurons, originating from the ventricular zone (VZ), migrate radially towards the surface of the brain to form the preplate (PP). Most of these neurons migrate on radial glial processes represented in the diagram by vertical lines. For simplicity, the cell body of these glial cells was not represented. The PP is then split into the marginal zone (MZ) and the subplate (SP) by successive migrating neurons that form the cortical plate (CP) in between. Layers II-VI are then formed at the CP by an inside-out migratory sequence. IZ, intermediate zone

either by intraventricular injection of the neurotrophin or by creating transgenic mice that overexpress BDNF, produces neuronal heterotopias (collections of cells) in the MZ (Brunstrom et al., 1997; Ringstedt et al., 1998; Alcantara et al., 2006). Injection of NT-4 has an even greater effect in the production of heterotopias, as compared with the injection of BDNF, while infusion of NT-3 has no effect (Brunstrom et al., 1997). This accumulation of cells in the MZ of the developing cortex appears to be the result of increased neuronal migration and it is at least partially due to a decrease in the expression of reelin (Brunstrom et al., 1997; Ringstedt et al., 1998). Reelin, which is produced by the Cajal–Retzius cells of the MZ, is required for normal lamination of the cortex during embryonic development and is downregulated postnatally as maturation occurs (reviewed in Marin and Rubenstein, 2003). This downregulation is dependent on BDNF since it is delayed in mice lacking this neurotrophin (Ringstedt et al., 1998). Therefore, BDNF appears to act as a cortical maturation factor that, by being expressed in high amounts ahead of time, induces cortical lamination defects. Does this imply, however, that endogenous BDNF plays a role in migration in the neocortex? Preliminary analysis of the BDNF-deficient mouse brain revealed a reduction in the thickness of the cortical layers (Jones et al., 1994). A similar phenotype was seen in TrkB conditional mouse mutants where TrkB expression is reduced during embryogenesis being virtually eliminated following birth (Medina et al., 2004). These mice show a delay in migration of newly born cortical neurons that assume altered positioning in the newly formed cortical layers (Medina et al., 2004). However, it is not clear whether the reduction in TrkB expression during embryogenesis and early postnatal life, achieved in these conditional mutants, is enough to reveal the full plethora of defects that the lack of this receptor would cause in the context of neuronal migration. It would be interesting to study in more detail the brain architecture of embryonic and early postnatal TrkB and BDNF/NT-4 double mutant mice to clarify the role of these neurotrophins in neuronal migration. In support of a role for endogenous TrkB ligands in migration to the appropriate cortical layer TrkB expression is seen in migratory neurons in the CP in vivo, during the period of maximal neuronal migration (Behar et al., 1997). In addition, a gradient of BDNF transcripts is observed in the neocortex, with the CP

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expressing higher amounts of this neurotrophin as compared to the ventricular zone from where cortical neurons originate (Behar et al., 1997). This may be the reason why BDNF, and not the more highly expressed NT-3, is important for neuronal migration. By being present in low amounts in discrete regions, which allows the formation of a gradient, BDNF may direct the migration of TrkB-expressing neurons toward regions of higher BDNF concentration.

3.1.2 Tangential Migration Tangential migration of GABAergic interneurons into the cortex is also promoted by NT-4 and BDNF. Exogenous application of these neurotrophins to brain slices increased the number of MGE neurons that migrate into the cortex and the rate at which they migrate (Polleux et al., 2002). NT-3 was again without effect on the migration of these interneurons. The role of endogenous neurotrophins in this process was demonstrated by a reduction in the number of MGE neurons that migrate into the cortex in brain slices of TrkB deficient mice and in slices treated with the Trk inhibitor K-252a (Polleux et al., 2002). Interestingly, transactivation of Trk receptors by endocannabinoids has been reported to induce interneuron migration in vitro (Berghuis et al., 2005).

3.1.3 Neurotrophin Signaling Pathways Involved The Trk signaling pathways involved in the modulation of cortical neuronal migration by neurotrophins have only recently been studied. The PI3K pathway was suggested to mediate the effects of TrkB ligands on radial migration as well as interneuron tangential migration, by pharmacological approaches (Polleux et al., 2002; Yoshizawa et al., 2005). However, both the Shc-binding site, which leads to the activation of PI3K and ERK, and the PLCg-binding sites on the TrkB receptor need to be mutated to induce a delay in radial migration similar to that observed in conditional TrkB mouse mutants (Medina et al., 2004). Mice with single point mutations at either site show normal CNS development (Medina et al., 2004). The PI3K pathway was also suggested to lead to the activation of P-Rex1, a Rac guanine nucleotide-exchange factor (GEF) that plays an important role in BDNF-induced migration of cortical neurons (Yoshizawa et al., 2005). A dominant negative form of P-Rex1 was shown to inhibit the migration of cortical neurons toward higher concentrations of BDNF in vitro and to give rise to radial migration impairments in vivo (Yoshizawa et al., 2005). Other molecules such as the Src-family kinases, which have been involved in Trk signaling and function (Alema et al., 1985; Kremer et al., 1991; Tsuruda et al., 2004; Rajagopal and Chao, 2006), were recently shown to be important for neuronal migration. Double mutant mice lacking both Src and Fyn show a defect in cortical lamination similar to that observed in Reelin
/
mice (Kuo et al., 2005). However, it is unknown whether these molecules play a direct role in neuronal migration in the context of neurotrophin receptor signaling. Future studies will be necessary to uncover the relative contribution of several signaling pathways to neurotrophin-induced migration in the cortex.

3.2 Modulation of Neuronal Migration in the Cerebellum by BDNF and NT-3 The characteristic layered structure of the cerebellum is formed by the coordinated migration of two principle classes of neurons: Purkinje cells and granule neurons. Purkinje cells, the major output neurons of the cerebellar cortex, originate from the ventricular zone. Once they reach the roof of the developing cerebellar anlage and become postmitotic they migrate inwards along the radial glial fibers and settle on a broad area just above the deep cerebellar nuclei (Altman and Bayer, 1985; Hatten, 1999). The precursors of cerebellar granule neurons, which originate from the rhombic lip (Alder et al., 1996), first migrate in a dorsorostral pathway to cover the surface of the emerging anlage, forming the external germinal layer (EGL; Hatten, 2002). As they become postmitotic, granule neurons migrate radially, past the Purkinje cell layer, to

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form the internal germinal layer (IGL; Hatten, 1999). This leads to the establishment of the three layers of the cerebellum: the outer molecular layer (ML), composed of granule cell axons and Purkinje cell dendrites, the Purkinje cell layer and the IGL. Neurotrophins appear to be important for the proper migration of cerebellar granule neurons during development. Both TrkB and TrkC are expressed, although to various degrees, by the principal neurons of the developing cerebellum (Klein et al., 1990; Ernfors et al., 1992). NT-3 mRNA levels peak in the IGL at the time of granule neuron migration and decrease afterwards (Rocamora et al., 1993). Although BDNF mRNA was not detected in the IGL in the first two postnatal weeks (Rocamora et al., 1993), when granule neurons migrate radially to form that layer, BDNF protein was detected at P7, both in the IGL and the ML (Borghesani et al., 2002). While this apparent controversy remains unsolved, it is interesting to note that BDNF protein levels are low in the EGL, the starting point of granule neuron radial migration, and high in the ML and IGL, where these neurons migrate to. Additionally, TrkB immunoreactivity in the developing cerebellum is highest in migratory granule neurons when they are passing through the ML (Gao et al., 1995). Moreover, BDNF is acutely motogenic for granule cells in culture and when these cells are exposed to a gradient of this neurotrophin they migrate toward higher concentrations of BDNF (Borghesani et al., 2002). A role for BDNF and TrkB in the migration of cerebellar granule neurons was confirmed in vivo by studying the development of the cerebellum from BDNF and TrkB mouse mutants. The BDNF-deficient mice exhibit an increase in the thickness of their EGL, at P14, while their IGL is sparser and thinner (Schwartz et al., 1997; Borghesani et al., 2002). The EGL abnormally persists until later in development, in BDNF mutants, being still present at P17-P21 (Jones et al., 1994; Schwartz et al., 1997). These phenotypes are believed to be the result of a delay rather than a deficit in migration since at P23 the EGL is already not apparent in the BDNF-deficient animals (Jones et al., 1994). Indeed, BDNF-deficient granule neurons show a decreased migratory index: the number of cells that migrate is lower although the speed of migration is similar to wildtype (Borghesani et al., 2002). Therefore, BDNF appears to be important for the initiation of cerebellar granule neuron migration (Borghesani et al., 2002). TrkB conventional and conditional mouse mutants also displayed a delay in granule cell migration, in similarity to the BDNF-deficient mice (Minichiello and Klein, 1996; Rico et al., 2002). However, TrkB/TrkC double mutants (TrkB
/
; TrkC+/
or TrkB+/
; TrkC
/
) exhibit a more pronounced enlargement of the EGL as compared with the single mutants (Minichiello and Klein, 1996). This suggests that both NT-3 and BDNF may contribute to the proper migration of cerebellar granule neurons. Analysis of NT-3 conditional mutant mice failed to detect a defect in the layering of the cerebellum (Bates et al., 1999). Nevertheless, this study was performed at P8, when the layering of BDNF-deficient cerebellum is also not yet different from the wildtype (Schwartz et al., 1997). On the other hand, the NT-3-deficient cerebellum presents a similar foliation defect to that seen in BDNF mouse mutants, which may or may not be related with a problem in migration (Schwartz et al., 1997; Bates et al., 1999). In conclusion, neurotrophins appear to be important for the proper timing of neuronal migration in both the cerebral cortex and the cerebellum. However, the overlapping effects of different neurotrophins in these brain regions may be preventing the clarification of the exact role of these molecules in neuronal migration. A more comprehensive analysis of conventional and conditional single and double neurotrophin mutants could undoubtedly further clarify the promising role of neurotrophins in neuronal migration.

4

Neurotrophins and CNS Dendritic Development

Soon after neurons reach their appropriate location, or even during migration, they extend neurites that eventually differentiate into dendrites and axons. This polarization sets the stage for the formation of an elaborate network of synaptic connections, in which neurite outgrowth, axonal guidance, and synaptogenesis take part. Although no evidence was yet presented for a role of neurotrophins in neuronal polarization, these molecules are established regulators of dendritic growth and complexity while a role in axonal guidance and synaptogenesis is also emerging (McAllister, 2001; Gillespie, 2003). On the last section of this chapter, we will focus on the most documented aspect of neurotrophin function on central neuron differentiation: the regulation of dendritic growth and complexity.

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Neurons in the CNS evolve from immature cells with just a few processes to bearers of highly complex dendritic arbors, which integrate most of the afferent input received by these neurons. To achieve this level of intricate dendritic morphology neurons appear to follow an intrinsic growth program, capable of generating a basic dendritic arborization, and to respond to extrinsic signals that shape their dendritic tree into a final mature form. Neuronal activity, in response to afferent neuronal connections, has been regarded as a key factor in the regulation of dendritic morphology by controlling intracellular Ca2+ concentration. Alterations in the concentration of this ion can activate signaling pathways that may locally induce cytoskeletal reorganization and direct the synthesis of new proteins needed for dendritic growth (reviewed in Dijkhuizen and Ghosh, 2005b). Neurotrophins and their receptors are highly expressed in the developing CNS during the time period when dendritic growth is occurring (reviewed in McAllister et al., 1999). Moreover, neurotrophins are secreted in response to neuronal activity and therefore appear as plausible candidates to mediate activitydependent dendrite plasticity (Ghosh et al., 1994; Blochl and Thoenen, 1995; Goodman et al., 1996; Wang and Poo, 1997; Hartmann et al., 2001). Indeed, neurotrophins, and in particular BDNF, have been extensively described as crucial regulators of dendrite number, length and branching (reviewed in Dijkhuizen and Ghosh, 2005b; McAllister, 2001).

4.1 Effects of Exogenous Neurotrophins on Pyramidal Neuron Dendrites Early studies using ferret visual cortex organotypic slice cultures showed that all the neurotrophins have the potential of modulating dendritic morphology of pyramidal neurons (McAllister et al., 1995). These and other types of organotypic slice culture systems have been extensively used in the study of dendritogenesis since the basic cytoarchitecture of the tissue is conserved and dendritic development can be followed for extended periods in culture. In this system, all neurotrophins, and more effectively BDNF, NT-3 and NT-4, were shown to increase dendritic complexity of cortical pyramidal neurons by increasing dendritic length and branching and the number of primary dendrites (McAllister et al., 1995). Basal dendrites appear to be more responsive to neurotrophins than apical dendrites, and to be maximally stimulated by a given neurotrophin according to the specific cortical layer at which the neuron is located (McAllister et al., 1995). Moreover, BDNF and NT-3 oppose each other’s actions on dendritic growth. In layer 4, NT-3 blocks BDNF-induced increase in dendritic growth while BDNF inhibits the effects of NT-3 on layer 6 pyramidal neuron dendrites (McAllister et al., 1997). The positive effects of exogenous BDNF, NT-3 and NT-4 on pyramidal neuron dendritic length and number were also demonstrated to occur in rat brain, cortical and hippocampal organotypic cultures, with region-specific and basal versus apical variations (Baker et al., 1998; Niblock et al., 2000; Schwyzer et al., 2002; Wirth et al., 2003). The effects mentioned above on dendrite morphology were obtained by exposing young organotypic slice cultures, where neurons are still elaborating their dendritic arbors, to neurotrophins, for 36 h to a few days. BDNF treatment of older ferret visual cortex organotypic slices, where pyramidal neurons have already established their mature morphology, increases the number of basal dendrites at the expense of distal branches, in just 24 h (Horch et al., 1999). Dendrite dynamics and instability were increased by BDNF, with new basal dendrites being rapidly added and lost, in contrast to control neurons that rarely loose basal dendrites (Horch et al., 1999). The effect of BDNF on dendrite morphology was demonstrated to be extremely localized, occurring within 4.5 mm from the neurotrophin source (Horch and Katz, 2002).

4.2 Effects of Exogenous Neurotrophins on the Dendrites of Non-Pyramidal Interneurons and Other Neuronal Types Non-pyramidal interneuron dendrites are also responsive to neurotrophin treatment. BDNF was shown to increase total dendritic length and the number of dendritic branch points both in organotypic cortical cultures and in dissociated cortical neurons in culture (Jin et al., 2003; Kohara et al., 2003; Wirth et al.,

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2003). NT-4 was also able to increase the same dendritic parameters on non-pyramidal interneurons in cortical organotypic slice cultures (Wirth et al., 2003), while NT-3 was without effect (Baker et al., 1998). However, it is not certain whether the differential effects of these neurotrophins were not due to different experimental conditions, such as differences in the developmental stage of the culture, duration of neurotrophin treatment, and brain region analyzed. Other types of central neurons that show increased dendritic growth in response to neurotrophins include rat Purkinje cells and mouse parvalbumin-positive interneurons of the olfactory bulb, which are responsive to BDNF (Hirai and Launey, 2000; Berghuis et al., 2006), rat dopaminergic mesencephalic neurons, which are affected by NT-4 (DeFazio et al., 2000), and mouse cerebellar granule neurons and basilar pontine nuclei neurons that are responsive to both neurotrophins (Gao et al., 1995; Rabacchi et al., 1999). An interesting effect of exogenous BDNF was reported in the Xenopus visual system, where microsphere-coupled BDNF increases retinal ganglion cell (RGC) dendritic complexity in vivo when injected in the optic tectum, their target area, while having the opposite effect when injected in the retina, where RGC dendrites are located (Lom and Cohen-Cory, 1999; Lom et al., 2002). These results indicate that local effects of neurotrophins in different subcellular domains of central neurons may exert distinct outcomes on dendrite differentiation.

4.3 Role of Endogenous Neurotrophins in Dendritic Differentiation The role of endogenous neurotrophins on neuronal dendritic development has been addressed through the study of transgenic animals or the use of neurotrophin-neutralizing antibodies and the Trk receptor fusion proteins TrkA-IgG, TrkB-IgG, and TrkC-IgG. The later, which bind to the respective Trk ligands preventing activation of the corresponding neurotrophin receptor, have been shown to inhibit dendritic development in the ferret visual cortex (McAllister et al., 1997). While TrkA-IgG has minor effects on dendritic growth, TrkB-IgG causes retraction of existing basal dendrites in layer 4 pyramidal neurons and TrkC-IgG reduces dendritic complexity of layer 6 neurons (McAllister et al., 1997). However, in rat visual cortex organotypic slices, neutralizing antibodies against NT-4, but not those against BDNF, decrease dendritic length and complexity (Wirth et al., 2003). In addition, activity-deprived organotypic cultures, which have decreased BDNF mRNA levels, show normal dendritic parameters (Wirth et al., 2003). These results argue in favor of NT-4 being the most important TrkB ligand in early dendritic development of the visual cortex pyramidal neurons. Interestingly, analysis of a forebrain-restricted early onset BDNF
/
mouse strain showed that a decrease in primary dendrite number and branching is only seen in animals older than 3–5 weeks, after the peak of primary dendrite development (Gorski et al., 2003). Therefore, endogenous BDNF appears to be important for the maintenance but not the initial development of cortical pyramidal dendrites. The need for TrkB ligands in the maintenance of cortical neuron dendritic arbors was confirmed in late onset TrkB mutant mice, which show fewer and shorter branches at 6 weeks of age (Xu et al., 2000). For other neuronal types, such as the cerebellar Purkinje cells, the role of endogenous neurotrophins in dendritic development is still controversial. Although one report shows that BDNF increases Purkinje cell dendritic differentiation, while TrkB-IgG has the opposite effect (Hirai and Launey, 2000), another study shows no effect of either BDNF or TrkB-IgG on Purkinje cell dendritic parameters (Shimada et al., 1998). On the other hand, of two different strains of BDNF-deficient mice, one shows altered Purkinje cell dendritic arbors with smaller and more numerous primary dendrites (Schwartz et al., 1997), while the other has normal Purkinje cell dendritic arbors (Jones et al., 1994). A conditional TrkB mutant mouse strain that lacks TrkB in the cerebellum also shows normal Purkinje cell dendritic development (Rico et al., 2002). Purkinje cell dendrites were also unaffected in organotypic cultures of BDNF-deficient mouse cerebella or wildtype cultures treated with BDNF or K-252a (Adcock et al., 2004). A transgenic mouse bearing a single nucleotide polymorphism (SNP) in the bdnf gene, which leads to a valine to methionine substitution at position 66 of the BDNF prodomain, was recently described to have abnormal dendritic morphology (Chen et al., 2006). This polymorphism, which reduces depolarization-evoked BDNF secretion, leads to decreased dendritic complexity of dentate gyrus neurons

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in 8-week-old mice. These mice also exhibit behavior abnormalities such as increased anxiety and decreased contextual-dependent memory, in similarity to the BDNF heterozygous mice (Chen et al., 2006). These are important findings since this SNP was found to decrease hippocampal-dependent memory in humans (Egan et al., 2003), thus associating dendritic development deficits with a behavior-impairing human polymorphism.

4.4 Neurotrophin Receptors and Downstream Signaling Pathways Involved in Dendritic Development An important issue in neurotrophin-mediated dendritic development is to identify the neurotrophin receptors and downstream signaling molecules that are involved. Trk neurotrophin receptors have been shown to mediate many of the effects of endogenous and exogenously added neurotrophins on dendritic morphology, by the inhibitory actions of the Trk inhibitor K-252a (Morfini et al., 1994; Horch et al., 1999; Jin et al., 2003; Wirth et al., 2003). Additionally, transfection of ferret visual cortex organotypic slice cultures with full-length TrkB induces an increase in proximal dendritic complexity similar to what was observed with neurotrophin treatment (Yacoubian and Lo, 2000). NT-4 and BDNF potentiate the increase in dendritic complexity observed after full-length TrkB transfection. Interestingly, transfection with the truncated TrkB receptor T1 increases distal dendritic mass by net elongation of preexisting dendrites, which is similar to the effect of K-252a on its own (Yacoubian and Lo, 2000). Analysis of two different strains of p75-deficient mice revealed an increase in the dendritic complexity of hippocampal pyramidal neurons, while overexpression of p75 had the opposite effect (Zagrebelsky et al., 2005). This suggests that the outcome of neurotrophin action on dendritic development may depend on a balance of competing signals from Trk receptors, which promote dendritic growth, and p75 receptors, which inhibit growth. However, a positive effect of p75 on dendritic growth and complexity in response to BDNF, NT-3, and NGF was reported to occur in young cultures of subventricular zone cells that later become responsive to BDNF only, through the activation of TrkB receptors (Gascon et al., 2005). The identity of the Trk downstream pathways involved in dendritic development has only recently been addressed by studies using pharmacological approaches. Inhibitors of the ERK and Akt pathways were shown to prevent the BDNF-induced increase in primary dendrite number and dendritic complexity in dissociated cortical neurons and cortical slices in culture (Dijkhuizen and Ghosh, 2005a), respectively. PLCg inhibition had only a slight inhibitory effect. Expression of a constitutively active form of PI3K was able to mimic the effect of BDNF on primary dendrite numbers, while constitutively active MEK (mitogen activated protein kinase), the kinase that activates ERK, was without effect (Dijkhuizen and Ghosh, 2005a). This confirms an involvement of the Akt pathway in dendritic development. Interestingly, the PI3K-Akt pathway controls dendritic size in cultured rat hippocampal CA3/CA1 pyramidal neurons, while a coordinated activation of this pathway and the ERK signaling cascade is necessary to regulate dendritic complexity (Kumar et al., 2005). Also suggesting a role for the ERK pathway in dendritic development, the small GTPase Rap1, which is known to lead to sustained ERK activation downstream of Trk receptors in PC12 cells (York et al., 1998), is required for depolarization-induced dendritic growth and branching in rat cortical cultures (Chen et al., 2005). In rat olfactory GABAergic neurons in culture, both PLCg and ERK inhibitors reduce the BDNFinduced increase in neurite branching and elongation. Further studies will be necessary to clarify which Trkactivated signaling pathway or pathways play the most relevant role in neurotrophin-regulated dendritic development, and whether this is a common mechanism among different types of neurons and different regions of the brain. An interesting molecule downstream of TrkB is Tiam1, a guanine-nucleotide exchange factor that activates the Rho family GTPases Rac1 and Cdc42. TrkB directly activates Tiam1 by phosphorylating a specific tyrosine residue on this protein (Miyamoto et al., 2006). TrkB-phosphorylated Tiam1 is capable of activating Rac1 in vitro. Treatment of cultured cortical neurons with BDNF induces the association of TrkB with Tiam1, as well as Tiam1 tyrosine phosphorylation and activation. Moreover, BDNF-induced increase in neurite number and length is inhibited by siRNA knockdown of Tiam1 and by transfection with mutant Tiam1 carrying a single point mutation on the tyrosine residue that is phosphorylated by TrkB (Miyamoto

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et al., 2006). Tiam1 is therefore a promising new TrkB effector, suggesting the existence of a simple signaling pathway with a few intermediaries that closely links TrkB activation and actin cytoskeleton dynamics, which are crucial for dendritic remodeling.

4.5 Interplay Between Neurotrophins and Neuronal Activity As we previously mentioned, neuronal activity is an established regulator of dendritic development. What is the relationship between neurotrophins and activity in this context? Only a few reports address this question and do not offer a consensual answer. One of the initial studies on the effects of neurotrophins on dendritic differentiation, using the ferret visual cortex organotypic cultures, showed that blocking activity, with NMDA or AMPA receptor antagonists or voltage-gated Na+ or Ca2+ channel inhibitors, prevents most of BDNF-induced dendritic growth (McAllister et al., 1996). However, in rat brain organotypic slices, NMDA and AMPA receptor antagonists were not able to inhibit NT-3-induced increase in pyramidal neuron dendritic length and number (Baker et al., 1998). A lack of effect of activity blockade on BDNF-induced increase in GABAergic interneuron dendritic complexity was also demonstrated in mouse organotypic cortical slices (Jin et al., 2003). On the other hand, the effect of BDNF on interneuron dendrite morphology was mimicked by KCl depolarization, which was blocked by anti-BDNF antibodies (Jin et al., 2003). This suggests that neuronal activity induces the release of BDNF, which is responsible for at least some of the activity-induced effects on dendritic differentiation. A similar notion was suggested in studies using cerebellar Purkinje cells and granule neuron co-cultures, where activity-induced release of BDNF by granule neurons was proposed to mediate Purkinje cell dendrite development (Hirai and Launey, 2000). This would be in agreement with previous reports that show that BDNF is secreted in response to activity (Ghosh et al., 1994; Blochl and Thoenen, 1995; Goodman et al., 1996; Wang and Poo, 1997; Hartmann et al., 2001). Whether neurotrophins increase activity, act in conjunction with activity or are released by activity in order to exert their effects on dendritic development is an extremely important issue that needs to be clarified. Judging from the studies mentioned above, the three scenarios may co-exist and the comparative weight of each one of them for neurotrophin-induced dendritic growth and complexity probably depends on the type of neuron analyzed, among other factors. Understanding the relationship between neuronal activity and neurotrophins, and the consequences on higher order function and behavior, is perhaps the most exciting direction in the study of neurotrophins on CNS neuronal differentiation.

Acknowledgments The authors are supported by grants from NIH (NS21072 and HD23315). D.B.P. is supported by a postdoctoral fellowship from ‘‘Fundac¸a˜o Portuguesa para a Cieˆncia e a Tecnologia.’’

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Nerve Growth Factor Regulated Gene Expression

L. A. Greene . J. M. Angelastro

1 Overall Aim and Content of This Chapter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2 The Role of Regulated Gene Expression in the NGF Mechanism—What Genes Does NGF Regulate and Why Do We Want to Know? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3 Regulation of Gene Expression by NGF—A View from the Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 4 Long‐Term Regulation of Gene Expression by NGF—Results of a SAGE Study with PC12 Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 5 Closing Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52

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2008 Springer ScienceþBusiness Media, LLC.

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Nerve growth factor regulated gene expression

Abstract: This chapter provides a current accounting of the identities of genes and gene products that are subject to long‐term regulation by nerve growth factor (NGF). We provide tabular listings of (a) NGF‐ responsive genes and proteins that are reported in the literature and (b) transcripts found by comprehensive serial analysis of gene expression (SAGE) to be differentially expressed in PC12 cells before and after long‐term NGF exposure. Transcriptional changes are key elements in the mechanisms by which NGF affects neuronal differentiation, function, protection, and repair. The information provided here is thus meant to serve as a resource for those interested in the molecular mechanisms and consequences of trophic factor actions. List of Abbreviations: ATF5, activating transcription factor 5; EST, expressed sequence tag; NGF, nerve growth factor; SAGE, serial analysis of gene expression

1 Overall Aim and Content of This Chapter The aim of this chapter is to provide a current account of what we know about the identities of the genes and gene products that are subject to long‐term regulation by NGF. Our objective is like that of traditional handbooks of chemistry and physics, that is, to list information without including extensive interpretive rhetoric. We hope that the reader will find this information to be a useful resource that will inspire further experimental and intellectual advances regarding the development, function, and repair of the nervous system. There are two principal portions of this chapter. One is a tabular listing of the genes and gene products that have been identified in the literature as being regulated by NGF. The second is a tabular listing of identified NGF regulated transcripts that we have detected in a large‐scale SAGE comparison of the transcriptomes of naı¨ve and long‐term NGF‐treated PC12 cells. In each case, the regulated genes have been subdivided into categories based on the current information about their functional properties. Part of the appeal of putting together such a chapter is the opportunity provided by current electronic technology to easily and swiftly update and extend the information herein and to make such modifications rapidly accessible to readers. We intend to continue to add to this chapter new findings in the literature as well as the results of our continuous efforts to fully match the output of our SAGE study with known genes. We will also continue to take into account new findings about the functional activities of the various genes listed here. An additional aspect is that, such a format will permit us to correct errors and omissions that we anticipate have not only occurred, but will be (and we encourage this) pointed out to us by readers. Finally, as noted earlier, the present chapter includes genes and gene products subject to ‘‘long‐term’’ regulation by NGF. By long term, we mean to the exclusion of immediate early genes and potentially, to some genes that are only transiently regulated by NGF. Should the opportunity arise, we will endeavor to add such genes in the future.

2 The Role of Regulated Gene Expression in the NGF Mechanism—What Genes Does NGF Regulate and Why Do We Want to Know? Though we now largely take for granted the notion that neurotrophic factors such as NGF act in part by regulating gene expression, this was not the case always. The capacity of NGF to promote neuron survival as well as differentiation led to an early debate over whether the factor was merely permissive for differentiation or whether it truly possessed instructive actions. Moreover, the relative stability of NGF‐regulated transcripts and proteins and the capacity of NGF to promote neurite outgrowth in explanted ganglia led to the hypothesis that the factor acted by a mechanism that was independent of transcription (Partlow and Larrabbee, 1971; Mizel and Bamburg, 1976). The advent of cell systems such as PC12 cells without a requirement for NGF or without prior exposure to the factor (in contrast to dissected neurons) permitted the first demonstrations that NGF does regulate genes (McGuire et al., 1978 ; Greene, 1981; Greenberg et al., 1985) and that such effects play essential roles in neuronal differentiation and function (Burstein and Greene, 1978; Greene et al., 1983). Moreover, the introduction of means to culture adult sensory neurons, which respond to NGF, but do not require it, has extended our capacity to uncover effects of the factor on gene expression (Lindsay, 1988; Lindsay and Harmar, 1989). Additional advances in neuron culture, in

Nerve growth factor regulated gene expression

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introducing NGF in vivo, and in generating animals that are null for key cell death genes and in which neurons can consequently survive in absence of trophic support, have further widened the opportunities to identify NGF‐regulated genes and gene products. Finally, molecular technologies have greatly improved the sensitivity and efficiency with which NGF‐dependent changes in gene expression can be monitored. If NGF acts in part by regulating genes, then the question naturally arises about their identities. Knowing the identities of NGF‐regulated genes is important for several reasons. NGF participates in a number of fundamental activities including promotion of differentiation (itself a multifaceted response), survival, plasticity, repair, neuroprotection, and neurite outgrowth. Many of the genes regulated by NGF are key regulators and participants in these major activities and their identification thus provides fundamental insight into the molecular mechanisms by which they occur. Furthermore, understanding the mechanisms by which NGF acts, must include a clear description of the genes subject to its regulation. In this context, knowledge about the genes that NGF regulates not only permits construction of mechanistic pathways, but also provides candidates for experimental extension of, and intercalation within, these pathways. Confronting ourselves (and our colleagues) with the knowledge that there are large numbers of NGF‐regulated genes for which we understand neither function nor potential roles, is not only humbling, but also spurs us to understand how they contribute to the NGF mechanism of action. Finally, NGF is one of many neurotrophic factors. If we can identify and understand the functions of the genes that NGF regulates, we will have an enormous insight into the role of such genes in the actions of other trophic factors as well.

3 Regulation of Gene Expression by NGF—A View from the Literature > Table

2‐1 is a listing based on a search of the literature (up to July 2004), of genes and gene products that have been reported to be subject to regulation by NGF. Though we have attempted to be comprehensive, there are likely to be unintentional omissions and readers are invited to point out these for future inclusion. Multiple entries for the same gene/gene product are given in cases in which the findings have been made in different experimental systems, in which observations regarding RNA and protein were in different papers, or in cases in which different authors have reported contrasting results. We have not attempted to list all authors or papers that report similar observations in the same experimental system or necessarily those that were published first. Where sufficient information was given about the molecule in question, we have listed an NCBI accession number. On the basis of information provided within the cited papers, the general literature, and NCBI genome listings, we have subdivided the various genes/proteins into functional categories. In some cases, the same gene is listed under multiple functional headings. We have not quantified the reported changes (and in many cases, neither did the authors); hence, there is no threshold for degree of regulation other than the author’s interpretation that the reported changes were significant. Finally, a word about the definition of gene regulation in the context of this chapter. By this we mean changes in the levels of cellular proteins and transcripts. We are aware that such changes are not necessarily due to altered gene transcription and may arise from changes in stability. However, few of the responses to NGF have been studied at this level; hence, we follow this broader interpretation.

4 Long‐Term Regulation of Gene Expression by NGF—Results of a SAGE Study with PC12 Cells Although the list of NGF‐regulated genes in the literature is quite substantial, we felt that it was likely to be far short of the numbers that are actually responsive to NGF. To address this, in 2000 we carried out a comprehensive comparison, using SAGE technology, of transcripts expressed by rat PC12 pheochromocytoma cells before and after 9days of treatment with NGF (Angelastro et al., 2000). PC12 cells proliferate in serum‐containing medium and do not require NGF for survival. In response to NGF under the same conditions, PC12 cells exit the cell cycle and over a time‐course of days undergo differentiation into neurite‐ bearing, electrically excitable cells that resemble sympathetic neurons. Thus, they represent a convenient system in which to identify NGF‐promoted changes in gene expression. SAGE is an unbiased and highly

23

24

2

Nerve growth factor regulated gene expression

. Table 2-1 NGF regulated changes in gene expression Gene name

Access. #

Up/ Down RNA/Prot Antioxidant

System

References

Catalase (Cat)

NM_012520

Up

R

PC12

Cat Glutathione peroxidase

NM_012520

Up Up

R R

Striatum in vivo PC12

Glutathione‐S‐transferase mu

Up

P

PC12

Glutathione‐S‐transferase pi

Up

P

PC12

AJ277828

Down

R, P

PC12

NM_053800

Up

R, P

PC12

Sampath et al. (1994) Frim et al. (1994) Sampath et al. (1994) Nur‐E‐Kamal et al. (2000) Nur‐E‐Kamal et al. (2000) Naranjo‐Suarez et al. (2003) Bai et al. (2003)

Hypoxia inducible factor 2 alpha (Hif‐2a) Thioredoxin

Calcium binding Calbindin 1 (Calb1)

NM_031984

Up

P, R

PC12

Up

R

PC12

NM_019905

Up

P, R

PC12

NM_031114

Up

R

PC12

Calmodulin Calpactin I heavy chain/ Annexin II S‐100 related protein, clone 42C/calpactin I light chain

Iacopino et al. (1992) Bai and Weiss (1991) Jacovina et al. (2001) Masiakowski and Shooter (1988)

Cytoskeleton and neurite outgrowth Alpha actinin

Up

P

PC12

Alpha‐tubulin/T alpha 1 alpha‐ tubulin (Tuba1) Tuba1

NM_022298

Up

R

PC12

NM_022298

Up

R

Tuba1

NM_022298

Up

R

Adenomatous polyposis coli (APC) Beta‐1 adducin

NM_012499

Up

P

Sympathetic neurons in vivo Adult DRG in vitro PC12

AF130338

Up

R

Cultured DRG’s

Gamma‐actin

X52815

Up

P

PC12

Growth associated protein 43 (Gap43) Gap43

NM_017195

Up

P, R

PC12

NM_017195

Up

R

Up

P

Adult sensory neurons in vitro PC12

Gelsolin Integrin alpha 1 (Itga1)

NM_030994

Up

R

PC12

Microtubule‐associated protein 1b (Map1b)

AF079778

Up

P

PC12

Sobue and Kanda (1989) Miller et al. (1987) Mathew and Miller (1990) Mohiuddin et al. (1995) Dobashi et al. (1996) Ghassemi et al. (2001) Chang et al. (1986) Costello et al. (1990) Mohiuddin et al. (1995) Furnish et al. (2001) Danker et al. (2001) Greene et al. (1983)

Nerve growth factor regulated gene expression

2

. Table 2-1 (continued) Gene name Microtubule‐associated protein tau (Mapt) Microtubule‐associated protein 2 (Mtap2) Neurofilament, light polypeptide (Nfl) Nef3 neurofilament 3, medium/Nfm Neuronal kinesin heavy chain

Access. # NM_017212

Up/ Down Up

RNA/Prot P, R

System PC12

NM_013066

Up

P, R

PC12

NM_031783

Up

P, R

PC12

NM_017029

Up

P, R

PC12

Up

P, R

Peripherin 1 (Prph1)

NM_012633

Up

P, R

PC12, neuroblastoma in vitro PC12

Stathmin 1 (Stmn1)

NM_017166

Up

R

PC12

Stathmin‐like 2/SCG10 (Stmn2) Thymosin beta‐4

NM_053440

Up

R

PC12

NM_031136

Up

R

PC12

Tropomyosin 1 (Tpm1), alpha/ alpha‐tropomyosin (TMBr‐1) Tropomyosin 1 (Tpm1), alpha/ alpha‐tropomyosin (TMBr‐3)

M34135

Up

R

PC12

M34136

Up

R

PC12

Up

P

PC12

Down

P

PC12

NM_199501

Down

P

PC12

NM_053593

Down

P

PC12

NM_080782

Up

P, R

PC12

NM_171991

Down

P

PC12

Cyclin D1 (Ccnd1)

NM_171992

Up

P, R

PC12

Cyclin D2 (Ccnd2)

D16308

Down

P

PC12

Cyclin F (Ccnf)

XM_340763

Down

P, not R

PC12

Up

P

PC12

References Drubin et al. (1985) Fischer et al. (1991) Lindenbaum et al. (1987) Lindenbaum et al. (1987) Vignali et al. (1996) Aletta et al. (1988) Takekoshi et al. (1998) Stein et al. (1988) Leonard et al. (1987) Weinberger et al. (1993) Weinberger et al. (1993)

Cell cycle APC

NM_012499

Cdc2/Cdk1 Cyclin dependent kinase 2 (Cdk2) Cyclin‐dependent kinase 4 (CDK4) Cdkn1a cyclin‐dependent kinase inhibitor 1A/Waf1 Cyclin B1 (Ccnb1)

Cyclin G Deleted in colorectal carcinoma (Dcc)

U68725

Up

R

PC12

E2F transcription factor 1 (E2f1)

D63165

Down

P

PC12

Dobashi et al. (1996) Buchkovich and Ziff (1994) Buchkovich and Ziff (1994) Yan and Ziff (1995) Yan and Ziff (1997) Yan and Ziff (1995) Yan and Ziff (1995) Tamaru et al. (1994) Movsesyan et al. (1996) Gollapudi and Neet (1997) Lawlor and Narayanan (1992) Persengiev et al. (1999)

25

26

2

Nerve growth factor regulated gene expression

. Table 2-1 (continued) Up/ Down Up

RNA/Prot P

System PC12

Down

P

PC12

Up

P

PC12

XM_342209

Down

P

PC12

NM_053484

Up

P

PC12

NM_031094

Up

P, R

PC12

NM_022381

Down

P

PC12

XM_344434

Down

P

PC12

Gene name E2F transcription factor 2 (E2f2) E2F transcription factor 3 (E2f3) E2F transcription factor 4 (E2f4) E2F transcription factor 5 (E2F5) Growth arrest specific 7 (GAS7) Retinoblastoma‐like 2 (Rbl2); p130 Proliferating cell nuclear antigen (Pcna) Retinoblastoma 1 (Rb1)

Access. #

References Persengiev et al. (2001) Persengiev et al. (2001) Persengiev et al. (2001) Persengiev et al. (2001) Chao et al. (2003) Paggi et al. (2001) Yan and Ziff (1995) Persengiev et al. (2001)

Bcl2l1 Bcl2‐like 1/Bcl‐xl

NM_031535

Up

R

PC12

Bcl2l1 Bcl2‐like 1/Bcl‐xs

NM_031535

Up

R

PC12

Bcl2l11 BCL2‐like 11 (apoptosis facilitator)/BIM Caspase 3 (Casp3)

NM_022612

Down

P

PC12

NM_012922

Up

R

PC12

Clusterin (Clu)

NM_053021

Up

R

PC12

ADP‐ribosyltransferase 1 (Adprt)

U94340

Down

R, P

PC12

Amyloid beta (A4) precursor protein (App) App

NM_019288

Up

P, R

PC12

Villa et al. (2001)

NM_019288

Up

R

Prion protein (Prnp)

NM_012631

Up

R

Developing brain PC12

Prnp

NM_012631

Up

R

Mobley et al. (1988) Wion et al. (1988) Mobley et al. (1988)

Death and survival Rong et al. (1999) Rong et al. (1999) Biswas and Greene (2002) Rong et al. (1999) Gutacker et al. (1999) Taniguchi et al. (1988)

Disease related

Developing brain

Growth factors and receptors/signal transduction Epidermal growth factor receptor (EGFR) Fibroblast growth factor receptor 1 (FGFR1) Interleukin 1 alpha (Il1a)

NM_031507

Down

R

PC12

NM_024146

Up

R

PC12

NM_017019

Up

R, P

PC12

Transforming growth factor, beta 1 (Tgfb1)

NM_021578

Up

R, P

PC12

Shibutani et al. (1998) Meisinger et al. (1996) Alheim et al. (1996) Kim et al. (1994)

Nerve growth factor regulated gene expression

2

. Table 2-1 (continued) Gene name Tgfb1

Access. # NM_021578

Up/ Down Up

RNA/Prot R, P

Vascular endothelial growth factor (Vegf)

NM_031836

Down

R

Ca channel, voltage‐ dependent, N type, alpha 1B subunit Cacna1c Ca channel, voltage‐ dependent, L‐type alpha 1C Kcnb1 K voltage gated channel, Shab‐related, member 1 Cacna1d Ca channel, voltage‐ dependent, L‐type alpha 1D K small conductance Ca‐ activated channel N1 (Kcnn1) Proton gated cation channel DRASIC Na channel, voltage‐gated, type 1, a polypeptide (Scn1a) Na channel, voltage‐gated, type 2, a1 polypeptide (Scn2a1) Na channel, voltage‐gated, type 2, a1 polypeptide (Scn2a1) Na channel, voltage‐gated, type III, a polypeptide (Scn3a) Na channel, voltage‐gated, type 9, a polypeptide (Scn9a) Na channel, voltage‐gated, type 10, a polypeptide (Scn10a) Na channel, voltage‐gated, type I, beta polypeptide (Scn1b)

NM_147141

Up

NM_012517

System Chromaffin cells in oculo PC12

References Forander et al. (2001) Naranjo‐Suarez et al. (2003)

R

PC12

Colston et al. (1998)

Down

R

PC12

NM_013186

Up

P, not R

PC12

Colston et al. (1998) Sharma et al. (1993)

NM_017298

Down

R

PC12

NM_019313

Up

P

DRG in vitro

NM_173135

Up

R

NM_030875

Up

R

NM_012647

Up

R

Sensory neurons DRG neurons in vitro PC12

NM_012647

Up

R

DRG neurons in vitro

Zur et al. (1995)

NM_013119

Down

R

NM_133289

Up

R

Adult DRG in vitro PC12

NM_017247

Up

P

Adult DRG in vitro

Black et al. (1997) D’Arcangelo et al. (1993) Black et al. (1997)

NM_017288

Up

R

DRG neurons in vitro

Zur et al. (1995)

Kuklinski et al. (2003) Pesheva et al. (2000)

Ion channels

Colston et al. (1998) Boettger et al. (2002) Mamet et al. (2003) Zur et al. (1995) Mandel et al. (1988)

Lectins Lectin, galactose binding, soluble 3 (Lgals3) Lgals3

NM_031832

Up

P, R

PC12

NM_031832

Up

P

DRG in vitro

Miscellaneous þ þ

ATPase, Na K transporting, alpha 1 Collagenase 1 CTP: phosphocholine cytidylyltransferase beta2

NM_012504

Up

P

PC12

Up

R

PC12

Up

R

PC12

Kurihara et al. (1994) Vician et al. (1997) Carter et al. (2003)

27

28

2

Nerve growth factor regulated gene expression

. Table 2-1 (continued) Up/ Down Down

RNA/Prot R

System PC12

Up

R

PC12

NM_017043

Up

P, R

PC12

NM_031967

Up

P

PC12

Up

P

PC12

Up

P

PC12

Up

R

PC12

Up

R

PC12

NM_053601

Down

R

PC12

Ornithine decarboxylase 1 (Odc1) Peripheral myelin protein 22

NM_012615

Up

P, R

PC12

NM_017037

Up

R

PC12

Plaur plasminogen activator, urokinase receptor Serine (or cysteine) proteinase inhibitor, member 1 Similar to polypyrimidine tract binding protein 2/PTBLP‐L Similar to polypyrimidine tract binding protein 2/PTBLP‐S Similar to REN

NM_017350

Up

R

PC12

NM_012620

Up

R

PC12

Down

R

PC12

Up

R

PC12

XM_343923

Up

R

Tff3 trefoil factor 3/ITF

NM_013042

Up

P, R

PC12, TC‐1S cells PC12

Brain derived neurotrophic factor (Bdnf) NGF receptor (Ngfr) (TNFR superfamily, member 16)/p75 Ngfr/p75

NM_012513

Up

R

NM_012610

Up

P, R

NM_012610

Up

R

Ngfr/p75

NM_012610

Up

R

Trk precursor

NM_021589

Up

R

Trk precursor

NM_021589

Up

R

Gene name Lactic Dehydrogenase type M (LDH‐M) Low density lipoprotein receptor related protein LRP1B/LRP‐DIT Prostaglandin‐endoperoxide synthase 1 (Ptgs1) N‐myc downstream regulated 4 (Ndr4) Neuroendocrine‐Specific Protein‐A (NSP‐A) Neuroendocrine‐Specific Protein‐C (NSP‐C) NID67 putative small membrane protein Nin283/Znfr1

Access. #

Nnat neuronatin

NM_173126

References Calissano et al. (1985) Bu et al. (1998)

Kaplan et al. (1997) Nakada et al. (2002) Hens et al. (1998) Hens et al. (1998) Vician et al. (2001) Araki et al. (2001) Joseph et al. (1996) Feinstein et al. (1985) De Leon et al. (1994) Farias‐Eisner et al. (2000) Vician et al. (1997) Ichikawa et al. (2002) Ichikawa et al. (2002) Gallo et al. (2002) Probst et al. (1997)

Neurotrophins and receptors Adult DRG’s in vivo Cholinergic neurons in vivo

Apfel et al. (1996) Cavicchioli et al. (1989)

DRG neurons in vitro Sympathetic neurons, PC12 Adult DRG in vivo Forebrain neurons in vitro

Lindsay et al. (1990) Miller et al. (1991) Mearow (1998) Kojima et al. (1994)

Nerve growth factor regulated gene expression

2

. Table 2-1 (continued) Gene name

Access. #

Up/ Down RNA/Prot Neuropeptides

System

References

Adenylate cyclase activating polypeptide 1 (Adcyap1)/ PACAP Adcyap1/PACAP

NM_016989

Up

R

PC12

Hashimoto et al. (2000)

NM_016989

Up

R

PACAP receptor 1 (Adcyap1r1) Adrenomedullin (Adm)

NM_133511

Up

R

Adult DRG in vivo PC12

NM_012715

Down

P, R

PC12

Angiotensin II receptor, type 2 (Agtr2)

NM_012494

Down

R

Bradykinin receptor b2 (Bdkrb2) Calcitonin/calcitonin‐related polypeptide, alpha (Calca) Galanin (Gal)

NM_173100

Up

P, R

NM_017338

Up

P, R

NM_033237

Up

R

Gal

NM_033237

Down

R

Neuropeptide Y (Npy) Neurotensin/neuromedin N gene Tachykinin 1 (Tac1)

NM_012614 M21187

Up Up

P, R P, R

Cultured hypothalamus/ brainstem Cultured adult DRG Cultured adult DRG Basal forbrain in vivo DRG, in vitro and in vivo PC12 PC12

Jongsma Wallin et al. (2001) Cavallaro et al. (1995) Kobayashi et al. (2004) Huang et al. (1997)

NM_012666

Up

P, R

Vanilloid receptor (Vr1)/Trpv1

AF327067

Up

R

Neuropeptide Y receptor Y1 (Npy1r)

XM_344502

Up

R

Acetylcholinesterase (Ache)

NM_172009

Up

Adenosine A2a receptor (Adora2a) Agrin (Agrn)

NM_053294 NM_175754

DRG, trigeminal neurons in vitro Adult DRG neurons in vitro PC12

Lee et al. (2002) Lindsay and Harmar (1989) Planas et al. (1997) Corness and Hokfelt (1998) Allen et al. (1987) Caillaud et al. (1995) Vedder et al. (1993) Winston et al. (2001) Bournat et al. (2001)

Neurotransmission

Choline acetyltransferase (ChAT) Cholinergic receptor, muscarinic 1 (Chrm1) Cholinergic receptor, muscarinic 2 (Chrm2) Cholinergic receptor, muscarinic 3 (Chrm3) Cholinergic receptor, muscarinic 4 (Chrm4) Chrm4

PC12

Down

R (stabilized) P, R

Up

R

PC12

Up

R

NM_080773

Down

R

NM_031016

Up

R

NM_012527

Down

R

XM_345403

Up

R

XM_345403

Up

R

Basal forebrain in vivo Telencephalic neurons in vitro Striatal neurons in vitro Telencephalic neurons in vitro Telencephalic neurons in vitro PC12

PC12

Deschenes‐Furry et al. (2003) Arslan et al. (1997) Smith et al. (1997) Higgins et al. (1989) Eva et al. (1992) Ebstein et al. (1993) Eva et al. (1992) Eva et al. (1992) Lee and Malek (1998)

29

30

2

Nerve growth factor regulated gene expression

. Table 2-1 (continued) Gene name Chromogranin A (Chga)

Access. # NM_021655

Up/ Down Up

RNA/Prot R

System PC12

Cholinergic receptor, nicotinic, alpha polypeptide 3 (Chrna3) Chrna3

NM_052805

Up

R

PC12

NM_052805

Down

R

PC12

Cholinergic receptor, nicotinic, alpha polypeptide 5 (Chrna5) Chrna5

NM_017078

Up

R

PC12

NM_017078

Up

R

PC12

Chrna5

NM_017078

Down

R

PC12

Cholinergic receptor, nicotinic, alpha polypeptide 7 (Chrna7) Cholinergic receptor, nicotinic, beta polypeptide 2 (Chrnb2) Cholinergic receptor, nicotinic, beta polypeptide 3 (Chrnb3) Chrnb3

NM_012832

Up

R

PC12

NM_019297

Up

R

PC12

AY574259

Up

R

PC12

AY574259

Down

R

PC12

Cholinergic receptor, nicotinic, beta polypeptide 4 (Chrnb4) Chrnb4 Chrnb4

NM_052806

Up

R

PC12

NM_052806 NM_052806

Up Down

R R

PC12 PC12

Dopamine beta hydroxylase (Dbh) Dopa decarboxylase (Ddc) Dystrophin Dp71

NM_013158

Down

R

PC12

NM_012545

Down Up

R P, R

PC12 PC12

Glutamate receptor, metabotropic 1 (Grm1) GTP cyclohydrolase 1 (Gch)

NM_017011

Up

P, R

PC12

NM_024356

Up

R

PC12

Gch

NM_024356

Up

R

Nitric oxide synthase 1, neuronal (Nos1) Rab3a: RAB3A, member RAS oncogene family Nitric oxide synthase 1, neuronal (Nos1) Secretogranin 2 (Scg2)

NM_05279

Up

P,R

Sympathetic neurons in vitro PC12

NM_013018

Up

R

PC12

NM_05279

Up

R

NM_022669

Up

P, R

Cholinergic neurons in vivo PC12

Synuclein, alpha (Snca)

NM_019169

Up

P, R

PC12

Solute carrier family 6, member 2/noradenalin transporter

NM_031343

Down

R

PC12

References Mahata et al. (1999) Henderson et al. (1994) Rogers et al. (1992) Takahashi et al. (1999) Henderson et al. (1994) Rogers et al. (1992) Henderson et al. (1994) Rogers et al. (1992) Takahashi et al. (1999) Rogers et al. (1992) Henderson et al. (1994) Avila et al. (2003) Rogers et al. (1992) Badoyannis et al. (1991) Li et al. (1997) Cisneros et al. (1996) Kane et al. (1998) Anastasiadis et al. (1996) Hirayama and Kapatos (1995) Sheehy et al. (1997) Sano et al. (1989) Holtzman et al. (1996) Laslop and Tschernitz (1992) Stefanis et al. (2001) Ikeda et al. (2001)

Nerve growth factor regulated gene expression

2

. Table 2-1 (continued) Gene name Synapsin 1 (Syn1)

Access. # NM_019133

Up/ Down Up

RNA/Prot P

System PC12

Synaptophysin (Syp)

NM_012664

Down

P

PC12

Synaptotagmin 1 (Syt1)

XM_343205

Up

P

PC12

Tyrosine hydroxylase (Th)

NM_012740

Up

P

Th

NM_012740

Up

R

Th

NM_012740

Up

P

Sympathetic ganglia in vitro Sympathetic neurons in vitro PC12

Solute carrier family 18, member 3 (Slc18a3)/VAChT VGF nerve growth factor inducible (Vgf)

NM_031663

Up

R

NM_030997

Up

R

References Romano et al. (1987) Vetter and Betz (1989) Lah and Burry (1993) Max et al. (1978) Ma et al. (1992)

Septal neurons in vitro PC12

Osaka and Sabban (1997) Oosawa et al. (1999) Levi et al. (1985)

Signal transduction Aldehyde dehydrogenase family 1, subfamily A2 (Aldh1a2) Dual specificity phosphatase 6 (Dusp6)/MKP‐3 Guanine nucleotide binding protein, alpha o (Gnao) G beta

NM_053896

Up

R

Adult DRG in vitro

Corcoran and Maden (1999)

NM_053883

Up

R

PC12

NM_017327

Up

P

PC12

Up

P

PC12

Gnai2: GTP‐binding protein (G‐ alpha‐i2) Guanylate cyclase 1, soluble, alpha 3 (Gucy1a3) Guanylate cyclase 1, soluble, beta 3 (Gucy1b3) Neurofibromatosis type I (NF1) Opioid receptor, sigma 1 (Oprs1) Phospholipase D1 (Pld1)

NM_031035

Up

P

PC12

NM_017090

Down

P, R

PC12

Mourey et al. (1996) Zubiaur and Neer (1993) Zubiaur and Neer (1993) Zubiaur and Neer (1993) Liu et al. (1997)

NM_012769

Down

P, R

PC12

Liu et al. (1997)

Up

R

PC12

NM_030996

Up

P

PC12

NM_030992

Up

R

PC12

Protein kinase C, alpha (Prkca) Protein kinase C, beta 1 (Prkcb1) Phospholipase D2 (Pld2)

XM_343975 NM_012713

Up Up

P P

PC12 PC12

Metheny and Skuse (1996) Takebayashi et al. (2002) Hayakawa et al. (1999) Min et al. (2001) Min et al. (2001)

NM_033299

Up

P, R

PC12

Protein tyrosine phosphatase, non‐receptor type 16/MKP‐1 Protein tyrosine phosphatase, receptor type, R (Ptprr)

NM_053769

Up

R

NM_053594

Transient Up

R

Sympathetic neurons in vitro PC12

Gibbs and Meier (2000) Peinado‐Ramon et al. (1998) Sharma and Lombroso (1995)

31

32

2

Nerve growth factor regulated gene expression

. Table 2-1 (continued) Gene name Ptprr

Access. # NM_053594

Protein tyrosine phosphatase, receptor type, epsilon (Ptpre) Retinoic acid receptor beta2

XM_341950

Thioredoxin

NM_053800

Up/ Down Down

RNA/Prot R

System PC12h

Transient Up Up

R

PC12

P, R

PC12

Up

R, P

PC12

References Shiozuka et al. (1995) Mukouyama et al. (1997) Cosgaya and Aranda (2001) Bai et al. (2003)

Substrate interactions Integrin alpha 1 (Itga1)

NM_030994

Up

R

PC12

Neural cell adhesion molecule L1 (NcamL1) Neural cell adhesion molecule 1 (Ncam1) Matrix metalloproteinase 3 (Mmp3)/stromelysin1 Thymus cell antigen 1, theta (Thy1)

NM_017345

Up

P, R

PC12

NM_031521

Up

P, R

PC12

NM_133523

Up

R

PC12

NM_012673

Up

P,W

PC12

Achaete‐scute complex homolog‐like 1 (Ascl1)/ MASH1 AP‐2

NM_022384

Activating transcription factor 5 (ATF5) DNA (cytosine‐5‐)‐ methyltransferase 1 dnmt1 Estrogen receptor

Danker et al. (2001) Grant et al. (1996) Prentice et al. (1987) Machida et al. (1989) Doherty et al. (1988)

Transcriptional regulators Down

R

PC12

Grumolato et al. (2003)

Up

P

PC12

NM_172336

Down

P, R

PC12

NM_053354

Down

P, R

PC12

Up

P, R

PC12

Up

R

PC12

NM_012953

Up

P

PC12

NM_012954

Up

P

PC12

NM_145880

Up

R

DRG in vitro

AJ277828

Down

R, P

PC12

AF370447

Paggi et al. (2001) Angelastro et al. (2003) Deng and Szyf (1999) Sohrabji et al. (1994) Rhodes et al. (2003) Cosgaya and Aranda (2001) Cosgaya and Aranda (2001) Jameson and Lillycrop (2001) Naranjo‐Suarez et al. (2003) Jameson and Lillycrop (2001) Uittenbogaard and Chiaramello (2002) Chen et al. (1994)

Fak1/fetal Alzheimer antigen/ falz Fos‐like antigen 1/FRA‐1 (Fosl1) Fos‐like antigen 1/FRA‐ 2 (Fosl2) LIM homeobox protein 1 (Lhx1)/Rlim Hypoxia inducible factor 2 alpha (Hif‐2a) LIM homeobox protein 3 (Lhx3) Math2/Nex1/Neurod6

Up

R

DRG in vitro

XM_001059051 Up

P

PC12

C‐myc

Y00396

R, P

Human neuroblastoma lines

Down

Nerve growth factor regulated gene expression

2

. Table 2-1 (continued) Gene name Mycn v‐myc viral related oncogene, neuroblastoma derived P48ZnF (transcription factor)

Access. # XM_234025

Up/ Down Down

RNA/Prot R

System Neuroblastoma

References Woo et al. (2004)

AY377983

Up

R

PC12

POU domain, class 2, transcription factor 2/Oct2 Sox21

XM_341802

Up

R, P

Down

P

Sensory neurons in vitro PC12

Tumor protein p53 (Tp53)

NM_030989

Up

P

PC12

Wilms tumor 1 (Wt1)

NM_031534

Down

R

PC12

Heese et al. (2004) Wood et al. (1992) Ohba et al. (2004) Gollapudi and Neet (1997) Liu et al. (2001)

Eukaryotic translation initiation factor 2B (eIF2B)

Z48225

Translational regulators Up

P

PC12

Kleijn et al. (1998)

sensitive approach to defining cellular transcriptomes (Velculescu etal., 2000). Put briefly, each transcript recovered from the cell of interest is converted into a defining SAGE ‘‘tag,’’ which includes the most 30 CATG in the transcript followed by the next 11 bases. The relative number of tags representing a given transcript that are recovered in the analysis is directly proportional to its relative abundance in the cell. If a large number of SAGE tags are analyzed to compare the same cell in two different states (in this case
NGF treatment), this provides a very comprehensive view of the changes that occur in gene regulation. In our study, we analyzed large libraries of approximately 80,000 tags each (76,280 without NGF; 87,004 with NGF). The results revealed that approximately 4% of the 22,000 transcripts detected in PC12 cells responded to NGF by undergoing changes in expression of sixfold or greater. One of the challenges of interpreting SAGE data is to match the various tags with known transcripts. Thus far, we have, by a variety of informatic means (Angelastro etal., 2000), matched to known transcripts approximately half of the tags representing genes that are regulated by sixfold or more in response to long‐term NGF exposure. > Table 2-2 lists the NGF‐responsive transcripts thus far (up to September 2004) identified from our SAGE study. The fold changes in expression are listed either as fold increase after NGF treatment (positive numbers) or as fold decrease in expression after NGF treatment (negative numbers). When no tags were detected, for the purposes of calculating a fold change, the tag number was set to 1 (to avoid a ratio of infinity). Thus, some of the fold changes may be underestimated. The fold changes reported reflect normalization for the differences in tag numbers for the two libraries. With the sixfold cutoff that we have chosen, all reported changes are significant with a p value of 0.05 or less. As in the previous table, we have subdivided the regulated genes into functional categories and in some cases, we have listed the same gene under multiple categories. We have also included relevant accession numbers. The present table updates and substantially expands the lists we published in Angelastro etal. (2000) and such additions reflect the enormous increase in genome information available since that time. We have done our very best to ensure the accuracy of the data presented in the table, but acknowledge that errors are possible. Readers are encouraged to provide feedback in this respect. We wish to note that because of the considerable delay between preparation of > Table 2-1 (September, 2004) and publication of this chapter, the Table does not reflect recent advances in annotation of the rat genome. Nevertheless, the accession numbers given in the Table should permit the interested reader to find the current annotation for each of the listed transcripts. It is our intention to provide updates for the Table in future electronic versions of this chapter. Readers may also directly request the lastest version of this table by email ([email protected]).

33

34

2

Nerve growth factor regulated gene expression

. Table 2-2 NGF‐PROMOTED long‐term changes in gene regulation in PC12 cells as detected by SAGE Tag

(þ) NGF

(;) NGF

Fold change

AAGGTTCACTC

19

1

17

GCATACGGCGC

18

0

16

AGCTTGATTAA

16

1

14

GTGGCCCACTT

23

2

10

AATAAAAGTTC

15

2

7

TATCCAAACAG

7

1

6

CCAAGGAAAAC TACCATCTTTC

51 1

8 6

6 7

TACTAGAAAAG

1

7

8

ATCCAAGTCGC

1

8

9

AGGTCGCTTGG

1

8

9

CCCGACTGGGT

5

66

15

GCTGGAATTGA AAGGGTCCCCG

10 8

1 0

9 7

TCTGTCCTGCT

8

0

7

ATTTGCTTCTT

8

0

7

TTTCAGCAGTG

7

0

6

GGCCCCCAAGT

1

5

6

GCTTTAATGGA

6

31

6

GACAATGAAAA

2

12

7

GGAGGACCTCG

2

12

7

Identity Energetics

Accession #

Similar to Mtch1 protein (mitochondrial carrier homolog 1) Atp5k: ATP synthase, Hþ transporting, mitochondrial F1F0 complex, subunit e Similar to RIKEN cDNA 2410011G03O; 0rtholog of human NADH: ubiquinone oxidoreductase Cox17: cytochrome c oxidase, subunit XVII assembly protein homolog (yeast) Atp5a1: mitochondrial Hþ‐ATP synthase alpha subunit Pdha1: pyruvate dehydrogenase E1 alpha 1 Ldha: lactate dehydrogenase A Similar to integral membrane protein CII‐3/ Sdhc: succinate dehydrogenase complex, subunit C Similar to NADH dehydrogenase (ubiquinone) Fe–S protein 2 Similar to Cytochrome oxidase biogenesis protein OXA1, mitochondrial precursor (OXA1‐ like protein) (OXA1Hs) Similar to xylulokinase homolog; xylulokinase (H. influenzae) homolog Homolog of human LOC56901: ubiquinone oxidoreductase MLRQ subunit homolog Metabolism

XM_215358

Farnesyl diphosphate synthase (Fdps) Prpsap1: phosphoribosylpyrophosphate synthetase‐associated protein (39kDa) Degs: degenerative spermatocyte homolog (Drosophila); transmembrane protein involved in meiosis in fly; lipid metabolism Similar to ectonucleotide pyrophosphatase/ phosphodiesterase 5 Hprt: hypoxanthine guanine phosphoribosyl transferase Ortholog of murine BC004012: cDNA sequence BC004012; putative inorganic polyphosphate/ ATP‐NAD kinase Similar to 2310047E01Rik protein; encodes ortholog of murine Car12: carbonic anyhydrase 12 Isocitrate dehydrogenase 3 (NADþ), gamma (Idh3g) Dimethylarginine dimethylaminohydrolase 2 (Ddah2)

NM_080481 XM_216880

NM_053540 NM_023093 XM_343787 NM_017025 XM_2139361

XM_213940 XM_214182

NW_000361 NM‐020142

NM_031840 NM_022545 XM_346454

XM_236956 NM_012583 BC004012

XM_343416

X74125 XM_215315

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag TTGGGGGGTGA

(þ) NGF 2

() NGF 12

Fold change 7

GACCCCTCAAA TGTCCAGCTGG

1 1

7 7

8 8

CTGGGTGGGGG

4

32

9

AGACCTAGGAA CTTGTGACAGG TTTACAGCTGC

1 0b 0

8 9 10

9 10 11

ACCTACAGGAT

1

12

14

ATCCCTCCCCA

1

15

17

AATGTGAGTCA TGCACAGTGCT AAATCCTTTCA TGTAGCTCAAT

14 14 12 11

0 0 0 1

12 12 11 11

ACTCGGAGCCA

10

0

9

AGACACTTCCT ATTCTGTGCTG GGTGTGCCAGG

10 10 10

1 1 0

9 9 9

AAGCCTTGCTG

9

0

8

TTTTGTGATGG

9

0

8

TCAGGCATTTT CCCCCTGGATC CTGCCATCCCT

9 26 8

0 3 0

8 8 7

TAGTCCAGGCT

8

0

7

TCCTGTGTCCT CTTCCAAATGT AATGCCCCCAG CGCGCGCGCGC

8 15 7 7

1 2 1 0

7 7 6 6

CTGTTAGGTGG

7

0

6

Identity Hadha: hydroxyacyl‐Coenzyme A dehydrogenase/3‐ketoacyl‐Coenzyme A hiolase/enoyl‐Coenzyme A hydratase (trifunctional protein), a subunit Glutaredoxin 2 (Glrx2) (thioltransferase) Similar to thymidine kinase 2, mitochondrial; thymidine kinase 2 Similar to dehydrogenase/reductase (SDR family) X chromosome Similar to ecto ADP‐ribosylhydrolase Monoglyceride lipase (Mgll) Similar to cysteine‐tRNA ligase isoform b; cysteine translase; cysteine‐tRNA synthetase Branched chain alpha‐ketoacid dehydrogenase subunit E1 alpha (Bckdha) Ortholog of human COMMD1: copper metabolism (Murr1) domain containing 1

Accession # NM_130826

XM_213890 XM_226211 XM_213723 XM_342918 NM_138502 XM_215134 J02827 NM_152516

Signaling 14‐3‐3 protein gamma‐subtype S100a4: S100 calcium‐binding protein A4 Pleiotrophin Guanine nucleotide binding protein, alpha o (Gnao) Calmodulin 1 (Calm1) (phosphorylase kinase, delta) Anxa2: calpactin I heavy chain/ANNEXIN 2 Cd9: CD9 antigen (p24) Similar to RIKEN cDNA 5730466P16; similar to protein PM1; putative receptor protein Growth factor receptor bound protein 2 (Grb2) Similar to RIKEN cDNA 2010107K23 encoding protein with MAGE domain; Homolog to human MAGE‐H1 Similar to Ras‐related protein Rab‐1B S100a6 calcium binding protein A6 (calcyclin) LOC294900: similar to PC‐1; Tpd52: tumor protein D52; possible roles Ca‐mediated signaling and proliferation LOC291675: similar to RIKEN cDNA 2010001M09—encodes protein similar to human PACAP Inositol 1, 4, 5‐triphosphate receptor 3 (Itpr3) Rtn4: Nogo‐A Presenilin‐2 (Psen2) Ptpns1: protein tyrosine phosphatase, non‐ receptor type substrate (Bit, SHPS‐1) Nicastrin Component of the gamma‐secretase complex (Ncstn)

NM_019376 NM_012618 NM_017066 NM_017327 NM_031969 NM_019905 X76489 NM_003876 NM_030846 XM_346291

X13905 NM_0533485 XM_215524

AI028965

NM_013138 NM_031831 NM_031087 NM_013016 NM_174864

35

36

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TAGGTGTCAAA GCGAATACAAG GCTACAGGGAG

(þ) NGF 7 7 7

() NGF 0 0 1

Fold change 6 6 6

ACGTGCATCAT

1

5

6

GACTGGAAGCT

1

5

6

AGTCCCTCCCG

1

5

6

CGCGCGCTAGT

1

5

6

CCAGCCAGCGT

1

5

6

CTGCTAGCACC

1

5

6

CCCCACACTGG

1

5

6

TGGAGGAGGCG

1

5

6

GGCACCTCCTA

2

11

6

CACACAAAAAA

1

5

6

CTGTTAGAACT

1

5

6

CTGCTGTGTGG

2

10

6

CTGTCAAGACC

0

5

6

TTTGTGGGAGG ACCACACCGGC

1 0

5 5

6 6

CTTTCTGTAAT

0

5

6

GGTTGATTCTG

0

5

6

TCTCACCCACT TAGAGTGTAAA

0 1

6 6

7 7

Identity Mir16: membrane interacting protein of RGS16 Tspan‐2: Tspan‐2 protein; tetraspan protein Similar to rod outer segment membrane protein 1; tetraspanin family RGD:628676: protein phosphatase 1G (formerly 2C), magnesium‐dependent, gamma isoform Ortholog of murine1700019B16Rik: RIKEN cDNA 1700019B16 gene putative G protein receptor Protein kinase LYK5; STRAD; activates tumor suppressor LKB1 involved in cell polarity LOC292887: similar to lobe homolog‐like; mouse ortholog¼Akt1s1, AKT1 substrate 1 (proline‐rich), binds 14‐3‐3 Crcp: calcitonin gene‐related peptide‐receptor component protein Similar to Epidermal growth factor receptor pathway substrate 8‐like protein 2; Eps8l2; PTB domain/Eps8 actin regulator Ortholog of murine Ssh3: slingshot homolog 3 (Drosophila); Slingshot family phosphatases that dephosphorylate cofilin LOC308453: similar to aarF domain containing kinase 4 XM_218358 Similar to Serine/threonine‐protein kinase SNK (Serum inducible kinase) Ortholog of murine Gmip: Gem‐interacting protein putative Rho‐GAP activity Protein phosphatase 4, regulatory subunit 1 (Ppp4r1) Aip: aryl‐hydrocarbon receptor‐interacting protein/XAP2; may play positive role in AHR‐ mediated signaling LOC293783: similar to 4933402K05Rik protein; ortholog of murine Lpxn: leupaxin; homologous to paxillin Serine threonine kinase pim3 Inositol 1,4,5‐trisphosphate 3‐kinase C (Itpkc); conversion IP 1,4,5 to IP 1,3,4,5 Ortholog of human FGF11: fibroblast growth factor 11 Ortholog of human PDE4D phosphodiesterase 4D, cAMP‐specific (phosphodiesterase E3 dunce homolog, Drosophila) Haspp28: kinase substrate HASPP28 3‐Phosphoinositide dependent protein kinase‐1 (Pdpk1)/PDK1

Accession # NM_032615 NM_022589 XM_219564 NM_147209 NM_028829

NM_182820 XM_238103

NM_053670 XM_341958

NM_198113

XM_218358 XM_234920 NM_198101 NM_080907 NM_172327

XM_215158

NM_022602 NM_178094 NM_004112 NM_006203

NM_022595 NM_031081

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag TGCAATAGGGA

(þ) NGF 1

() NGF 6

Fold change 7

GCAAGGGGTGG GCGAGGAGTCC

0 0

6 6

7 7

TGCCTCTTCCG ATGGAGCAGTC

3 2

19 13

7 7

GACCCAGCTCT

0

7

8

TGCACACACTG

0

7

8

TACCTCGATGG

1

8

9

TTCTGCCTCCA

1

8

9

CTACAGTTCCT

1

13

15

TGGAACCTTGC

16

1

14

AGTTGATGCAA AGTTTGCTGAT GAAAAATAGTC TATTTGTTTTG AGCTTTCCTGT CCACACTGTCT

32 43 11 10 10 9

2 3 0 1 1 1

14 13 10 9 9 9

CAGTATCCCTA GACTGTGCCAA GAGGAGGGGGA CCCTTCCCTGC GCAATAAATGG GCTTAGCCATT CTGCTAGCACC

9 8 8 7 7 13 1

1 1 0 0 0 2 5

8 7 7 6 6 6 6

ACCTTCTTGGT GAAGGAGAACT

2 0

11 6

6 7

GCAGTGGGCTC

1

7

8

Identity Ortholog of human PPP1R12C: protein phosphatase 1, regulatory (inhibitor) subunit 12C/myosin binding subunit 85 Ortholog of murine Efna2: ephrin A2 Stk25: serine/threonine kinase 25 (STE20 homolog, yeast); (oxidant stress response kinase 1 Similar to tetraspanin similiar to uroplakin 1 Similar to NAKAP95: neighbor of A‐kinase anchoring protein 95; binds regulatory subunit (RII) of PKA and to DNA LOC303259 similar to Map4k6‐pending protein Similar to protein kinase BRPK/protein kinase BRPK /PINK1 (PTEN induced putative kinase 1) Acid nuclear phosphoprotein 32 (leucine rich) (Anp32); may be involved in signal transduction Serine‐threonine kinase 16 (STK16)/F52/EDPK, Krct, PKL12 CL1BA: CL1BA protein/Latrophilin/CIRL/CL1; 7 transmembrane domain receptor of secretin family

Accession # NM_017607

BC048697 NM_184049

XM_230297 XM_216820

XM_239248 XM_216565

NM_012903

D86220 NM_022962

Cytoskeleton Dncli2: LIC‐2 dynein light intermediate chain 53/55 Nfl: neurofilament, light polypeptide Ortholog of murine Tagln2: transgelin 2 Nfl: neurofilament, light polypeptide Prnp: Prion protein, structural Cortactin isoform B (Cttnb) Ortholog of murine Capza1: capping protein (actin filament) muscle Z‐line, alpha 1 Msn: Moesin Pin: dynein, cytoplasmic, light chain 1 Ortholog of murine Tuba6: tubulin, alpha 6 Ctxn: cortexin Dbn1: drebrin 1; actin binding protein Microtubule‐associated protein 4 Similar to Epidermal growth factor receptor pathway substrate 8‐like protein 2; Eps8l2; PTB domain/Eps8 actin regulator Add1: adducin 1, alpha Add3: Adducin 3, gamma; Actin capping protein Coronin relative protein; actin binding protein

NM_031026 NM_031783 BC009076 NM_031783 NM_012631 NM_021868 NM_009797 NM_030863 NM_053319 NM_009448 L15011 NM_031024 XM_345984 XM_341958

NM_016990 NM_031552 NM_139115

37

38

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TGACTAGTGTC

AGAGGTCTGAG

(þ) NGF 0

1

() NGF 10

16

Fold change 11

18

Identity Similar to hypothetical protein MGC37888; encoded protein¼Eml2: echinoderm microtubule associated protein like 2 similar to coactosin‐like 1; coactosin‐like protein, actin‐binding protein that may link 5‐lipoxygenase to actin

Accession # XM_215147

XM_341700

DNA binding proteins and transcription factors GAGGCAGCTGG

10

0

9

GTTCTACCCCA

9

0

8

CCTTTAATCCT

9

0

8

CCCTTCACCTC

8

1

7

CGAAGTCAGGC

8

1

7

TTCCCCACACA

8

1

7

TGAATGGCCTA

7

0

6

CAAATAAGTTT

7

0

6

TGATCTTTTTG AGAAGCGCAAG

2 2

11 11

6 6

TGAGTGAAGAG

3

15

6

TTCCAGACGGA

1

5

6

TTGTGGTAACC

0

5

6

CTGAGCAGTGG

0

5

6

CACCTTGAGTG

0

5

6

GAAAAATCCAC

0

5

6

GGGATGCTGCT

0

5

6

Similar to PEA3: polyomavirus enhancer activator 3, transcription factor Ortholog of murine Rnf14: ring finger protein 14/TRIAD2; transcriptional coactivator Similar to Cyclic‐AMP‐dependent transcription factor ATF‐6 alpha (Activating transcription factor 6 alpha) Similar to RIKEN cDNA 2310074H19; Homolog of human DRAP1: Dr1‐associated protein 1; transcriptional corepressor Ssrp1: Structure specific recognition protein 1; DNA binding protein Tgfb1i4: Transforming growth factor beta stimulated clone 22; transcription factor LOC299113: similar to RIKEN cDNA 2310022K15; Klhdc2 kelch domain containing 2; putative transcriptional regulator Ortholog of murine 2310042L19Rik: RIKEN cDNA 2310042L19 gene which encodes protein highly homologous to human pirin, a transcriptional activator Atf4: activating transcription factor ATF‐4 RGD:621323: aristaless (Drosophila) homeobox, Arix; Phox2a; Control noradrenergic phenotype Ortholog of human SOX12: SRY (sex determining region Y)‐box 12 LOC313666: similar to polyomavirus late initiator promoter binding protein; Zbtb17; Lp‐1; Miz1; mZ13; Zfp100 Ortholog of murine Zik1: zinc finger protein interacting with K protein 1; transcriptional repressor RGD:727889: v‐rel reticuloendotheliosis viral oncogene homolog A; Rela Nr1h2: nuclear receptor subfamily 1, group H, member 2 nuclear orphan receptor; may interact with RXR AZF1: zinc finger protein 1; Putative DNA binding protein Ortholog of murine Zhx3: zinc fingers and homeoboxes 3

XM_340910 NM_020012 XM_222871

XM_215177

L08814 NM_013043 XM_216721

XM_136134

NM_024403 NM_053869 NM_006943 XM_233676

NM_009577

NM_199267 NM_031626

XM_342745 NM_177263

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag TCTCTTCTCGA

(þ) NGF 0

() NGF 5

Fold change 6

CCACCTACATC

0

6

7

GAGAACATCAC

0

6

7

TCCCCAACGGC

0

6

7

CTGCTGAGCCT

0

6

7

TTGGCCAGAAT

1

6

7

AAAAAGAATAA

0

6

7

CGGAAATGATG

1

6

7

CCCCAACCCTA

1

7

8

GAGAGAAGTGG

0

7

8

CGGGAGTGCCT

1

8

9

TACTTGGGGGC

0

9

10

TGACAGTCAGG

0

9

10

CCGGGAGTGTG GACAGTGGAGA

0 1

9 9

10 10

AATAACTTTAA

1

10

11

GCTGGGCAAGG

1

10

11

TGCTCCGTGTA

1

10

11

GGAGCAGGAAC

0

10

11

Identity LOC305471: similar to transcription factor MAZR; ZNF278; ZSG; MAZR; PATZ; RIAZ; ZBTB19; transcription repressor Similar to Transcription factor E3; X‐linked b‐HLH zip transcriptional activator Similar to RIKEN cDNA 5730434I03 gene: homolog of human similar to RNA polymerase B transcription factor 3 Ascl1: achaete‐scute complex homolog‐like 1 (Drosophila)/Mash1 Similar to helix‐loop‐helix protein; nescient helix loop helix 1/Hen1, Nscl, Tal2; implicated in neuronal differentiation. Similar to transcription elongation regulator 1; transcription factor CA150; TATA box binding protein‐associated factor Gata2: GATA‐binding protein 2; Zn finger transcription factor Usf2: transcription factor USF2 (UPSTREAM STIMULATORY FACTOR 2) (HLH zipper transcriptional activator via AP1 sites Orthololg of murine RIKEN cDNA C630022N07 gene; similar or¼to mafG Similar to RIKEN cDNA 2400009B11 gene; encodes homolog to PUTATIVE TRANSCRIPTIONAL REGULATORY PROTEIN HRC1putative Ortholog of murine Zfp61: zinc finger protein 61; putative DNA binding protein LOC293513 hypothetical LOC293513: encodes protein that matches murine Snf2‐related CBP activator protein Similar to Meis3 (myeloid ecotropic viral integration site‐related gene 3); homeodomain family member regulating gene expression Similar to fork head‐related protein like A Similar to methyl‐CpG binding domain protein 2; transcription regulator Cnbp: cellular nucleic acid binding protein; Zn‐finger protein potentially regulating transcription and translation Similar to KIAA0138 gene product; encodes protein with RNA and DNA binding sites that homologous to scaffold protein B. Supt5h: suppressor of Ty 5 homolog (S. cerevisiae) Lisch7: Liver‐specific bHLH‐Zip transcription factor

Accession # XM_223592

XM_228760 XM_345561

NM_022384 XM_222898

XM_225983

NM_033442 AB047556

NM_032711 NM_025886

BC079015 XM_238138

XM_341796

XM_343526 XM_214544 NM_022598

XM_238571

XM_218382 NM_032616

39

40

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag GTGTGTAGGGG

(þ) NGF 1

() NGF 11

Fold change 12

TGATTGGTAGA

1

13

15

GCGGCCGGCTT

1

15

17

AGAACCTAGTC

5

130

30

TCTGGCTCCTT AAGCTGGTTTA

11 9

0 0

10 8

ATGAGGAACTT GTACCAGGACA

8 8

0 1

7 7

AGGATGCTTTG

7

1

6

TTCTGTCCTAT

1

5

6

CCATCGAGGAG

3

17

6

AATAAAGTTGT

2

10

6

TCGGTGCAGGC

0

5

6

GAAATGTAAGA

2

12

7

CGGAAGGAATT

2

13

7

GTAGGAACATA

0

6

7

TTGATCGAAGT AGACAAGCTGG

0 2

6 13

7 7

GCCCGAAAGAT

1

6

7

TCCAGGGCCTT

0

7

8

CACAACTGTGA

0

7

8

CTTGGAGAACG

0

7

8

Identity Nfat5: nuclear factor of activated T‐cells 5, tonicity‐responsive Ortholog of murine Tef: thrytroph embryonic factor; transcription factor member of PAR family of bZip transcription factors Cebpb: Liver activating protein; LAP, NF‐IL6, nuclear factor‐IL6, previously designated TCF5, sfb ilencer factor B, CEB/PB Atf5: activating transcription factor 5

Accession # XM_226436 NM_017376

NM_024125

NM_172336

RNA binding, splicing, processing, and stability Similar to RNA binding motif protein 5 Snrpb: small nuclear ribonucleoprotein polypeptides B and B1: splicing factor Similar to U1 snRNP‐specific protein C Similar to hypothetical protein MGC14151; encodes snRNP Sm related protein Similar to RIKEN cDNA 2610528E23; member DEAD box helicase family Ortholog of murine Rpo1–3: RNA polymerase 1–3 LOC290660: similar to R27090_2; DEADc; DEAD‐box helicase Ortholog of human PABPN1: poly(A) binding protein, nuclear 1; role poly adenylation Similar to murine Tdrd9: tudor domain containing 9 HELICc, Helicase superfamily c‐terminal domain; Tudor domain Ortholog of murine Pcbp2: poly(rC) binding protein 2; RNA binding protein Similar to UPF3 regulator of nonsense transcripts homolog A isoform hUpf3p; mRNA surveillance Similar to splicing factor U2AF homolog ‐ mouse Similar to poly(A) polymerase V Sfrs5: splicing factor, arginine/serine‐rich 5 (SRp40, HRS) Ortholog of murine Sfrs16: splicing factor, arginine/serine‐rich 16 (suppressor‐of‐white‐ apricot homolog, Drosophila) Homolog of murine Tia1: cytotoxic granule‐ associated RNA binding protein 1 Similar to splicing factor, arginine/serine‐rich 2, interacting protein; SC35‐interacting protein 1; RNA splicing Similar to MADP‐1 protein; HAS RNA RECOGNITION MOTIFS;

XM_217263 M29295 XM_342101 XM_213347 XM_213638 NM_009087 XM_214290 NM_004643 XM_127120

XM_128023 XM_341460

XM_218195 XM_234508 L13635 NM_016680

AK033792 XM_231361

XM_343320

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag ACAGAGGCATT

(þ) NGF 1

() NGF 8

Fold change 9

TTGCTGGCTTT

1

8

9

GAAGAGTGTAA

2

15

9

AGCAAACCCCC

1

8

9

GTCGCTTCTGA

1

9

10

GATCGAGCAAG

1

9

10

GAAGGATGGCT ATGACTTGGGT

0 0

10 10

11 11

GCTGGAGAGCC

0

10

11

CCTGCCCTGTG

0

11

12

GGCTTCACGGG

1

14

16

ACTGAGTGCTT

12

1

11

GTCCAGGAAAA CAGTGCTGGGT

9 9

1 1

8 8

CTTTGGGTACA GAGTGAAGAAT

8 1

0 5

7 6

TCCTATCTGCA

1

6

7

TTTCCAAAGTT

0

6

7

GACAGGGAGGT

0

6

7

ACCAGAGCAAC

2

14

8

CCATCAGTGGG

2

15

9

CGCAAGAAGGT

1

9

10

CAAACTGCATT GGGCAGACAGG

0 1

9 9

10 10

Identity Ortholog of murine Sfrs6: splicing factor, arginine/serine‐rich 6 Ortholog of human RBM9: RNA binding motif protein 9; transcription Ptb: pyrimidine tract binding protein; pre‐mRNA splicing Similar to DAZ associated protein 1 isoform b; deleted in azoospermia associated protein 1 Similar to U2 auxiliary factor 26; RNA binding protein Ddx24: DEAD (Asp‐Glu‐Ala‐Asp) box polypeptide 24 Cirbp: cold inducible RNA‐binding protein Ortholog of murine Rbm18: RNA binding motif protein 18 Similar to RNA‐binding protein Raly ‐ hnRNP‐associated with lethal yellow Similar to polymerase (RNA) III (DNA directed) (155kD) LOC363134: similar to U3 snoRNP‐associated protein; nucleolar protein Proliferation Similar to putative oral cancer suppressor; (DOC1)/CDK2‐associated protein 1 Ccnd1: Cyclin D1 Similar to NimA‐related protein kinase; NEK9: NIMA (never in mitosis gene a)‐ ortholog; regulates mitotic progression Cyclin G1 Phb: prohibitin; suppresses proliferation and activates p53 Similar to Cyclin K; CCNK: member of the cyclin family and may regulate PolII Similar to ring‐box 1; ring‐box protein 1; ubiquitin ligases component‐possible cell cycle protein Similar to PISSLRE; ortholog of murine Cdk10: cyclin‐dependent kinase (CDC2‐like) 1; cell cycle progression Similar to prostate tumor over expressed gene 1 (Ptov1); Promotes S phase entry Similar to centromere autoantigen B; CENP‐B: Centromeric protein B; centrosome assembly protein Pold1: DNA polymerase delta, catalytic subunit; DNA replication and repair Cdk4: cyclin‐dependent kinase 4 Rpa2: p32‐subunit of replication protein A; DNA replication and repair

Accession # NM_026499 XM_086858 NM_022516 XM_343164 XM_341828 NM_199119 NM_031147 NM_026434.2| XM_215880 XM_341388 NXM_343469

XM_341076 D14014 XM_216755

NM_012923 NM_031851 XM_234516 XM_216991

XM_341712

XM_214944 XM_342521

NM_021662 NM_053593 X98490

41

42

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TCGGAGAAGAG

(þ) NGF 0

() NGF 10

Fold change 11

Identity Ptms: parathymosin/Zn binding protein; nuclear Zn binding protein possibly involved in proliferation

Accession # NM_031975

Protein synthesis CTCAGACAGTG CTCAAACACCA

14 1

1 6

12 7

TGTATGAAGCA

1

7

8

TTCGTGGTCAA ATCCTCGCTGA

2 1

16 9

9 10

ACAGAAAGTGG

1

9

10

Similar to 40S ribosomal protein S27 LOC363023: similar to mitochondrial ribosomal protein L4 isoform a; Mrpl4 Metap2: methionine aminopeptidase 2/Initiation factor 2 associated 67 kDa protein (Amp2); protein synthesis Eef2: eukaryotic translation elongation factor 2 LOC287126: similar to TCE2; Ortholog of murine Mrps34 mitochondrial ribosomal protein S34 Similar to mitochondrial ribosomal protein L18

X59375 XM_343354 NM_022539

NM_017245 XM_213234

XM_214751

Proteasomal pathway GTGCTGGACCT

10

1

9

AGAGGAAGTGG

10

1

9

AGACGCCTGTG

10

0

9

CCTTACACTTG

9

1

8

AAAACACCTTG

7

1

6

AAGTAGCTGGA

7

0

6

TGATGTCTCTC

0

6

7

CAGGGCGAGAT

1

6

7

CTCCTCCTGAT

1

6

7

GACCTTGGAGT

0

11

12

Psme2: protease (prosome, macropain) 28 subunit, beta; activator of proteasome Similar to F‐box protein FBL2; related to skp2 so putative involvement in proteasomal targeting Similar to 26S proteasome‐associated pad1 homolog Similar to tetratricopeptide repeat domain 3; Ortholog of murine Ttc3; putative ubiquitin ligase Nedd4a: neural precursor cell expressed, developmentally down‐regulated gene 4; E3 UBIQUITIN PROTEIN LIGASE Similar to RIKEN cDNA 1300013G12; encoded protein very similar to murine Ubxd2: UBX domain containing 2 Ortholog of human COP1: constitutive photomorphogenic protein; E3 ligase that can target p53 Similar to RIKEN cDNA 6330414O09; ortholog of Irf2bp1: interferon regulatory factor2 binding protein1; E3 ligase domain Similar to Ubiquitin carboxyl‐terminal hydrolase 12/USP12 ubiquitin specific protease 12 Ortholog of murine Rnf26: ring finger protein 26; putative ubiquitin‐protein ligase

NM_017257 XM_217519

XM_215745 XM_340973

XM_343427

XM_222627

NM_022457

XM_218405

XM_341033

NM_153762

Lysosomal function CTGATCCCCAT

3

17

6

Lamp1: Lysosomal associated membrane protein 1 (120 kDa); membrane glycoprotein

NM_012857

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag GGTAAGTCATC

(þ) NGF 1

() NGF 6

Fold change 7

AATCGGAACAA GTTCACCGACG

0 0

7 8

8 9

AGCCTCCCTTG

12

1

11

GATTGTCTTGA GGTTTGATTCC AGTTCTGCTTG TGTGCAGTGAA TGGGTTAGACC GCGCGCGTTTA

12 9 17 17 8 8

0 1 2 2 1 1

11 8 7 7 7 7

GTATTGGCCAG

0

5

6

CTCTCACCCCT

0

5

6

CCTGGTTATAC

0

5

6

GGCCTAAGGCA ATTGGGAAGCT

1 1

5 5

6 6

GTATTGGCCAG

0

5

6

ACCTTGCCCTC

3

18

7

ACGAGCTTTAA

1

6

7

TCTTACTGGCA

1

7

8

TCATCTTTAAC

1

7

8

Identity Ortholog of murine Cln2: ceroid‐lipofuscinosis, neuronal 2; lysosomal serine protease Dnase2: deoxyribonuclease II; lysosomal DNAse Man2b1: alpha‐D‐mannosidase; lysosomal enzyme

Accession # NM_009906 NM_138539 NM_010764

ER, molecular chaperones Similar to carboxy ter of Hsp70‐interacting protein Similar to 25 kDa FK506‐binding protein Ortholog of murine Canx: calnexin Plp2: proteolipid protein 2 Similar to signal peptidase 12kDa Similar to prefoldin 1 Ortholog of human VBP1: von Hippel‐Lindau binding protein 1/PREFOLDIN SUBUNIT 3 / VHLBINDING PROTEIN‐1) (VBP‐1); Chaperone Similar to SREBP cleavage activating protein: SCAP; Required for sterol‐regulated transport of SREBPs from ER to Golgi Ortholog of murine Caþþ activated nucleotidase 1 (Cant1);Ca2þ‐dependent ER nucleoside diphosphatase/apyrase 1 Ortholog of murine Dnaja4: DnaJ (Hsp40) homolog, subfamily A, member 4; heat shock protein, DNAJ‐like 4 LOC291671: similar to grp75 Ortholog of murine 1110021N07Rik: RIKEN cDNA 1110021N07 gene; encodes protein derlin‐1 involved in ER transport Similar to TRAM1: translocating chain‐ associating membrane protein Similar to signal sequence receptor, beta; translocates newly synthesized polypeptides across ER membrane Similar to FK506‐binding protein: molecular chaperone Similar to HRD1 protein; synoviolin 1; involved in ER‐associated degradation Calr: calreticulin; ER chaperone

XM_213270 XM_216717 AK017254 XM_217597 XM_214276 XM_341596 NM_003372

XM_217279

AK081118

NM_021422

XM_214583 NM_024207

XM_232596 XM_215619

XM_215758 XM_341999 NM_022399

Trafficking, vesicular transport TGTTGTTGATC

11

1

10

TGTGAAGTAGC

17

2

7

TGGTGACTAAG

7

1

6

Similar to evectin‐2 (postGolgi vesicular membrane protein) Arf1: ADP‐ribosylation factor 1; vesicular transport Similar to ADP‐ribosylation factor binding protein GGA2 (Golgi‐localized, gamma ear‐ containing, ARF‐binding protein 2)

XM_217372 NM_022518 XM_215045

43

44

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag GACTTCTGTCA

(þ) NGF 1

() NGF 5

Fold change 6

AAGATCATCGA

1

5

6

TCTCTGGGCCA

1

5

6

TCAGCTGACCA

1

5

6

TTAATTCATTT

1

5

6

GTCTTTTCAGA

1

6

7

GAAGGCAGTTT

1

7

8

AACTGGGTCTG

4

27

8

CCCCATTCCCA

0

7

8

TCAGCTGAATA

1

8

9

Identity Similar to leptin receptor overlapping transcript‐like 1 Ortholog of murine Ap1m2: adaptor protein complex AP‐1, mu 2 subunit RGD:621591: tumor specific antigen 70 kDa. Ortholog of human and mouse Coronin 7; vesicular trafficking Ortholog of murine Aspscr1: alveolar soft part sarcoma chromosome region, candidate 1 / TUG; traps intracellular GLUT4 Timm23: translocase of inner mitochondrial membrane 23; intracellular protein transporter Rab10: ras‐related protein rab10; putative role in protein transport/neurotransmitter release Ortholog of human SCFD2: sec1 family domain containing 2; vesicle dependent protein transport Arl3: ADP‐ribosylation factor‐like 3; binds GTP and may regulate intracellular transport Similar to zinedin; A calmodulin‐binding, WD repeat protein with putative role in membrane trafficking Gosr2: golgi SNAP receptor complex member 2; vesicle transport from the cis/medial to the trans‐Golgi/TGN

Accession # BC058504 NM_009678 XM_220167

NM_026877

NM_019352

NM_017359 NM_152540

X76921

XM_218432

NM_031685

Carbohydrate binding and metabolism GCGGCGGATGG TTCAGAGGGGC

215 14

15 0

13 12

TGCTCCTGTGA ATCTAAGCCAG

14 10

0 1

12 9

GTTCCCCTCAC ATTTTCCCCCG

8 7

0 0

7 6

GAGACCTCTGG

1

7

8

GAGACGGCATC

1

10

11

ACTCCTGTCAG TACAGAAGGAG

8 1

0 7

7 8

Galectin 1 Similar to 106 kDa O‐GlcNAc transferase‐ interacting protein Hexa: hexosaminidase A Lgals3: lectin, galactose binding, soluble 3/ galectin3 Similar to glucuronosyltransferase I Similar to Stromal cell‐derived factor 2 precursor (SDF‐2) Ortholog of murine Fn3k: fructosamine 3 kinase Similar to Vesicular integral‐membrane protein VIP36 precursor; lectin family Endocytosis Cltb: clathrin, light polypeptide (Lcb) Ortholog of human ITSN2: intersectin 2, involved in clathrin mediated endocytosis

M19036 XM_236715 XM_217144 NM_031832 XM_238155 XM_213377 NM_022014 XM_214428

NM_053835 XM_039680

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag CTGTCTGACTC

(þ) NGF 0

() NGF 12

Fold change 14

Identity Ap2b1: adaptor‐related protein complex 2, beta 1 subunit/beta‐adaptin; beta‐chain clathrin associated protein complex AP‐2; component of complex linking clathrin to receptors in coated pits and vesicles, intracellular protein transport

Accession # NM_08083

Transporters TTCTAGCATAT

11

0

10

TGCTACCACAC

7

1

6

GTGGGCCAAAC

2

10

6

TAGAAAAATGG GTGGGCCAAAC

1 1

5 5

6 6

GGGCAGACACA

1

5

6

GACATAGCCCA GTGGCCAATCA TTGTATAATAG

2 0 1

10 6 6

6 7 7

CAGTGGGTGGG ATTCTCTGGAT

5 1

33 7

8 8

GGCTTGCTCCT

1

8

9

CTGGAGCTGGG

1

9

10

ACAGTGAAGGG

3

26

10

AACGCTGACCA

1

13

15

TTGGTGAGGTA

1

16

18

Atp1b1: ATPase Naþ/Kþ transporting beta 1 polypeptide HesB protein; contains Hesb domain involved in Inorganic ion transport and metabolism Similar to solute carrier family 39 (zinc transporter), member 1; zinc‐iron regulated transporter‐like gene; Slc16a1: solute carrier family 16, member 1 Ortholog of murine Slc39a1: solute carrier family 39 (zinc transporter), member 1/Zip1 Timm44: translocator of inner mitochondrial membrane 44 NM_017267 CHOT1: choline transporter NM_017348 LOC291840: amino acid transporter Ant2: Adenine nucleotide translocator 2, fibroblast isoform (ATP‐ADP carrier protein) LOC287642: galactose transporter Atp2a2: ATPase, Caþþ transporting, cardiac muscle, slow twitch 2 LOC290673: similar to cation‐transporting atpase Slc25a10: solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter), member 10 Slc3a2: solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 LOC288919: similar to arsenic resistance ATPase Slc7a8: solute carrier family 7 (cationic amino acid transporter, Yþ system), member B/ LAT4

NM_013113 NM_181626 XM_342286

NM_012716 NM_013901 NM_017267 NM_017348 NM_1003705 D12771 NM_199081 NM_017290 XM_214310 NM_133418

NM_019283

XM_213848 NM_053442

Neurotransmission/Synapses CTGGAGGTGTG CCGCTATAACA CTAGACACCTG AGTAATTTTAG

22 8 8 7

2 0 1 1

10 7 7 6

GCACACTGTGT

7

0

6

ACATTTCAATT

1

5

6

Homolog of murine Syn2: synapsin II Syn2: synapsin II Scg3: secretogranin III Acp1: acid phosphatase 1, soluble; possible role synaptic transmission Sh3d2c1: SH3 domain protein 2 C1; implicated in vesicle recycling Similar to gamma‐aminobutyric acid (GABA(A)) receptor‐associated protein‐like 1

NM_013681 NM_919159 NM_053856 NM_021262 NM_031238 XM_216288

45

46

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TCCAGACCAGG

(þ) NGF 1

() NGF 5

Fold change 6

TCTCACTGCAG

1

5

6

GCCACAAGCTT

0

5

6

GTGTAAGGGAG

2

11

6

TGATGGAACCA

1

10

11

GCTCAGATCCA

0

5

6

GAGAGCTAACA

1

6

7

GAAATGTCTGA

0

11

13

TTGGGCTGGTT

0

12

14

GTGATAGACAA AGCTGGGACTT

0 0

6 7

7 8

Identity RGD: 621786: myc box dependent interacting protein 1; Bin‐1; may play role in synaptic vesicle endocytosis RGD: 63135: shank‐interacting protein; sharpin; interacts with the ankyrin Shank; at PSDs Ortholog of murine Unc13b: unc‐13 homolog B (C. elegans); Munc13‐1; Involved in exocytosis and synaptic transmission. Syt4: synaptotagmin; vesicular trafficking and exocytosis Ortholog of murine Nptxr: neuronal pentraxin receptor; synaptic protein uptake

Accession # NM_053959

NM_031153 NM_021468

NM_03169 XM_128199

Ion channels Kcnh2: potassium voltage‐gated channel, subfamily H (eag‐related), member 2 Nnat: neuronatin; possibly regulates ion channels during brain development Similar to hypothetical protein MGC27385; ortholog of murine Kctd6: K channel tetramerization domain containing 6 Asic4: SPASIC protein: Non‐inactivating proton‐gated ion channel in brain

NM_053949 U08290 XM_223921

NM‐022234

Antioxidant action Peroxiredoxin 6 (Prdx6); antioxidant enzyme Sod2: Superoxide dismutase 2, mitochondria

NM_053610 X566001

Cell death related GACAGCACAAG

1

5

6

CTGCCGCCTCA

1

5

6

TTTGTTAAAAC

0

5

6

ACATCCACCCA

0

6

7

GCTGAGGGAGA

1

12

14

TGCCCAATAAA

0

15

17

Similar to Apoptosis regulatory protein Siva (CD27‐binding protein) (CD27BP) Adprt: ADP‐ribosyltransferase 1; poly(ADP‐ ribose) polymerase PARP‐1 Ddit4: DNA‐damage‐inducible transcript 4; Hypoxia‐Inducible Factor 1‐Responsive Gene, RTP801 Similar to direct IAP binding protein with low PI (DIABLO); SMAC Ortholog of human paternally expressed 3 (PEG3) protein with KRUPPEL ZINC FINGER PROTEIN/PW1; LOC246273: kinase; novel kinase induced during PC12 cell death

XM_343117 NM_013063 NM_080906

XM_213814 XM_042345

NM_144755

Neurodegenerative disease associated GAAGTCAGCCA CACGCACAGTC

1 0

5 15

6 17

AGTGGAGGGAA

1

26

30

SMN1: survival of motor neuron 1, telomeric Drpla: dentatorubral pallidoluysian atrophy (atrophin‐1) LOC361649: similar to ataxin 2 related protein isoform A; ataxin‐2 domain protein; ATXN2L: ataxin 2‐like

NM_022509 NM_017228 XM_341928

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag

(þ) NGF

() NGF

Fold change Identity Extracellular matrix effects

CTCTGACTTTA

27

4

6

TTAAGACCAAG

7

0

6

ACCCAGCTCAG

0

5

6

CCTCCGCCTCC

1

5

6

Bsg: Basignin/Ox47 antigen/CE‐9/EMMPRIN; extracellular matrix metalloproteinase inducer Serpinb6: Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 6 A disintegrin and metalloproteinase domain 15 (Adam15) (metargidin). Lu: Lutheran blood group (Auberger b antigen included) Sequence; may act as an adhesion molecule and receptor for laminin [RGD]

Accession # NM_012783 NM_199085 AJ251198 NM_031752

Peroxisomal function TGCTTGCCACA

0

7

8

Similar to peroxisomal acyl‐CoA thioesterase 2B; likely ortholog of mouse peroxisomal acyl‐ CoA thioesterase 2B

XM_234398

DNA repair/damage response CTGAGGAGGGG

10

1

9

TATGCACAGGC

8

1

7

CTGCCGCCTCA

1

5

6

TGATCTGCCTG

0

5

6

DNA‐damage inducible transcript 3 (Ddit3)/ GADD153/Chop10 X‐ray repair cross‐complementing group 1 protein (Xrcc1) Adprt: ADP‐ribosyltransferase 1; poly(ADP‐ ribose) polymerase PARP‐1 MGC5178: hypothetical protein MGC5178; encoded protein has nuclease domain putatively involved in DNA repair

U36994 NM_053435 NM_013063 BC000803

Miscellaneous TAGAGCGTGCT

11

0

10

GTTTTGCTACC

8

0

7

GAACGCACACC

7

0

6

GTCTAGGTCAC

7

0

6

ACACGGAGGAG

0

5

6

CTCAGCAAAAC

0

5

6

GTGCGGTACCT

0

5

6

GGAAGCTGCAA

1

5

6

GTAGCAGCCAG

1

5

6

Similar to nucleosome assembly protein 1‐like 4; nucleosome assembly and gene expression Similar to NIPSNAP1 protein: 4‐nitrophenylphosphatase domain and non‐ neuronal SNAP25‐like protein homolog 1 (C. elegans) Similar to erthyrocyte band 7 integral membrane protein, protein 7.2B, stomatin Similar to hepatitis B virus x‐interacting protein; HBx‐interacting protein; HBixP Haptoglobin (Hp); has serine‐type endopeptidase activity LOC317431: similar to RIKEN cDNA 2610028I09; ortholog of murine Ribc1: RIB43A domain with coiled‐coils 1 LOC300222: similar to microspherule protein 1 P78; MCRS1; MSP58; ICP22BP; putative cell cycle/transcription regulator LOC361425: similar to hypothetical protein COX4AL ortholog of murine Noc4: neighbor of Cox4 LOC296731: similar to NEDD8 ultimate buster‐ 1; NUB1; binds NEDD8 a ubiquitin‐like protein

XM_341967 XM_341249

XM_216045 XM_215674 NM_012582 XM_228840

XM_217048

XM_341703

XM_231280

47

48

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag CAAATTACTAA

(þ) NGF 0

() NGF 6

Fold change 7

CGCTGCAGAAA

1

6

7

GTGTGTGGTGC

1

6

7

GCCATTTGGTG

0

6

7

CAAATTACTAA

0

6

7

GCAATATTGGC

0

6

7

GGATGATGGTC

1

8

9

AAACATTGGGG

2

16

9

ACCTTGTTGAT

3

30

11

CAGGTTCTCCT

1

15

17

GACTGAAAAAG

0

16

18

GCAGCAAGAAG

0

19

22

Identity Ortholog of murine RIKEN cDNA A530089I17 gene; encodes SAC3: Sac domain‐containing inositol phosphatase 3 (Lipid transport and metabolism) Nurim (Nrm) (nuclear envelope membrane protein) LOC362571: similar to prion protein interacting 1 like (30.8kDa) (4B10); appears to be in exonuclease family; prnpip LOC361035 similar to methyltransferase like 3; putative methyltransferase; m6a methyltransferase Ortholog of murine RIKEN cDNA A530089I17 gene; encodes SAC3: Sac domain‐containing inositol phosphatase 3 (Lipid transport and metabolism) Similar to RIKEN cDNA 1200009I24 gene (LOC289323); Nuclear valosin‐containing protein‐like; AAA ATPase family LOC292306: similar to Ribonuclease 6 precursor; Rnaset2; extracellular ribonuclease LOC309475: similar to transmembrane protein TM9SF3 D‐dopachrome tautomerase (Ddt); converts D‐ dopachrome to 5,6‐dihydroxyindole; melanin synthesis Stellate cell activation‐associated protein (Staap); cytoglobin with peroxidase activity LOC309161: similar to CG17265‐PA; Ortholog of human DIPA: hepatitis delta antigen‐ interacting protein A LOC361682: similar to tumor‐suppressing subchromosomal transferable fragment 4; TSSc4

Accession # NM_133999

NM _212508 XM_342890

XM_341310

NM_133999

XM_213963

XM_214769 XM_220013 NM_024131

NM_130744 XM_219510

XM_341965

Transcripts encoding novel proteins of unknown function TGCTCTGCATA

11

0

10

GTGACCGGCCC

10

0

9

CTCTGTGGGTT

9

1

8

GTGCCCACTGG

9

1

8

ATGCTTCCTGT

9

0

8

Ortholog of murine Fin14 fibroblast growth factor inducible 14 Hypothetical LOC294291 encoding hypothetical protein of unknown function LOC294362: similar to DNA segment, Chr 10, ERATO Doi 214, expressed; has Zn finger domain Ortholog of murine DNA sequence BC020184 encoding novel protein member of TB2/DP1, HVA22 family. Similar to chromosome 11 open reading frame; ortholog of murine Tmem16f: transmembrane protein 16F

AK002917 XM_215355 XM_215378

XM_128892

XM_235640

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag CGTTCCACCAG TAGGGGTGGAG

(þ) NGF 9 9

() NGF 0 1

Fold change 8 8

TGCACAGATGT

68

8

7

AAGAAAGTCGC

8

0

7

GTTTCCTGCTT TGGTACACGGA

8 8

1 1

7 7

TATACAGAGCG

8

0

7

TCTCAAAGAAC GAGCAGCCACC GTTCCGACAGT

8 8 8

0 1 0

7 7 7

CACCACTGGAT

8

1

7

GCTATATTCCA

8

0

7

AGCCGTGTATA

7

1

6

TTTAGTGACGT

7

1

6

ATTGTCTTTTC

7

0

6

CCTGGCCAGCC

7

0

6

CTACTTCTGTA GGCCTGGCTTA CCCAGACTGAA CCCCTTCACCC GGATGTGAACT

7 7 7 13 7

0 0 0 2 0

6 6 6 6 6

TGGGGAGAAAT TTAAGTCCTTG

3 1

16 5

6 6

TATGCAACCTG

2

10

6

GTGTTCCTCCG

0

5

6

GCAGCCCCATA TGCAGAATCCA

0 0

5 5

6 6

Identity Similar to Protein C22orf5 Ortholog of murine 2610017J04Rik: RIKEN cDNA 2610017J04 gene of unknown function Growth and transformation‐dependent protein; Encodes novel gene of unknown function LOC287828: similar to HN1; hematological and neurological expressed sequence 1 LOC361797: LOC361797 Similar to DNA segment, Chr 5, Brigham & Womens Genetics 0834 expressed Ortholog of human DKFZP564D166: putative ankyrin‐repeat containing protein Similar to hypothetical protein MGC45400 Similar to RIKEN cDNA E030034P13 Similar to CDV‐3B (carnitine deficiency‐ associated gene expressed in ventricle 3) Transcribed sequence with weak similarity to protein pir:T43483 (H.sapiens) translation initiation factor IF‐2 homolog Ortholog of murine 1110061A14Rik: RIKEN cDNA 1110061A14 gene of unknown function Ortholog of human C9orf25: chromosome 9 open reading frame 25 Ortholog of murine 9430029L20Rik RIKEN cDNA 9430029L20 gene Ortholog of murine Tub: tubby candidate gene Ortholog of murine 2010004M13Rik: RIKEN cDNA 2010004M13 gene encoding product of unknown function Similar to 0610010K06Rik protein Similar to hypothetical protein FLJ20627 Similar to RIKEN cDNA 2010315L10 Similar to hypothetical protein 4933417N17 Similar to Ser/Thr‐rich protein T10 in DGCR region Putative ISG12(b) protein; interferon inducible LOC309763: similar to DKFZP586B0923 protein LOC296565: similar to hypothetical protein MGC36831 LOC288557: similar to RIKEN cDNA 1190005J19; MOSPD3 motile sperm domain containing 3 LOC304649: similar to CG2662‐PA LOC313668: similar to RIKEN cDNA 1700027M01

Accession # XM_343289 AI463119 M17412

XM_213527 XM_347003 XM_222140 XM_044366 XM_346349 XM_340760 XM_236579 AI228578

AK004333 AK022819 AK079127 XM_207983 AK008102

XM_223020 XM_214758 XM_214304 XM_214392 XM_341010 XM_238467 XM_228159 XM_216002 XM_213738

XM_222452 XM_233612

49

50

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TGCAGGCGACA

(þ) NGF 0

() NGF 5

Fold change 6

CGTATTCAGAA CGCCCTTGAGC

1 1

5 5

6 6

CGGTGGAGATA

0

5

6

AGCAGAGAATG

0

5

6

ATGCCAAACAC

0

5

6

GTGCTCAAACC

1

5

6

AGCCTCCAGGG

0

5

6

ACTCTAGCCAG ATGCCAAACAC

0 0

5 5

6 6

GCCAGCCAGCA

0

5

6

GCCTTCCCTCA GTATTTGCAAA

1 1

5 5

6 6

GTGGGTTTCTG

1

5

6

GGGGTACCTGG TACTGGAGTAT

3 0

17 6

6 7

ACTGTAGCTTC

1

6

7

AGAGACCCTGC

0

6

7

ATGTGAAACTG CTCCTAAACCT GAAGCCTGTAG GACCAGCAGGG CCTGGCCCTTT

0 1 1 1 0

6 6 6 6 6

7 7 7 7 7

GCCAGCACAGC GAGTCACGGAG TGGAGCCTCAA

1 0 1

6 6 6

7 7 7

GGGGTGGGGGG

1

6

7

Identity MGC94549 similar to cDNA sequence BC005632 Clone UI‐R‐FJ0‐cqb‐f‐24‐0‐UI unknown mRNA. Ortholog of murine 2610528K11Rik: RIKEN cDNA 2610528K11 gene 7530403E16Rik: RIKEN cDNA 7530403E16 gene; mRNA Sequence NM_175184 Ortholog of murine 1700020I14Rik: RIKEN cDNA 1700020I14 gene LOC363077: similar to expressed sequence AV340375 Ortholog of human JMJD3: jumonji domain containing 3 Ortholog of murine 1110002E23Rik: RIKEN cDNA 1110002E23 gene LOC296751: similar to KIAA1897 protein; LOC362914: similar to hypothetical protein FLJ10204 Similar to putative zinc finger protein (LOC301122) Similar to RIKEN cDNA 5730469M10 Ortholog of murine 1810006K23Rik: RIKEN cDNA 1810006K23 gene; similar to CaMKII inhibitor protein alpha Ortholog of murine D130072O21Rik: RIKEN cDNA D130072O21 gene Putative ISG12(b) protein; interferon inducible LOC308795: similar to MESDC1: mesoderm development candidate 1 Ortholog of murine 2300002D11Rik: RIKEN cDNA 2300002D11 gene; encodes novel protein of unknown function Ortholog of murine RIKEN cDNA 1600010O03 gene Similar to mKIAA0236 protein (LOC316524) Similar to leucine zipper domain protein Similar to hypothetical protein MGC2494 Ortholog of murine RIKEN cDNA 2610510D13 Ortholog of murine clone C230062K19; full insert sequence Similar to mKIAA1930 protein Similar to KIAA1193 protein Ortholog of murine LOC209064: RIKEN cDNA 1810055D05 encoding novel protein with a DNAJ domain Otholog of murine expressed sequence AW049604; encodes Tafa5, a secreted brain‐ specific protein

Accession # XM_216853 AY724538 NM_175184 BC051225 XM_488956 XM_343408 XM_043272 NM_025365 XM_216060 XM_343244 XM_217318 XM_341406 AI835402

NM_175322 XM_213523 XM_218853 XM_130600

BC002221 XM_237291 XM_213213 XM_213267 AK012090 AK048777 XM_214666 XM_343172 XM_124946

NM_134096

Nerve growth factor regulated gene expression

2

. Table 2-2 (continued) Tag CCTGGAATCTC

(þ) NGF 1

() NGF 6

Fold change 7

TCCAACTCTAG

1

6

7

GTGTCAGCAAG TCCCTATAGTC

1 0

6 7

8 8

TCCTTTTTCAC

1

7

8

TCAGAGCCTCA

0

7

8

AGGAGAAGGTG GAGAGACTTTC

0 0

7 7

8 8

GCACGAACATC GGCCAGGACAG

1 1

7 8

8 9

GTTGGAACACC

1

8

9

TAGACTGTGCA

1

8

9

TTCGTGTGTCT TGGCCAGTAAC GCACTCCTCCT

0 1 1

9 10 10

10 11 11

GTGAAAAAGGA

1

11

13

AGCCTGGAGAG

0

11

13

TGCTGGTGGGT

1

11

13

CACACCTCAGG

1

13

15

GCCGGCCGGAC

1

19

22

Identity Brain‐enriched SH3‐domain protein Besh3 (Besh3) Ortholog of murine RIKEN cDNA 2610510H01 gene Similar to CG31635‐PA LOC291609: similar to Nedd4 WW domain‐ binding protein 5 Ortholog of murine Mea1: male enhanced antigen 1 LOC314598: similar to wizL; Widely‐interspaced zinc finger motifs Similar to oriLyt TD‐element binding protein 7 Similar to RIKEN cDNA 4921536I21 (LOC362056); encodes protein similar to spermatogenesis associated protein 1 Similar to CG14977‐PA Ortholog of murine RIKEN cDNA 1600002K03 gene Ortholog of murine LOC328644: hypothetical gene supported by AK045595 mRNA Bladder cancer associated protein (Blcap); novel protein of unknown function Similar to hypothetical protein MGC10120 Wmp1: Fertility related protein WMP1 Ortholog of murine 1110021J02Rik: RIKEN cDNA 1110021J02 gene encoding protein of unknown function Ortholog of murine similar to hypothetical protein FLJ30213; encoded protein has putative YIPPEE Zn binding domain Ortholog of murine PERQ amino acid rich, with GYF domain 1 (Perq1) Similar to KIAA0540 protein; Beige/BEACH domain LOC361550 similar to RIKEN cDNA 1300003M23 LOC293514: similar to BCL7C; unknown function

Accession # NM_139334 C019606 XM_218397 XM_214564 NM_010787 XM_234841 XM_345879 XM_342358

XM_341044 AK005397 NM_198629 NM_133582 XM_215245 NM_138862 XM_128573

NM_145008

NM_031408 XM_236649 AY171575 XM_215076

ESTs GTATTAAATAG AGCTAGAGCTG TAAAGTACTCA TGTAATGAGAT GCTCCAGCTAC ATTGTCTTTTC TGGGCACTGGG TGTTCTATAGG

11 9 9 8 8 7 7 7

1 1 1 0 1 0 0 1

10 8 8 7 7 6 6 6

ESTs ESTs ESTs ESTs EST ESTs EST ESTs

BQ192965 BF392924 BQ202150 BE117509 AA858996 AI548927 AA818499 AI060317

51

52

2

Nerve growth factor regulated gene expression

. Table 2-2 (continued) Tag TGTTCACTTGT GAATACAGCCT CATTTTAGAAT ACCACAGGCCT GCTGCCACACA GATAGCCATAG TAGCCCAACCC TTGTGGTAACC TTAAATAATTG ATGGTGGTGAT TCTTTAACCCC TTTGGTAACTG AACGTGTACAC GGCCACATTAG CACGCACACAC CAAGGAGGAAC CAGGCAAACCC CAAGCAAAACA GAGGCAGAGAA TCTCGTCCTAG AGGGGAGGGGA GCCCCACAGCA

(þ) NGF 7 2 0 0 0 0 0 0 1 0 0 0 1 0 3 1 0 1 3 0 0 0

() NGF 1 11 5 5 5 5 5 5 5 6 6 6 6 6 20 7 7 7 30 10 11 12

Fold change 6 6 6 6 6 6 6 6 6 7 7 7 7 7 8 8 8 8 11 11 12 14

Identity ESTS ESTs ESTs ESTs ESTs ESTs ESTs ESTs ESTs ESTs EST ESTs ESTs ESTs EST ESTs EST EST ESTs ESTs EST EST

Accession # CB715527 BQ194973 AW527014 AW528651 BQ195365 BF398152 BQ209689 AW532812 CK843774 BE117794 AI576247 AI711568 BI287446 CR474415 BF412923 AI070392 BE097768 XM_214182 BG378614 AI385367 BF407565 BE105351

Though our aim here is not to interpret or speculate the significance of the various regulated genes, a few generalizations seem warranted. One is that although the findings in > Tables 2-1 and > 2-2 do overlap to some extent, they also include many genes that do not. In many cases, this reflects the difference in fold‐ change required to reach significance in that changes of only greater than sixfold are reported for the SAGE study. In other cases, due to the absence of 30 sequence, it may not be presently possible to verify the SAGE tag corresponding to the genes given in > Table 2-1. Another point of interest is that the SAGE study has uncovered many regulated transcripts that encode novel proteins of unknown function. Likewise, a number of additional regulated transcripts encode proteins of at least partially defined function, but that have no obvious or previously known roles in neuronal differentiation or function. This suggests that we have a very long way to go in our quest to fully understand how NGF works and that there are many opportunities to fit new proteins into the NGF mechanism. One example of this is our initial work on ATF5, a transcription factor that is 30‐fold downregulated by NGF and that previously had no known role in the nervous system. Functional studies inspired by our SAGE findings revealed that ATF5 is highly expressed in neuroprogenitor cells but not mature neurons and glia and that it must be downregulated by trophic factors such as neurotrophins to permit neuroprogenitor cells to exit the cell cycle and to differentiate (Angelastro etal., 2003; Angelastro et al., 2005; Mason et al., 2005).-

5 Closing Remarks In summary, we provide here information about the identities of genes and gene products that are subject to regulation by NGF. We anticipate that many such genes will be regulated by other neurotrophic factors in multiple cellular settings. In contrast, others will show both cell and factor‐specific regulation. We hope that

Nerve growth factor regulated gene expression

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perusal of the information here will permit the reader to formulate overarching ideas about how NGF and other trophic factors influence the properties of their target cells and will perhaps inspire them to choose among the regulated genes their own favorites for further study.

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Sampath D, Jackson GR, Werrbach‐Perez K, Perez‐Polo JR. 1994. Effects of nerve growth factor on glutathione peroxidase and catalase in PC12 cells. J Neurochem 62: 2476-2479. Sano K, Kikuchi A, Matsui Y, Teranishi Y, Takai Y. 1989. Tissue‐specific expression of a novel GTP‐binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC‐12 cells. Biochem Biophys Res Commun 158: 377-385. Sharma E, Lombroso PJ. 1995. A neuronal protein tyrosine phosphatase induced by nerve growth factor. J Biol Chem 270: 49-53. Sharma N, D’Arcangelo G, Kleinlaus A, Halegoua S, Trimmer JS. 1993. Nerve growth factor regulates the abundance and distribution of Kþ channels in PC12 cells. J Cell Biol 123: 1835-1843. Sheehy AM, Phung YT, Riemer RK, Black SM. 1997. Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells. Brain Res Mol Brain Res 52: 71-77. Shibutani M, Lazarovici P, Johnson AC, Katagiri Y, Guroff G. 1998. Transcriptional down‐regulation of epidermal growth factor receptors by nerve growth factor treatment of PC12 cells. J Biol Chem 273: 6878-6884. Shiozuka K, Watanabe Y, Ikeda T, Hashimoto S, Kawashima H. 1995. Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase. Gene 162: 279-284. Smith MA, Fanger GR, O’Connor LT, Bridle P, Maue RA. 1997. Selective regulation of agrin mRNA induction and alternative splicing in PC12 cells by Ras‐dependent actions of nerve growth factor. J Biol Chem 272: 15675-15681. Sobue K, Kanda K. 1989. Alpha‐actinins, calspectin (brain spectrin or fodrin), and actin participate in adhesion and movement of growth cones. Neuron 3: 311-319. Sohrabji F, Greene LA, Miranda RC, Toran‐Allerand CD. 1994. Reciprocal regulation of estrogen and NGF receptors by their ligands in PC12 cells. J Neurobiol 25: 974-988. Stefanis L, Kholodilov N, Rideout HJ, Burke RE, Greene LA. 2001. Synuclein‐1 is selectively up‐regulated in response to nerve growth factor treatment in PC12 cells. J Neurochem 76: 1165-1176. Stein R, Orit S, Anderson DJ. 1988. The induction of a neural‐ specific gene, SCG10, by nerve growth factor in PC12 cells is transcriptional, protein synthesis dependent, and glucocorticoid inhibitable. Dev Biol 127: 316-325. Takahashi T, Yamashita H, Nakamura S, Ishiguro H, Nagatsu T, et al. 1999. Effects of nerve growth factor and nicotine on the expression of nicotinic acetylcholine receptor subunits in PC12 cells. Neurosci Res 35: 175-181. Takebayashi M, Hayashi T, Su TP. 2002. Nerve growth factor‐ induced neurite sprouting in PC12 cells involves sigma‐1

Nerve growth factor regulated gene expression receptors: Implications for antidepressants. J Pharmacol Exp Ther 303: 1227-1237. Takekoshi K, Nomura F, Isobe K, Motooka M, Nammoku T, et al. 1998. Identification and initial characterization of stathmin by the differential display method in nerve growth factor‐treated PC12 cells. Eur J Endocrinol 138: 707-712. Tamaru T, Okada M, Nakagawa H. 1994. Differential expression of D type cyclins during neuronal maturation. Neurosci Lett 168: 229-232. Taniguchi T, Morisawa K, Ogawa M, Yamamoto H, Fujimoto S. 1988. Decrease in the level of poly(ADP‐ribose) synthetase during nerve growth factor‐promoted neurite outgrowth in rat pheochromocytoma PC12 cells. Biochem Biophys Res Commun 154: 1034-1040. Uittenbogaard M, Chiaramello A. 2002. Constitutive overexpression of the basic helix‐loop‐helix Nex1/MATH‐2 transcription factor promotes neuronal differentiation of PC12 cells and neurite regeneration. J Neurosci Res 67: 235-245. Vedder H, Affolter HU, Otten U. 1993. Nerve growth factor (NGF) regulates tachykinin gene expression and biosynthesis in rat sensory neurons during early postnatal development. Neuropeptides 24: 351-357. Velculescu VE, Vogelstein B, Kinzler KW. 2000. Analysing uncharted transcriptomes with SAGE. Trends Genet 16: 423-425. Vetter J, Betz H. 1989. Expression of synaptophysin in the rat pheochromocytoma cell line PC12. Exp Cell Res 184: 360-366. Vician L, Basconcillo R, Herschman HR. 1997. Identification of genes preferentially induced by nerve growth factor versus epidermal growth factor in PC12 pheochromocytoma cells by means of representational difference analysis. J Neurosci Res 50: 32-43. Vician L, Silver AL, Farias‐Eisner R, Herschman HR. 2001. NID67, a small putative membrane protein, is preferentially induced by NGF in PC12 pheochromocytoma cells. J Neurosci Res 64: 108-120. Vignali G, Niclas J, Sprocati MT, Vale RD, Sirtori C, et al. 1996. Differential expression of ubiquitous and neuronal

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Myelinating Cells in the Central Nervous System—Development, Aging, and Disease

J. Neman . J. de Vellis

1 1.1 1.2 1.2.1 1.2.2 1.2.3 1.3 1.4 1.5

Oligodendrocyte Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . General Developmental Stages/Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Embryonic Origins of Oligodendrocyte in the Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Motor Neuron–Oligodendrocyte Precursor (MNOP) Cell Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . Dorsal Spinal Cord Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glial-Restricted Precursor Cell Hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oligodendrocyte Development in the Forebrain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Oligodendrocyte Development from the SVZ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myelination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Axon–Myelin Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

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Aging and Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

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Abstract: Many neurological diseases are caused by myelin deficiencies, which can be the result of both genetic and environmental factors. The myelin forming cell, oligodendrocyte (OL), is a major target for the known causes of white matter diseases. During development, oligodendrocytes pass through a series of cell phenotypes from undifferentiated stem cells to mature myelin-forming cells. The general idea that there might be different subclasses of oligodendrocyte derived from different precursor subtypes is an area of active debate. Can cells that are born of progenitors in the different parts of the embryo, under the influence of different positional signals and expressing different sets of patterning genes, ever converge on precisely the same phenotypic endpoint? The following sections will review literature about controversy over oligodendrocyte origins and degenerative process resulting in demyelination in the postnatal aging brain and Alzheimer’s disease. List of Abbreviations: AD, Alzheimer’s Disease; AEP, anterior entopeduncular; APP, amyloid precursor protein; bFGF, basic fibroblast growth factor; CGE, caudal ganglionic eminence; CNS, central nervous system; Gsh2, genomic screened homeobox 2; LGE, lateral ganglionic eminence; MBP, myelin basic protein; MGE, medial ganglionic eminence; MNOP, motor neuron–oligodendrocyte precursor; NEP, neuroepithelial cells; NFT, neurofibrillary tangles; Nrcam, neuronal-adhesion molecules; NRG, neuregulin; NRP, neuron-restricted precursor; OL, oligodendrocyte; OLP, oligodendrocyte progenitor; PDGF, plateletderived growth factor; PDGFR/, platelet-derived growth factor receptor-/; PLP, proteolipid protein; PSA-NCAM, polysialylated form of the neural cell adhesion molecule; SHH, sonic hedgehog; SVZ, subventricular zone; VZ, ventricular zone

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Oligodendrocyte Development

1.1 General Developmental Stages/Scheme The oligodendrocyte (OL) is the myelin-forming cell of the central nervous system (CNS). Myelin, which is essential for the normal functioning of the mammalian CNS, is a fatty insulation composed of modified plasma membrane that surrounds axons and promotes the rapid and efficient conduction of electrical impulses. Many neurological diseases are caused by myelin deficiencies, which can be the result of both genetic and environmental factors. These white matter disorders occurring during the development of the CNS generally display hypomyelination or dysmyelination. Deficiencies of myelin may also occur after its formation in the postnatal brain through a degenerative process resulting in demyelination. Oligodendrocytes pass through a series of cell phenotypes from undifferentiated stem cells to mature myelin-forming cells (> Figure 3‐1). This sequential process of maturation of oligodendrocytes can be reproduced in culture. The oligodendrocyte cell lineage culture is an excellent system to study the influence of growth factors on cell lineage development. Each stage of oligodendrocyte lineage progression can be identified by its characteristic markers, and through the use of growth factors, experiments can be designed to increase or decrease the degree of proliferation and/or block, delay, or accelerate maturation of developing precursor cells. Treatment of oligodendrocyte progenitor (OLP) cells with growth factors known to be present in the developing CNS yields a complex set of ligand-dependent, phenotypic responses. Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are two of the key molecular signals controlling oligodendrocyte cell development. In the presence of PDGF, OLP cells are stimulated to divide with a short cell cycle length of 18 h; are highly motile and bipolar; and differentiate in a synchronous, symmetrical, clonal fashion with a time course similar to that in vivo. In the presence of bFGF, however, progenitor cells are stimulated to divide with a longer cell cycle length of 45 h; become nonmotile preoligodendrocytes with a multipolar shape; and are inhibited from expressing galactocerebroside glycolipid, proteolipid protein (PLP), or myelin basic protein (MBP). Progenitor cells treated with both bFGF and PDGF exhibit a third phenotypic response, remaining motile, bipolar progenitor cells that divide indefinitely and do not differentiate. This ligand-dependent, conditional ‘‘immortalization’’ can

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. Figure 3‐1 Model for oligodendrocyte development. The figure illustrates the characteristics that accompany the sequential differentiation within the oligodendrocyte lineage beginning from very early stem cells to the mature oligodendrocyte. Abbreviation for cell marker: SSEA1, stage specific embryonic antigen-1; A2B5, polysialic acidNCAM, ganglioside; CNP, 2,3-cyclic nucleotide-3-phosphohydrolase; GD3, ganglioside; GPDH, glycerolphosphate dehydrogenase; GC, galactocerebroside glycolipid; O4, O1, ganglioside; PLP, proteolipid protein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; Tf, transferrin; GSTpi, glutothione S-transferases pi; PLP, proteolipid protein; GPDH, glycerol 3-phosphate dehydrogenase

greatly expand the OLP cell population over extended periods of time. Upon removal of bFGF or both mitogens, the progenitor cells differentiate along the oligodendrocyte lineage. In addition, insulin-like growth factor 1 has been implicated in OLP proliferation and differentiation. While transforming, growth factor-beta 1 is also associated with differentiation because it inhibits the mitogen-induced proliferation of OLP through exit from the cell cycle and thus might have a role in triggering differentiation (Franklin, 2002). The morphology of oligodendrocytes varies according to the axons that they myelinate. Those that ensheath large-diameter axons have a large cell body that lies close to the axon and they may synthesize only a single internode’s worth of myelin, while other oligodendrocytes make as many as 30 internodes small axons. There are also molecular differences between oligodendrocytes on large- versus small-bore axons— for example, in their gap junction proteins (connexins). It is not known whether these are intrinsic differences or phenotypic variations of a single, plastic cell type. The general idea that there might be different subclasses of oligodendrocyte is derived from different precursor subtypes is an area of active

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debate. Can cells that are born of progenitors in different parts of the embryo—under the influence of different positional signals and expressing different sets of patterning genes—ever converge on precisely the same phenotypic endpoint? The following sections will review literature about controversy over oligodendrocyte origins (Richardson et al., 2006).

1.2 Embryonic Origins of Oligodendrocyte in the Spinal Cord The fundamental question in developmental neurobiology is how a relatively simple and undifferentiated neuroepithelium in the embryo can give rise to the remarkable cellular diversity and specialization of the mature CNS. Extrinsic signaling molecules and cell-intrinsic factors instruct multipotent progenitor cells, thereby restricting their potential to become specialized neurons, oligodendrocytes, or astrocytes. It is now clear that the initial specification of spinal cord oligodendrocytes takes place in the embryo. This process requires precise interplay between cell-intrinsic and regionally restricted extrinsic factors, which has much in common with the mechanisms that underlie the development of neuron. A substantial progress in understanding neural cell fate specification has resulted from a focus on the roles of transcription factors in the acquisition of progenitor subtype identity in the developing neural tube. An extensive body of work has shown how restricted patterns of transcription factor-encoding genes are crucial for the organization and initial fate choices of restricted sets of progenitor cells along the anteroposterior and dorsoventral axes of the developing neural tube. In this section, we will discuss how neuronal and glial progenitor domains are established in the spinal cord.

1.2.1 Motor Neuron–Oligodendrocyte Precursor (MNOP) Cell Hypothesis The MNOP hypothesis is based on observations that both motor neurons and oligodendrocytes arise in similar zone of the ventral spinal cord with similar concentrations of Sonic Hedgehog (SHH) required for the induction of both cell types. In addition, in vitro studies show that the induction of oligodendrocytes is frequently accompanied by the induction of motor neurons (Pringle et al., 1996; Orentas et al., 1999). Therefore, it seems logical to have motor neurons and oligodendrocytes developmentally related to each other since ensheating the axons of these neurons with myelin would increase the conduction velocity. Interestingly, in annelids and crustaceans, myelin-like membranes are associated with axons required for rapid escape responses. Therefore, it would be intuitive to ensure that motor neurons and the cells that ensheath them arise at the same place would be to derive both cells from the same precursor. In 2002, it was discovered by three separate groups that the Olig gene family of basic HLH transcription factors is involved in the developmental production of both motor neurons and oligodendrocytes (Lu et al., 2002; Takebayashi et al., 2002; Zhou and Anderson, 2002). Specifically, Olig1 and Olig2 genes are expressed in the developing mouse spinal cord within the specific region that appears to give rise to both oligodendrocytes and motor neurons. Forced expression of Olig1 or Olig2 in neuroepithelial stem cells induces expression of early markers of the oligodendrocyte lineage. Expression of Olig2 in conjunction with neurogenin2 appears to be critical for the generation of motor neurons. Moreover, the targeted disruption of Olig2 prevents oligodendrocyte and motor neuron specification in the spinal cord. Disruption of Olig1, in contrast, disrupted normal maturation of oligodendrocytes (Noble et al., 2004).

1.2.2 Dorsal Spinal Cord Development Initially, it was believed that oligodendrocytes in the spinal cord are only derived from ventral sources. However, recent new evidence shows that oligodendrocytes in the spinal cord are derived from both ventral and dorsal sources (Cai et al., 2005; Fogarty et al., 2005; Vallstedt et al., 2005) (> Figure 3‐2). The Nkx6 transcription factors are normally expressed in the ventral part of the embryonic spinal cord in progenitor domains p3, pMN, p2, and p1, and are activated by SHH signaling from the notochord and

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. Figure 3‐2 Oligodendrocyte development in the spinal cord. Neurons are formed before glia (astrocytes and oligodendrocyte precursors (OLPs)). In general, OLPs are formed before astrocytes and ventral cell types before dorsal. The figure shows the expression domains of several transcription factors. Dashed bars indicate that the expression domain boundaries shift during development, in the direction of the small arrows—for example, expression of the transcription factor Nkx2.2 expands dorsally, and expression of the developing brain homeobox gene Dbx1 contracts. Approximately 85% of all spinal cord oligodendrocytes are generated from pMN and the remainder from other progenitor domains. It is not known whether astrocytes are also generated from pMN but, if so, they are probably produced in small numbers relative to oligodendrocytes. Abbreviations: dP1–dP6, dorsal progenitor domains; Msx3, a homeobox gene; Olig2, oligodendrocyte lineage gene 2; Pax7, paired box gene 7; pMN and p0–p3, ventral progenitor domains. (Reproduced with permission from Richardson et al. (2006).)

floor plate at the ventral midline. Once activated, they in turn activate Olig2, which as stated before, is required for the generation of both motor neurons and oligodendrocyte precursors from progenitors in the ventral progenitor domain pMN. Therefore, in spinal cords of Nkx6-null mice (specifically Nkx6.1 and Nkx6.2) there is a loss of Olig2 expression in pMN leading to developmental blockage of both motor neurons and oligodendrocytes. However, oligodendrocyte precursors that express normal markers of platelet-derived growth factor receptor-/ (PDGFR/) and Olig2 continue to be produced in the dorsal spinal cord of Nkx6-null mice. The dorsal precursors coexpress paired box gene 7 (Pax7), confirming their dorsal origin. In wild-type mice, some oligodendrocyte precursors in the dorsal part of the cord are also found to express Pax7, which indicates that dorsal production is a normal phenomenon. These precursors were missed in previous studies presumably because they are generated after their ventrally derived counterparts and mingle with them unnoticed. There are fewer Pax7-expressing oligodendrocyte precursors in wild-type spinal cord than in Nkx6 mutant cord, which suggests that ventrally produced oligodendrocyte precursors normally suppress their dorsal counterparts, perhaps because they compete more effectively for essential proliferation and/or survival signals such as PDGF alpha. Generation of the ventral precursors starts a couple of days earlier than that of the dorsal ones (embryonic day (E) 12.5 compared with
E15), so that they have plenty of time to get preestablished (Richardson et al., 2006) (> Figure 3‐3).

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. Figure 3‐3 Oligodendrocyte developmental sequence in developing spinal cord and forebrain. (a) In the mouse spinal cord,
85% of oligodendrocyte precursors are generated from pMN in the ventral ventricular zones (1), starting at about embryonic day (E)12.5. At about E15, generation of a secondary wave of precursors starts in more dorsal regions by trans-differentiation of radial glia (2). (b) In the telencephalon, the ventral-most precursors in the medial ganglionic eminence are produced from about E12.5 (1), production of the lateral ganglionic eminence-derived precursors starts a few days later (2), and production of the cortex-derived precursors occurs mainly after birth (3). Diagram not to scale. (Reproduced with permission from Richardson et al. (2006).)

Further evidence for dorsally derived oligodendrocytes has been provided by Cre-lox fate-mapping experiments in transgenic mice under the control of regulatory elements surrounding the Dbx1 (developing brain) homeobox gene. In these mice, Cre expression mirrors the normal pattern of Dbx1 expression, which is restricted to neuroepithelial precursors in p1, p0, dP6, and dP5—that is, four progenitor domains centered on the dorsal–ventral midline. The Dbx1-derived oligodendrocytes comprised
3% of all oligodendrocytes in the spinal cord and were less widely spread than the majority—being mainly located in the lateral white matter radially opposite to their site of origin in the ventricular zone. Some Dbx1-derived, Olig2-positive cells retained a radial process and transiently coexpressed the radial glial cell marker RC2, which indicates that they are formed by direct interconversion from radial glia. In more recent fate-mapping studies with Msx3–Cre transgenic mice it appeared that 10–15% of all oligodendrocytes in the cervical spinal cord originate in the dorsal half of the cord. Many of these are concentrated in the dorsal funiculus where they contribute up to 50% of the oligodendrocytes (Richardson et al., 2006).

1.2.3 Glial-Restricted Precursor Cell Hypothesis At the same time that studies have been ongoing on origin and relation of motor neurons and oligodendrocytes, a separate line of investigation has raised questions about whether oligodendrocytes are developmentally more closely related to astrocytes rather than motor neurons. This latter investigation has led to the idea that during spinal cord development, the progression from pluripotent neuroepithelial stem cell to differentiated cell types requires prior generation of lineagerestricted precursor cells. Furthermore, it is thought that neurons and glial cells come from different lineage-restricted precursor cells, NRP and GRP, respectively. GRP cells, in response to local environmental signals, can generate either astrocytes or oligodendrocytes, with oligodendrocyte generation involving the intermediate generation of OLPs (Noble et al., 2004). Both GRP cells and neuron-restricted precursor (NRP) cells can be directly isolated from the developing rat spinal cord and grown as purified populations. Freshly isolated cells exhibit the same lineage restrictions as those cells derived from neuroepithelial stem cells in vitro. Clonal studies have demonstrated that GRP cells retain their tripotential nature even after weeks of in vitro expansion and several serial

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reclonings and also exhibit these same restrictions following transplantation in vivo. GRP cells generate both oligodendrocytes and astrocytes following transplantation into the brain or spinal cord, and do not generate neurons even when they migrate into such neurogenic zones as the rostral migratory stream and olfactory bulb. NRP cells, in contrast, generate only neurons (including motor neurons), even upon transplantation into such CNS regions as the adult spinal cord (Noble et al., 2004). GRP cells exist throughout the rostra caudal axis and were initially isolated from the developing rat caudal neural tube around E12 to E14 and can be distinguished from neuroepithelial cells (NEP) by the acquisition of A2B5 immunoreactivity and their initial lack of NG2 and PDGFRa expression, and their limited adhesion to laminin and preference for fibronectin. GRP cells therefore represent an early intermediate progenitor committed to glial lineage between NEP cells and fully differentiated glia and may be one of the earliest born glial precursors (Liu and Rao, 2004). NRP cells, in contrast, expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM) and were shown to give rise to multiple different kinds of neurons and not to glia (Noble et al., 2004).

1.3 Oligodendrocyte Development in the Forebrain However, just like the spinal cord, there are still ambiguities as to ventral and dorsal sources for oligodendrocytes in the forebrain as well. The controversy about the origins of oligodendrocytes extends to the forebrain as well. Here, too, there is evidence for a ventral source from the ventricular zone of the basal forebrain. Cells that express oligodendrocyte lineage markers such as Olig1, Olig2, Sox10, and PdgfR/ first appear in the neuroepithelium of the medial ganglionic eminence (MGE), and appear to migrate laterally and dorsally from there to all parts of the developing forebrain, including the cerebral cortex, before birth (Tekki-Kessaris et al., 2001). Recent fate mapping by William Richardson and colleagues has helped resolve some of the confusion. Using an Nkx2.1-Cre transgenic mouse line that marks neural progenitors in the basal forebrain (including the MGE, anterior entopeduncular (AEP), and pre-optic area), it was found that the first oligodendrocyte precursors migrate into the cortex about E16 from ventral territories. By E18, a second wave of oligodendrocyte precursors migration from the lateral ganglionic eminence (LGE) and/or caudal ganglionic eminence (CGE) that are genomic screened homeobox 2 (Gsh2) positive occurs. Therefore, at E18, all oligodendrocyte lineage cells in the cortex are ventral in origin. After E18, however, the contribution of ventral cells starts to decrease as they are joined by yet another wave of oligodendrocyte precursors that originate in the cortex itself (Emx1-positive neuroepithelium) (> Figure 3‐4). Therefore, similar to the spinal cord there are both ventral and dorsal sources depending on the stage of development studied. Furthermore, the original population of MGE/AEP-derived precursors disappears after birth, being rapidly eliminated from the cortex and more gradually from all other parts of the brain. Almost no trace can be found of the initial Nkx2.1-derived oligodendrocyte population anywhere in adults (Richardson et al., 2006). In addition, a subpopulation of OLPs exists in the telencelphelon, characterized by the expression of PLP/DM20 and does not depend on PDGFRa signaling for survival and proliferation (Spassky et al., 2001). These PDGF-AA-independent OLPs expressing PLP/DM-20 are detected in several regions of the embryonic brain prior to the emergence of PDGFRa-expressing cells. In addition, after birth PLP/DM-20 OLPs are also distinct from the population of PDGFRa cells in the subventricular zone (SVZ) of the cerebral cortex (Ivanova et al., 2003).

1.4 Oligodendrocyte Development from the SVZ As mentioned previously, forebrain oligodendrocytes originate first from progenitors in the embryonic ventral telencephalon. A second wave of oligodendrocytes originates later in development from the dorsal telencephalon. In addition, in the developing telencephalon, the neuroepithelieum gives rise to the SVZ,

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. Figure 3‐4 Sources of oligodendrocytes in the developing forebrain. (a) Three waves of oligodendrocytes generated from different lineages in the telencephalic ventricular zone. Cells from the Nkx2.1 population begin to migrate around E12.5, those from the Gsh2 population around E15.5, and those from the Emx1 population around P0. Nkx2.1 and Gsh2 expressions overlap in the ventricular zone overlying the medial ganglionic eminence. Also shown are presumed migratory pathways of oligodendrocyte precursors from each of these areas. (b) Sources of oligodendrocytes in the corpus callosum (square) at three time points. At P0, most of the cells arise from the ventral, Nkx2.1 and Gsh2 domains. By P10, cells from the Nkx2.1 lineage have mostly disappeared, replaced by a mix of Emx1 and Gsh2 cells. In the adult, the Emx1 and Gsh2 lineages make up most of the oligodendrocytes, but an unlabeled population is also present. (Reproduced with permission from Ventura and Goldman (2006).)

a secondary proliferative population of progenitors that simultaneously generates both neurons and glia. Progenitor cells specified to become glia migrate radially from the SVZ into the subcortical white matter, corpus collosum, striatum, and cerebral cortex, where they differentiate into astrocytes and oligodendrocytes (Marshall et al., 2003). In addition, oligodendrocytes continue to be produced in the adult brain and participate in myelin repair (Chari and Blakemore, 2002). Furthermore, work from James Goldman’s laboratory show that in the postnatal forebrain, oligodendrocytes are generated from progenitor cells that reside near the tips of the lateral ventricles (Levison et al., 1999). Yet, the primary progenitors for new oligodendrocytes born in the adult SVZ have not been identified. Recent work from Alvarex-Buylla and colleagues suggest that GFAP-positive type B astrocytes and a subpopulation of actively dividing type C cells in the SVZ express the transcription factor Olig2. They also show that type B cells generate a small number of nonmyelinating NG2-positive OLPs and mature myelinating oligodendrocytes. Olig2-positive, polysialylated neural cell adhesion molecule-positive, PDGFRa, and beta-tubulin-negative cells originating in the SVZ migrated into corpus callosum, striatum, and fimbria fornix to differentiate into the NG2-positive nonmyelinating and mature myelinating oligodendrocytes. Furthermore, primary clonal cultures of type B cells gave rise to oligodendrocytes alone or oligodendrocytes and neurons. Importantly, the number of oligodendrocytes derived from type B cells in vivo increased fourfold after a demyelinating lesion in corpus callosum, indicating that SVZ-derived oligodendrocytes participate in myelin repair in the adult brain (Ligon et al., 2006; Menn et al., 2006) (> Figure 3‐5).

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. Figure 3‐5 Olig2 contributes to ongoing oligodendrogenesis in progenitor cells of the adult SVZ. The cartoon depicts cells surrounding the lateral ventricle (V). Type B cells give rise to rapidly cycling type C progenitor cells, which about 95% of the time produce type A neuroblasts that enter the rostral migratory stream and become olfactory bulb interneurons (OBN). A small subpopulation (
5%) of type C cells express Olig2 and give rise to NG2+ OLP and mature oligodendrocytes. Abbreviation: E, ependymal layer. Reproduced with permission from Ligon et al. (2006).)

It would be interesting to know what the relationship between the embryonic and postnatal germinal zones is with respect to oligodendrocyte development. As suggested by Richardson and colleagues, when neurogenesis comes to an end during late embryogenesis, the forebrain ventricular zone (VZ) regresses until only a remnant remains at the cortico-striatal boundary, which remains active and continues to generate new oligodendrocytes after birth and into adulthood. The postnatal VZ and its neighboring SVZ is derived mainly from the embryonic LGE and lateral cortex, with no contribution from more ventral regions. Therefore, the most ventral, MGE/AEP-derived progenitors leave no descendants in the postnatal SVZ. This might contribute to the gradual loss of MGE/AEP-derived oligodendrocytes during postnatal life (Richardson et al., 2006).

1.5 Myelination Myelin, a multilamellar membrane, is formed by the spiral wrapping of glial (oligodendrocytes in the CNS and by Schwann cells in the peripheral nervous system (PNS)) plasma membrane extensions around the axon. The evolutionary need for the rapid and efficient conduction of action potentials in vertebrate neurons has resulted in the development of the myelin sheath. Neuron-derived signaling molecules can and do regulate the proliferation, differentiation, and survival of oligodendrocytes. Myelin is a lipid-rich membrane (lipids constitute 70% of dry myelin weight) that is highly enriched in glycosphingolipids and cholesterol. The major glycosphingolipids in myelin are galactosylceramide and its sulfated derivative sulfatide (20% of lipid dry weight). The two major CNS myelin proteins are MBP and the proteolipid proteins (PLP/DM20). During the active phase of myelination, each oligodendrocyte must produce as much as
5–50  103 mm2 of myelin membrane surface area per day (Pfeiffer et al., 1993). The timing of myelination is crucial because the ensheathment of axons must not occur before neurons signal to oligodendrocytes. In turn, signals from oligodendrocytes to neurons are necessary to cluster

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multiprotein complexes in the axonal membrane into distinct subdomains at the nodes of Ranvier—the gaps between myelinated segments of neurons. Therefore, the next event after differentiation of OLPs is the formation of myelin. Myelination is a multistep process requiring precise coordination of several different signals. Oligodendrocytes wrap their plasma membrane around axons that have a minimum axon caliber of 2.0 mm diameter. The physiological rationale for this requirement could be that saltatory conduction would not be significantly enhanced on smaller diameter fibers. The g-ratio is the ratio of the axonal diameter divided by the diameter of the axons and its myelin sheath. Most myelinated axons have an approximate g-ratio of 0.6. Therefore, larger axons have thicker myelin, while smaller ones have thinner myelin. The exception to the constant g-ratio is seen in axons that have been remyelinated and are therefore typically thinner than expected (Sherman and Brophy, 2005). The neuregulin (NRG)-ErbB receptor system has been shown to have considerable influence on the thickness of the myelin sheath. Recent studies show that NRG-1 type III on the axonal surface is required for the myelination by Schwann cells in the PNS (Taveggia et al., 2005). Transgenic mice with reduced NRG1 expression display hypomyelination, whereas over expression of NRG-1 induces increased myelin thickness (Nave and Salzer, 2006). Myelin-forming Schwann cells thus appear to use NRG-1 signals to know whether and to what extent axons require myelination. The signaling pathways involved in the CNS are not known. Whether this signaling system operates in CNS myelination remains an open question of major importance for human demyelinating diseases (Simons and Trajkovic, 2006). After oligodendrocytes have established proper contact with the axonal membrane, the process of myelination begins. One signal that seems to be required to trigger myelination is the electrical activity from the neuron that leads to secretion of promyelinating factors of adenosine from neurons and/or leukemia inhibitory factor from astrocytes (Demerens et al., 1996; Stevens et al., 2002; Ishibashi et al., 2006). Once the coordinated differentiation of the axon and myelination has occurred, the axonal membrane is differentiated into distinct molecular, structural, and functional domains. These domains include the nodes of Ranvier, the paranodal junction, the juxtaparanodes, and the internodal regions.

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Axon–Myelin Membrane

The Nodes of Ranvier are periodical interruptions in the myelin sheath that are spaced at intervals that are about 100 times the axonal diameter. These gaps are required for efficient and rapid propagation of action potentials. The nodes are flanked by paranodal loops that form septate-like junctions with the axonal membrane. Adjacent to them are the juxtaparanodal domains that lie under the compact myelin sheath. There are some structural differences between PNS and CNS. In peripheral nerves, the entire myelin unit is covered by a basal lamina, and the outermost layer of the Schwann cell extends microvilli that cover the nodes. The perinodal space that lies between the axolemma and the basal lamina contains the microvilli and is also filled with a filamentous matrix. In addition, in the CNS there are no basal lamina and the nodes are contacted by perinodal astrocytes (synantocytes) (Butt et al., 2005). The nodes are characterized by a high density of Na+ channels that are essential for the generation of the action potential during saltatory conduction. Nodes of Ranvier in the adult CNS and PNS mostly contain Nav1.6 (Yu et al., 2003). During development, both PNS and CNS nodes express Nav1.2, which is later replaced by Nav1.6 that may allow neurons to adapt to high-frequency firing (Kaplan et al., 2001). In addition, there exist two K+ channels at the nodes. Kv3.1 is mainly found in large axons in the CNS and only in few nodes in the PNS, whereas Kcnq2 is located in all PNS nodes and most CNS nodes (Devaux et al., 2003). Several transmembrane and cytoskeleton proteins have been identified at the node as well. They include the neuronal-adhesion molecules (Nrcam), neurofascin-186, and their ligand gliomedin (Eshed et al., 2005), the cytoskeleton adaptor/scaffolding protein ankyrin G, and the actin-binding protein spectrin Beta-IV (Garrido et al., 2003). In general, it is believed that interaction with neurofascin, NrCAM, and gliodemin recruits ankyrin G that contains binding sites for Na+ channels. Recruitment of stabilizing components such as bIV spectrin further enhances the clustering of Na+ channels by anchoring the complex more firmly to the axonal cytoskeleton (Simons and Trajkovic, 2006).

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The paranodal junction attaches the myelin sheath and serves as a fence that limits lateral diffusion of axolemmal proteins and to separate the electrical activity at the node of Ranvier from the internodal region under the compact myelin sheath. Both in the PNS and the CNS, the formation of the paranodes is dependent on axon–glia interactions. The interaction of the loops that form septate-like junctions with the axonal membrane is mediated by neurofascin-155 and contactin (Sherman and Brophy, 2005). The interaction of neurofascin-155 in glia to a complex of contactin and contactin-associated protein (Caspr) in axons initiates the assembly of the paranodal complex, which is then stabilized by interactions with a specialized axonal cytoskeleton consisting of proteins such as the membrane skeleton component protein 4.1B, ankyrin B, and a/bII spectrin (Ogawa et al., 2006). The juxtaparanode is located in a short zone just beyond the innermost paranodal junction. Caspr2, the second member of the Caspr family, interacts with heteromultimers of the delayed rectifier K+ channels of the Shaker family, Kv1.1, Kv1.2, and Kv2. In addition, Kv1.6 is present at this site, predominantly in small axons. These K+ channels may act as an active damper of reexcitation and to help in maintaining the internodal resting potential Two other proteins that are found at the juxtaparanodes are transient axonal glycoprotein-1 (Tag1), a GPI-anchored CAM that is related to contactin118, and connexin 29 (Cx29), which is found at the glial membrane. Two recent studies showed that Caspr2 and Tag1 form a juxtaparanodal complex, consisting of a glial Tag1 molecule and an axonal Caspr2/Tag1 heterodimer. This complex is essential for the accumulation of K+ channels in the juxtaparanodes, as targeted disruption of Caspr2 or Tag1 results in a striking reduction in the juxtaparanodal accumulation of these channels in both PNS and CNS axons (Poliak and Peles, 2003) (> Figure 3‐6 and > 3‐7).

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Aging and Alzheimer’s Disease

Several functions of the myelin-producing cells, the oligodendrocytes, make it an integral cell of the CNS and make it pertinent issue when discussing aging and pathology of Alzheimer’s Disease (AD). Most studies on the effects of aging on the nervous system have focused on the changes that occur as a consequence of AD. The age risk factor is present in nongenetic and genetic forms of AD; with increased amyloid beta peptides (Ab), deposition in the latter form. Myelin breakdown may be a contributing factor to the pathology of both aging and AD. In addition, these breakdowns have been observed early in disease in spite of infarction, Wallerian degeneration, or white matter amyloid angiopathy (Bartzokis et al., 2004). An attribute to this white matter breakdown maybe due to the accumulation Ab, reduced cholesterol, and myelin proteins (Roher et al., 2002). The dependence of the brain on oligodendrocyte-produced cholesterol has implications for CNS development and its continual functional plasticity. All brain cholesterol is synthesized de novo by oligodendrocytes. The human brain, which is approximately 2% of the body by weight, contains approximately 25% of the body’s membrane cholesterol. It is thus not surprising that myelin membrane changes are found to drive brain lipid changes with age as well as species differences in membrane composition. Cholesterol enrichment may contribute to the free exchange of cholesterol from oligodendrocytes to neurons and astrocytes with the aid of apolipoproteins. The low water binding produced by high cholesterol levels in myelin breakdown may promote the hydrophobic ends of Ab aggregates to preferentially interact with and damage myelin. As mentioned earlier, myelin is essential for normal brain function. Myelination results in salutatory conduction that results in increases signal transmission. Once myelin function in salutatory conduction is compromised, there is a decrease in transmission velocity and refractory period of the axon. These changes and disruption due to these delays can have impact on synchronization of impulses. The effects of this desynchronization would be most apparent on brain functions that involve encoding and retrieval of memories. Aging reduces 50% number of fast-conducting CNS axons in cats. Human stereological studies estimate that the total length of myelinated axons is reduced by 27–45% in old age, primarily through loss of fibers with small diameter that myelinate later in development and are most susceptible to Ab pathology. This aging-related myelin breakdown negatively impacts cognitive performance in primates and humans. The age-related loss of myelin function may also explain the conduction delays observed in aging animals,

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. Figure 3‐6 Myelinated axons. (a) Myelinating glial cells, oligodendrocytes in the central nervous system (CNS), or Schwann cells in the PNS form the myelin sheath by enwrapping their membrane several times around the axon. Myelin covers the axon at intervals (internodes), leaving bare gaps—the nodes of Ranvier. Oligodendrocytes can myelinate different axons and several internodes per axon, whereas Schwann cells myelinate a single internode in a single axon. (b) Schematic longitudinal cut of a myelinated fiber around the node of Ranvier showing a heminode. The node, paranode, juxtaparanode (JXP), and internode are labeled. The node is contacted by Schwann cell microvilli in the PNS or by processes from perinodal astrocytes in the CNS. Myelinated fibers in the PNS are covered by a basal lamina. The paranodal loops form a septate-like junction (SpJ) with the axon. The juxtaparanodal region resides beneath the compact myelin next to the paranode (PN). The internode extends from the juxtaparanodes and lies under the compact myelin. (c) Schematic cross-section of a myelinated nerve depicting the inner and outer mesaxons (IMA and OMA, respectively). (d) Drawing of the specializations found along the internodes. A strand composed of paranodal molecules (Caspr, Contactin; inner line) flanked by juxtaparanodal proteins (Caspr2, K+ channels and TAG-1; outer lines) extends along the internodal region (the juxtamesaxon) and below the Schmidt–Lanterman incisures (the juxtaincisure). In addition, Nf155 as well as connexins 29 and 32 are found at the glial side, opposite to these axonal strands. (Reproduced with permission from Poliak and Peles (2003).)

Myelinating cells in the central nervous system—development, aging, and disease

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. Figure 3‐7 Molecular composition of domains at the Nodes of Ranvier. Components at the nodes include neurofascin-186, NrCAM, and voltage-gated Na+ and K+ channels (Nav1.2, Nav1.6, Kv3.1, and Kcnq2), which are tethered to a complex containing ankyrin G and bIV-spectrin. The paranodes contain a complex of Caspr, contactin, and 4.1B at the axonal membrane, which binds to neurofascin-155 (NF155) on the septate junction of a paranodal loop. The multiprotein complex in the juxtaparanode contains a cis complex of Caspr2 and TAG-1, which interact with 4.1B and a PDZ domain-containing protein associated with the two shaker-type K+ channels (Kv1.1 and 1.2). This complex is linked through a trans interaction with TAG-1 to the glial membrane

humans, and patients with AD. Myelin loss may also underlie the reduced myelin staining in postmortem studies of aging and the aging-related loss of brain volume (Bartzokis, 2004). Since cholesterol- and lipid-synthesizing enzymes require iron to function, oligodendrocytes have the highest iron content of all and as much as 70% of brain iron is associated with myelin. Age-related increases in iron levels may contribute to the increased intracellular oxidation necessary to trigger oligodendrocyte precursors to differentiate. Inadequate iron levels result in poor myelination and mental deficiencies in children. Normal ferritin, a spherical protein in which more than of 90% of tissue nonheme iron is stored, can sequester and store iron and other transition metals. Many normal as well as pathological processes that have been shown to damage oligodendrocytes can also release iron from ferritin. Oligodendrocytes may be more vulnerable than other cells to such iron releases since in addition to containing the highest iron stores, their particular ferritin subunit composition makes iron available with greater ease than in other cells. Recent evidence suggests that elevated iron levels increase the production of amyloid precursor protein (APP) and that the soluble Ab can act as an iron chelator. However, iron and other transition metals such as copper and zinc can also promote Ab oligomerization. Oligomerization makes Ab toxic, thus making the homeostasis of iron and Ab critically important. Therefore, late-myelinating oligodendrocytes and their precursors are present at the cortical site of amyloid beta deposits observed in aging and AD (Xu et al., 2001). In addition, iron and Ab to promote the formation of reactive oxygen species (Butterfield, 2003). This could explain as to why myelin breakdown from older and AD patients has been attributed to increased levels of lipid peroxidation. Since myelination markedly reduces neuronal energy expenditure, the loss of axonal myelin would require an estimated increase of up to 5000-fold in neuronal energy expenditure in order to maintain neurotransmission levels. Approximately 2–3% of the oxygen consumed in normal mitochondrial respiration is obligatorily transformed into free radicals, and with aging, an increasing percentage of oxygen

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is converted to superoxide. The aging-related loss/dysfunction of myelin would result in a further increase in the production of damaging free radicals. Both neurons and especially oligodendrocytes are very susceptible to damage from free radicals. Free-radical (oxidative) damage has been shown to be strongly aging related and has been implicated in the pathophysiology of AD. An increase in neuronal free-radical production has also been postulated to contribute to AD tangle-related neuropathology. Oxidation of tau induces its dimerization and polymerization into insoluble filaments, the precursor to the intraneuronal neurofibrillary tangles (NFT), the second pathognomonic lesion observed in AD brain. These damaging oxidative processes are also observed in other neurodegenerative diseases and it is thus not surprising that many other neurodegenerative disorders manifest NFTs while normal individuals rarely do so (Bartzokis, 2004). Oligodendrocytes are markedly heterogeneous based on when in the protracted process of human brain development they differentiated into myelin-producing cells. Oligodendrocytes that differentiated late in life ensheath 30–50 smaller diameter axons as opposed to 1 oligodendrocyte per myelin segment of large CNS motor and primary sensory area axons. These late-differentiating cells cannot produce the same myelin thickness per axon segment as earlier-myelinating oligodendrocytes. The thinner, later-myelinating sheaths are more susceptible to functional impairment and destruction. In addition, later-differentiating oligodendrocytes have different lipid properties, may have a slower rate of myelin turnover, and reduced ability for myelin repair than earlier-differentiating cells. This development-dependent oligodendrocyte heterogeneity could contribute to reason as to why the neocortical regions of the brain are the most vulnerable to developing AD lesions that consist of extracellular amyloid neuritic plaques and intraneuronal NFT. Furthermore, the hippocampal and primary sensory and motor areas develop lesions only later on in the disease process. Interestingly, it is the late-myelinating neocortical regions that are the most vulnerable to AD lesions first, whereas the early-myelinating primary sensory and motor areas are affected last (Braak et al., 2006).

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Richardson WD, Kessaris N, Pringle N. 2006. Oligodendrocyte wars. Nat Rev Neurosci 7(1): 11-18. Roher AE, Weiss N, Kokjohn TA, Kuo YM, Kalback W, et al. 2002. Increased A beta peptides and reduced cholesterol and myelin proteins characterize white matter degeneration in Alzheimer’s disease. Biochemistry 41(37): 11080-11090. Sherman DL, Brophy PJ. 2005. Mechanisms of axon ensheathment and myelin growth. Nat Rev Neurosci 6(9): 683-690. Simons M, Trajkovic K. 2006. Neuron-glia communication in the control of oligodendrocyte function and myelin biogenesis. J Cell Sci 119(Pt 21): 4381-4389. Spassky N, Olivier C, Cobos I, LeBras B, Goujet-Zalc C, et al. 2001. The early steps of oligodendrogenesis: Insights from the study of the plp lineage in the brain of chicks and rodents. Dev Neurosci 23(4–5): 318-326. Stevens B, Porta S, Haak LL, Gallo V, Fields RD. 2002. Adenosine: A neuron-glial transmitter promoting myelination in the CNS in response to action potentials. Neuron 36(5): 855-868. Takebayashi H, Nabeshima Y, Yoshida S, Chisaka O, Ikenaka K, et al. 2002. The basic helix-loop-helix factor olig2 is essential for the development of motoneuron and oligodendrocyte lineages. Curr Biol 12(13): 1157-1163. Taveggia C, Zanazzi G, Petrylak A, Yano H, Rosenbluth J, et al. 2005. Neuregulin-1 type III determines the ensheathment fate of axons. Neuron 47(5): 681-694. Tekki-Kessaris N, Woodruff R, Hall AC, Gaffield W, Kimura S, et al. 2001. Hedgehog-dependent oligodendrocyte lineage specification in the telencephalon. Development 128(13): 2545-2554. Vallstedt A, Klos JM, Ericson J. 2005. Multiple dorsoventral origins of oligodendrocyte generation in the spinal cord and hindbrain. Neuron 45(1): 55-67. Ventura RE, Goldman JE. 2006. Telencephalic oligodendrocytes battle it out. Nat Neurosci 9(2): 153-154. Xu J, Chen S, Ahmed SH, Chen H, Ku G, et al. 2001. Amyloidbeta peptides are cytotoxic to oligodendrocytes. J Neurosci 21(1): RC118. Yu FH, Westenbroek RE, Silos-Santiago I, McCormick KA, Lawson D, et al. 2003. Sodium channel beta4, a new disulfide-linked auxiliary subunit with similarity to beta2. J Neurosci 23(20): 7577-7585. Zhou Q, Anderson DJ. 2002. The bHLH transcription factors OLIG2 and OLIG1 couple neuronal and glial subtype specification. Cell 109(1): 61-73.

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Sulfur‐Containing Amino Acids in the CNS: Homocysteine

D. K. Rassin

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 2 Overview of Methionine Metabolism in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 3 Homocysteine and Metabolic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 4 Homocysteine as a CNS Risk Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 5 Homocysteine: Potential Mechanisms of Action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 6 Methodological Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

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2008 Springer ScienceþBusiness Media, LLC.

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Sulfur‐containing amino acids in the CNS: Homocysteine

Abstract: The sulfur amino acid metabolic pathway plays an important role in supporting a number of functions within the CNS. The metabolic intermediate, homocysteine, sits at an important branch point in this pathway and disturbances related to this compound are related to detrimental outcomes for the CNS. Homocysteine first came to notice when biological dysfunction related to its excess was reported in individuals with the inherited metabolic defect homocystinuria. Subsequently, a number of biological dysfunctions have been associated with this amino acid unrelated to metabolic diseases. Increased homocysteine has been identified as an epidemiologic risk factor for neural tube defects, cardiovascular disease, cerebrovascular disease, Alzheimer’s disease and, potentially, several dysfunctions that occur in the newborn. The mechanisms by which homocysteine may exert detrimental effects on the CNS include oxidative damage, apoptotic induction, synthesis of the excitotoxic metabolite homocysteic acid, and inhibition of energy-related enzymes. Methodologic issues, related to its disulfide/sulfhydryl structure and to the small absolute increases that are associated with increased risk, make it difficult to define homocysteine’s role in the CNS. The potential importance of modulating the amount of this amino acid is tempered by the numerous unanswered questions about the mechanism of its functions. List of Abbreviations: CNS, central nervous system; GABA, g‐aminobutyric acid; MTHFR, N5,N10‐ methylenetetrahydrofolate reductase; NMDA, N‐methyl‐D‐aspartate; PARP, poly‐ADP‐ribose polymerase; SAH, S‐adenosylhomocysteine; SAM, S‐adenosylmethionine

1 Introduction The methionine metabolic pathway supports numerous functions within the central nervous system (CNS) beyond providing an essential amino acid precursor for protein synthesis. These functions include neurotransmitter synthesis, methyl group donation, polyamine precursor, osmotic protection, antioxidant synthesis, and DNA salvage synthesis. In previous editions of this Handbook the inherited genetic defects related to this pathway have been described (Gaull, 1973), and the general metabolism of methionine, especially as related to nutrition and development, has been reviewed (Tallan et al., 1983). In this chapter a general overview of the various constituents of this pathway and their role in the CNS are presented followed by a discussion of the role of homocysteine, as an agent that may have implications for potential damage to the brain. In the years since the first edition of this Handbook, there has been an evolution of our understanding of the role that homocysteine plays in biology. (Homocysteine exists in a variety of forms, see > Sect. 6, throughout this chapter the compound is presented with this spelling and discussed as if it represents the total of all forms unless stated otherwise.) Our appreciation of the functions of this amino acid has changed as investigations have progressed from viewing homocysteine as the potential toxic agent in a very rare inherited metabolic disease (homocystinuria) to a risk factor in several common human conditions, such as cardiovascular disease, cerebrovascular disease, and Alzheimer’s disease.

2 Overview of Methionine Metabolism in the CNS The importance that the pathway of sulfur amino acid metabolism has in the CNS was first stimulated by the findings of large concentrations of cystathionine in the brain (Tallan et al., 1958) and the association of mental retardation with homocystinuria (Carson and Neill, 1962; Gerritsen et al., 1962). These latter patients were found not to have cystathionine in the brain (Gerritsen and Waisman, 1964; Brenton et al., 1965). This latter finding stimulated the discussion for this, as for many other inherited metabolic diseases, of the role of toxic precursor metabolites versus deficient metabolic products in causing the mental retardation observed in inherited diseases of amino acid metabolism (Gaull et al., 1975). In addition, the finding that a number of the sulfur amino acids have neurotransmitter properties (cystathionine, cysteinesulfinic acid, cysteic acid, hypotaurine, and taurine) stimulated interest in this pathway (Curtis and Watkins, 1960; Werman et al., 1966).

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Methionine is the essential amino acid precursor for the sulfur amino acid pathway (> Figure 4-1) and is primarily metabolized via homocysteine either to taurine or is remethylated to methionine (a good example of the fact that a nutritionally essential amino acid may be synthesized in vivo). Methionine is supplied in the diet and enters the brain from the peripheral circulation via the large neutral amino acid transporter (Sershen and Lajtha, 1979). Methionine itself has not been shown to have neurotransmitter activity in the CNS; however, behavioral effects have been noted after its administration to schizophrenics. Individuals with schizophrenia consistently worsened after methionine loading (Pollin et al., 1961; Brune . Figure 4-1 Enzymes of the methionine metabolic pathway. 1 ¼ methionine adenosyltransferase, 2 ¼ numerous methylating reactions, 3 ¼ S‐adenosylhomocysteine hydrolase, 4 ¼ cystathionine b‐synthase, 5 ¼ g‐cystathionase, 6 ¼ cysteine oxidase, 7 ¼ cysteinesulfinic acid decarboxylase, 8 ¼ hypotaurine oxidase, 9 ¼ chemical oxidation, 10 ¼ cysteic acid decarboxylase, 11 ¼ N5‐methyl‐tetrahydrofolate: homocysteine methyltransferase, 12 ¼ serine hydroxymethyltransferase, 13 ¼ N5,N10‐methylene tetrahydrofolate reductase. THFA, tetrahydrofolic acid; B6, pyridoxal‐50 ‐phosphate cofactor; B12, methyl‐cobalamin cofactor

and Himwich, 1962), an effect that appeared to be associated with an increase in the synthesis of methylated products of biogenic amines (Spaide et al., 1968). This response reflects the first step in the metabolism of methionine to S‐adenosylmethionine (SAM), catalyzed by the enzyme methionine adenosyltransferase. SAM serves as the primary methyl donor in the brain (and elsewhere in the body) for biogenic amines, RNA, DNA, and phosphatidylcholine metabolism and formation. SAM is also the precursor for the polyamines (notably spermine and spermidine), metabolizing putrescine in the process catalyzed by the enzyme S‐adenosylmethionine decarboxylase. The polyamines have been proposed as regulators of DNA and RNA metabolism, but of particular interest relative to the CNS is their potential role in the regulation of neurogenesis (Malaterre et al., 2004). SAM has also been proposed as a nutritional supplement that may help you feel good by the dietary supplement industry; however, this advice ignores the fact that increased SAM may increase homocysteine resulting in greater health risks.

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The demethylated product of reactions that utilize SAM as a methyl donor is S‐adenosylhomocysteine (SAH). SAH is catabolized to homocysteine and adenosine, catalyzed by the enzyme S‐adenosylhomocysteine hydrolase. S‐Adenosylhomocysteine hydrolase appears to favor the reverse direction of synthesis of SAH (Schatz et al., 1981), which may be one explanation for the low to absent amounts of homocysteine usually observed in physiologic fluids and tissues. Homocysteine is the major branch point of the methionine metabolic pathway; approximately half the homocysteine that reaches this point is remethylated to homocysteine via the folic acid metabolic pathway and the remainder is metabolized via the transsulfuration pathway (Mudd and Poole, 1975). The transsulfuration pathway begins with the synthesis of cystathionine from homocysteine and serine catalyzed by the enzyme cystathionine b‐synthase. Cystathionine is an unusual sulfur ether compound that is found primarily in the brain (Tallan et al., 1958). Although this compound has been suggested to be a neurotransmitter (Werman et al., 1966), proof of this function has not been forthcoming. Cystathionine primarily serves as an intermediary in the transfer of a sulfur moiety from homocysteine to serine. This conversion is completed by the catabolism of cystathionine to cysteine, catalyzed by the enzyme g‐cystathionase. There is little measurable activity of g‐cystathionase in the brain (Shimizu et al., 1966; Brown and Gordon, 1971), which probably accounts for the accumulation of cystathionine in this organ. Cysteine, an amino acid required for protein synthesis, is a precursor for the major intracellular antioxidant glutathione (g‐glutamyl‐cysteinyl‐glycine) and is further metabolized to cysteinesulfinic acid catalyzed by cysteine oxidase. Cysteinesulfinic acid is decarboxylated to hypotaurine in a reaction catalyzed by cysteinesulfinic acid decarboxylase. Cysteinesulfinic acid may also be oxidized to cysteic acid, which may be decarboxylated to taurine, and hypotaurine also may be oxidized to taurine. Taurine may function as an inhibitory neurotransmitter; it is a structural analogue of g‐aminobutyric acid (GABA) but is more likely to be an important osmoregulator in the brain (Pasantes‐Morales and Schousboe, 1988). Cysteinesulfinic acid and cysteic acid are structural analogues of glutamic acid and have similar excitatory properties in the CNS; however, it is not clear whether or not they are excitatory neurotransmitters in their own right. Taurine appears to be the end product of the methionine metabolic pathway and is either excreted from the body via the urine or as one of the bile salt conjugates, for example, taurocholic acid. The enzymatic activities of g‐cystathionase and cysteinesulfinic acid decarboxylase are absent or low in the brain during early development, thus both cysteine and taurine must be supplied from exogenous sources (Sturman et al., 1970; Pascal et al., 1972; Gaull et al., 1977; Rassin, 1982). The remethylation of homocysteine to methionine is catalyzed via a remethylation reaction in the CNS by N5‐methyl tetrahydrofolate: homocysteine methyltransferase, an enzyme dependent upon a form of the cofactor of vitamin B12, methylcobalamin. N5‐Methyltetrahydrofolate (the methyl donor for homocysteine) is regenerated from the product of the remethylation reaction, tetrahydrofolic acid, in a sequence of two reactions. The first, catalyzed by serine hydroxymethyltransferase, adds a methylene group from the amino acid serine to tetrahydrofolic acid, subsequently producing the inhibitory neurotransmitter glycine and N5 N10‐methylenetetrahydrofolic acid. N5 N10‐Methylenetetrahydrofolic acid is converted to N5‐methyltetrahydrofolic acid in a reaction catalyzed by N5,N10‐methylenetetrahydrofolate reductase (MTHFR). Homocysteine may also be remethylated to methionine by a reaction catalyzed by betaine/homocysteine methyltransferase, utilizing betaine as the methyl donor. However, this reaction only appears to take place in the liver and the kidney (Sunden et al., 1997). Thus, methionine metabolic pathway is responsible for the synthesis of several putative neurotransmitters (cystathionine, cysteinesulfinic acid, cysteic acid, hypotaurine, taurine, and glycine), the major intracellular antioxidant (glutathione), the primary CNS methyl donor (SAM), the polyamines, and an important osmoregulator (taurine), in addition to providing the protein precursors, methionine and cysteine. The pivotal role of homocysteine in this pathway appears to be important for the health of the CNS.

3 Homocysteine and Metabolic Diseases As noted above, original interest in homocysteine began when the enzymatic defect in cystathionine b‐synthase was identified and shown to be associated with a number of clinical signs and symptoms,

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including mental retardation, and biochemical signs, including increased plasma methionine and homocysteine and decreased cysteine (Mudd et al., 2001). Subsequent to this discovery, it was found that increased plasma homocysteine occurred associated with defects in the enzymes N5‐methyltetrahydrofolate: homocysteine methyl transferase, N5,N10‐methylenetetrohydrofolate reductase, and in cobalamin transport and metabolism (Rosenblatt and Fenton, 2001). These diseases all are associated with CNS deficits, with the common biochemical feature being increased homocysteine in the blood and/or urine. It is interesting to note that two additional metabolic defects in the methionine metabolic pathway have been identified, one associated with methionine adenosyltransferase and the other with g‐cystathionase (Mudd et al., 2001). Neither of these latter defects are associated with increased homocysteine, though increased plasma methionine is a hallmark of methionine adenosyltransferase deficiency. Increased cystathionine in the plasma and urine is the primary biochemical sign observed in cystathioninuria. These two latter defects are not generally associated with functional CNS deficits, though demyelination has been observed in some patients with methionine adenosyltransferase deficiency (Surtees et al., 1991). Thus, the primary association with poor cognitive function in these inherited metabolic diseases is the presence of increased homocysteine concentrations in the blood and/or urine. The possibility that increased methionine, decreased cysteine, or a general imbalance of the various potential neurotransmitters in this pathway are responsible for the CNS deficits appear to be unlikely explanations.

4 Homocysteine as a CNS Risk Factor In searching for mechanisms to explain the neurologic deficits associated with homocysteine, the first finding that seemed to be plausible was related to the thromboembolic episodes seen in a large proportion of patients with cystathionine b‐synthase‐related homocystinuria. McCully (1969) suggested that homocysteine was responsible for initiating these events and developed a rabbit model that supported this hypothesis (McCully and Ragsdale, 1970). Although not all experimental results supported the finding by McCully and Ragsdale (1970) of increased atherosclerotic plaques in homocysteine‐treated rabbits (Donahue et al., 1974), these investigations stimulated further examination of the role of homocysteine in cardiovascular disease. Lead by the study of Wilcken and Wilcken (1976), a number of investigations identified mildly increased plasma homocysteine as a risk factor for cardiovascular disease (Wilcken et al., 1983; Reis et al., 1995; Graham et al., 1997). The finding by Mudd et al. (1981) that obligate heterozygotes for cystathionine b‐synthase deficiency were not at increased risk for thromboembolic episodes and the increased interest in the role of folic acid in prevention of neural tube defects (MRC Vitamin Study Group, 1991; Czeizel et al., 1992) suggested that the remethylation pathway of homocysteine might be of particular importance in understanding the pathophysiologic effects of this compound. The potential role of the remethylation pathway in the observed increased homocysteine concentrations was further supported by the finding that a common thermolabile variant of MTHFR existed (MTHFR 677 C!T) that was associated with an increased risk for cardiovascular disease (Kang et al., 1991). However, the association of the variant form of the enzyme itself with vascular disease has been inconsistent (Deloughery et al., 1996). The variant is associated with increased homocysteine and reduced folate (Deloughery et al., 1996). In another study that compared patients with acute ischemic stroke, high risk for atherosclerosis‐ related stroke, and elderly controls, no differences were observed amongst the groups related to either MTHFR variants or to increased homocysteine (Press et al., 1999). However, this elderly population did appear to have generally increased plasma homocysteine concentrations. The combination of findings regarding neural tube defects and the benefits of folic acid, and the possible role of homocysteine remethylation failure in cardiovascular disease, led to investigations of folate supplementation on homocysteine. The grain supply of the United States was fortified with folic acid starting in 1998 (Mills and England, 2001) and this fortification appears to have reduced the incidence of neural tube defects by 19% (Honein et al., 2001). Cereal fortification appears to result in a reduction in homocysteine concentrations (Malinow et al., 1998; Jacques et al., 1999). However, this food fortification has raised the issue that additional folic acid supplementation may not be effective in reducing homocysteine concentrations and

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their further role as a risk factor (Bostom et al., 2001, 2002). In addition, lifestyles (smoking and overweight) and aging are associated with increased homocysteine (Nurk et al., 2004). Increased homocysteine with age has been associated with increased risk of hip fractures (McLean et al., 2004). Patients with renal disease may not clear homocysteine well, so have increased homocysteine (Tamura et al., 1996). Given the results of these studies, it is not surprising that increased homocysteine has been associated with an increased risk for cerebrovascular disease, estimated to be between 2.0‐ to 2.5‐fold increases in risk (Boushey et al., 1995). In considering the above studies, it might be concluded that only vascular‐related diseases of the CNS would be associated with increased homocysteine. However, several studies have indicated that homocysteine is a risk factor for cognitive function in the normal aging process (Riggs et al., 1996), in geriatric patients with various dementias (Nilsson et al., 1996), and in patients with depression (Bell et al., 1992; Fava et al., 1997). Intense interest arose in the role of homocysteine in CNS dysfunction with publication of the study by Clarke and coworkers (1998) in which increased homocysteine was clearly associated with Alzheimer’s disease. This study has been considered particularly important because a subset of the patients had pathologically proven Alzheimer’s disease, and these patients had the highest plasma concentrations of homocysteine (Diaz‐Arrastia, 1998; Miller, 1999). The study by Clarke and coworkers (1998) also indicated that the progression of Alzheimer’s disease was accompanied by an increase in homocysteine concentrations. Patients with Alzheimer’s disease and also those with vascular dementia have increased plasma homocysteine and folate deficiency (Quadri et al., 2004). These findings suggested that early increased homocysteine may signal cognitive decline in the elderly. Data to date implicate homocysteine as a risk factor in both vascular and nonvascular diseases during the aging process (Parnetti et al., 1997), and suggests that homocysteine is associated with a cognitive decline in normal aging populations (Ravaglia et al., 2003; Lewerin et al., 2005). The unanswered questions related to these studies are whether or not supplementing the aged population with B vitamins and folic acid (cofactors at a number of steps in the methionine metabolic pathway) (see > Figure 4-1) will delay the cognitive decline (even if they reduce homocysteine) and what are the exact biological mechanisms that may explain the effects of homocysteine on the CNS (Vollset and Ueland, 2005). Further, few studies have actually measured homocysteine in the CNS, none was detected in the brain of homocystinuric patients (Brenton et al., 1965) or in the brain of a fetus carried by a mother homozygous for homocystinuria (Rassin et al., 1979). However, methodologic issues, especially protein binding as discussed below, may have influenced these results. While most of the data described above reflect homocysteine as a risk factor to the brain during aging or in disease processes usually found in the geriatric population, there is some evidence for similar risk associations during early development. Homocysteine plasma concentrations increase with age in childhood and this increase appears to be associated with a decrease in folate (van Beynum et al., 2005). The increased homocysteine was not strongly associated with the MTHFR 677 C!T polymorphism in this study. Increased homocysteine has been associated with poor pregnancy outcomes (Vollset et al., 2000; Hague, 2003), neural tube defects (Mills et al., 1995; Knott et al., 2003), and fetal death (Burke et al., 1992). A number of investigators have associated increased plasma homocysteine in children with ischemic stroke (Cardo et al., 1999; van Beynum et al., 1999) and venous thrombosis (Koch et al., 1999). Given these risk factors, it is of particular interest that mild hypoxia in neonatal rats may result in increased homocysteine when the animals are vitamin deficient (Blaise et al., 2005). This increased homocysteine may be a signal in the cascade that translates oxidative stress in neonates to cognitive dysfunction.

5 Homocysteine: Potential Mechanisms of Action A number of potential mechanisms have been proposed via which homocysteine may exert its toxic effects upon the CNS. These mechanisms include the direct toxic effect of homocysteine, interaction with the N‐methyl‐D‐aspartate (NMDA)–glutamate receptor, stimulation of the production of oxidative products, the production of neurotoxic products, inhibition of functional enzymes, and the induction of apoptosis or programmed cell death.

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Homocysteine administered to rodents induces convulsions in a manner analogous to those observed in animals treated with excitatory amino acids (Freed et al., 1980). Local application of homocysteine to rat CNS neurons resulted in an excitatory response similar to that observed with the administration of glutamate (Wuerthele et al., 1982). These investigations surmised that insufficient time or oxidation occurred in their experimental paradigm, for the effect to be explained by metabolism or chemical oxidation to the glutamate analogue, homocysteic acid (Wuerthele et al., 1982). The effect of homocysteine was blocked by the methyl donor, betaine (permitting an alternative methylation pathway), further suggesting the important role of remethylation in protecting against the adverse effects of homocysteine. The effects of homocysteine were blocked by the glutamate antagonist, glutamate diethylester, which suggested that homocysteine may act at the NMDA receptor. The potential direct interaction of homocysteine with the NMDA receptor was demonstrated in cultured cortical neurons. Homocysteine appeared to directly act as an agonist at the NMDA site, and possible metabolism to other excitotoxic analogues was ruled out in these experiments (Lipton et al., 1997). A number of experiments have implicated homocysteine in the induction of oxidative stress as a mechanism of action. Studies of transgenic mice expressing a human Cu/Zn superoxide dismutase mutation as a model of amyotrophic lateral sclerosis documented increased homocysteine immunoreactivity in the brain, especially the hippocampus (Chung et al., 2003). While the actual role of superoxide dismutase mutants in familial amyotrophic lateral sclerosis is unknown, its association with this disease (Al‐Chalabi and Leigh, 2000) implicates oxidative mechanisms and, thus, implies that homocysteine may be a component of the oxidative damage. Homocysteine also potentiates toxicity in primary neuronal cultures induced by copper and amyloid b peptide (potential causative agents in the development of Alzheimer’s disease) (White et al., 2001). In these latter experiments the toxicity of the combination of amyloidb, copper, and homocysteine was particularly notable but could be blocked by the presence of catalase. These investigators also found that homocysteine was associated with H2O2 production, which would explain why catalase could ameliorate this oxidative effect as this enzyme catalyzes the catabolism of H2O2 after its formation from oxygen radicals catalyzed by superoxide dismutase (White et al., 2001). In vitro experiments using homogenates of rat hippocampus have shown that homocysteine can increase thiobarbituric‐reactive substances (markers of oxidative stress) and reduce the radical‐trapping potential of this tissue, although antioxidant protective enzymes were not affected (Streck et al., 2003). Thus, homocysteine does appear to increase oxidative stress in the CNS. The role of the excitatory amino acid product, homocysteic acid, in insults observed in the CNS related to the presence of homocysteine is unclear. Data that indicate homocysteine exerts its neurotoxic effect via the NMDA–glutamate receptor (Kim and Pae, 1996) would suggest that perhaps conversion of homocysteine to homocysteic acid may be important. Homocysteic acid has been identified in the rat CNS, in amounts roughly a 1000‐fold less than glutamate (Kilpatrick and Mozley, 1986). However, as noted above, the effects associated with homocysteine may occur more quickly than could be accounted for by conversion to its acidic metabolite (Wuerthele et al., 1982). In addition, the formation of homocysteic acid in vivo has remained somewhat controversial (Waller et al., 1991) and its possible association with increased homocysteine unclear (Fritzer‐Szekeres et al., 1998). Several investigations by the same laboratory have presented evidence that homocysteine may exert its effects via inhibition of the Naþ/Kþ‐ATPase in brain, an enzyme important for the generation and maintenance of neuronal membrane potentials in the CNS (Streck et al., 2002a, b; Wyse et al., 2002). These studies demonstrated an in vitro inhibition of Naþ/Kþ‐ATPase but not Mg2þ‐ATPase by homocysteine and methionine but not by cysteine (Streck et al., 2002a). In vivo studies had similar results and also implicated homocysteine in antioxidant effects (Streck et al., 2002b; Wyse et al., 2002). Homocysteine may also exert its toxic effect via induction of programmed cell death. Studies in cultured human umbilical vein endothelial cells found that homocysteine induced an apoptotic type of cell death (marked by decreased cell viability, nuclear condensation, and caspase‐3‐dependent cell death) (Lee et al., 2005). The action of homocysteine appeared to be via induction of reactive oxygen species (it could be inhibited by superoxide dismutase plus catalase) and regulated by nitric oxide (Lee et al., 2005). An investigation in rat embryonic primary hippocampal cell cultures also documented the promotion of apoptosis by homocysteine (Kruman et al., 2000). These investigators suggested that the sequence of events

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initiated by homocysteine are: DNA damage, poly‐ADP‐ribose polymerase (PARP) activation, caspase activation, and activation of the tumor suppressor protein p53, followed by a decline in mitochondrial potential and finally nuclear disintegration (Kruman et al., 2000). These investigators also concluded that homocysteine may sensitize hippocampal neurons to the neurotoxic effects of excitatory amino acids such as glutamate (Kruman et al., 2000). Thus, there are a number of different possible pathways by which homocysteine may exert its toxic effects, with oxidation and interaction with the NMDA receptor being particularly interesting potential mechanisms.

6 Methodological Issues The measurement of homocysteine has been an issue because of its ability to bind to both proteins and other sulfur‐containing moieties via disulfide bonds. The protein‐bound pool of homocysteine was first described by Kang and coworkers (1979) and further characterized by Malloy and coworkers (1981a, b). Homocysteine (in a manner similar to that of both cysteine and glutathione) exists as the sulfhydryl, the homogeneous disulfide–homocystine, the mixed disulfide (with cysteine or glutathione), or as the protein– homocysteine disulfide (Rassin, 1996) (> Table 4-1). Indeed, the complexity of representing the multiple forms of homocysteine has been addressed in a paper specific to the problem (Mudd et al., 2000). Most of . Table 4-1 The forms of homocysteine Form Reduced (thiol, sulfhydryl) Oxidized (disulfide) Mixed disulfide

Name Homocysteine Homocystine Mixed disulfide

Protein bound (via disulfide bond) Sum of all but protein bound Sum of all forms

Protein bound Free homocysteine Total homocysteine

Abbreviation/Brief description Hcy–SH Hcy–S–S–Hcy R–S–S–Hcy (R ¼ cysteine, glutathione, protein) Protein–S–S–Hcy (Free) f Hcy (Total) t Hcy

The various forms of homocysteine found in biology. Adapted from Rassin (1996) and Mudd et al. (2000)

the published literature addressing the role of homocysteine as a risk factor has presented the data in terms of total homocysteine (usually measured by treatment of samples with reducing agents and then determining the total amount of the sulfhydryl form produced). However, it is not unreasonable to suggest that in vivo the free homocysteine is most available for transfer into the CNS and that pool, in equilibrium with that bound to proteins and other sulfhydryls, may determine the degree of damage that results from increased homocysteine. Also, homocysteine in blood and urine usually is present in the disulfide form, homocystine, which may alter its transport into tissues. Thus, homocysteine availability may be modified by its chemical form and by changes in the amounts of plasma proteins (particularly albumin), cysteine (with which homocysteine competes for protein binding), and other sulfhydryl‐containing compounds such as glutathione (Malloy et al., 1981a, b). A second methodological issue relates to the amount of homocysteine that circulates, and the changes that have been associated with its role as an increased risk factor. In five typical patients with cystathionine b‐synthase‐related homocystinuria, their total plasma homocysteines (the sum of free homocysteine, protein‐bound homocysteine, and mixed disulfide homocysteine) ranged from 101–186 mmole/L (Malloy et al., 1981a). In most of the epidemiologic studies of homocysteine as a risk factor, plasma homocysteine is in the range of 9–20 mmole/L, and often a statistically significant result will be obtained when a 10% reduction or increase is observed—in the order of 1 mmol/L. For example a reduction from 11.4
3.4 (mean
SD) to 9.7
2.3 mmol/L, an 11.0% change was very statistically significant (p < 0.001) in a study

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of the effect of folic acid supplementation in cereal on blood homocysteine (Malinow et al., 1998). The amounts of homocysteine observed in patients with Alzheimer’s disease were 13.2
4.0 mmole/L in controls, 15.3
8.4 mmole/L in clinically diagnosed and 16.3
7.4 mmole/L in histologically confirmed patients (Clarke et al., 1998). Some investigators have addressed the difficulty of assessing these quantitatively small changes by analyzing their data in terms of quartiles (McLean et al., 2004) or setting a possible abnormal limit, for example, total plasma homocysteine >13 mmole/L (Jacques et al., 1999). From a population perspective, monitoring plasma total homocysteine may be very useful but determining an individual’s risk factor in the light of these data may be considerably more troublesome; for example, how would one interpret a blood total homocysteine of 10.0 or 11.0 or 12.0 mmole/L—do any of these concentrations represent a specific risk?

7 Conclusion The sulfur amino acid metabolic pathway plays an important role in supporting a variety of functions within the CNS. Homocysteine sits at an important branch point in this pathway and disturbances of its equilibrium appear to be related to detrimental outcomes for the CNS. A number of questions remain to be answered regarding the role of this amino acid in the CNS. 1. What is the amount of plasma homocysteine that represents safety for the CNS? 2. How should homocysteine be best determined and expressed? 3. Does increased plasma homocysteine reflect increased brain homocysteine? 4. What is the most appropriate milieu relative to the availability of vitamins B6, B12, and folic acid? 5. What is the role of homocysteic acid in the toxicity of homocysteine? 6. Does the MTHFR677C ! T polymorphism determine the homocysteine risk factor? 7. Does homocysteine play a common role in the pathway leading to the degenerative processes observed in diseases such as Alzheimer’s, amyotrophic lateral sclerosis, atherosclerosis, and in the natural aging process?

References Al‐Chalabi A, Leigh PN. 2000. Recent advances in amyotrophic lateral sclerosis. Curr Opin Neurol 13: 397-405. Bell IR, Edman JS, Selhub J, Morrow FD, Marby DW, et al. 1992. Plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people. Acta Psychiatr Scand 86: 386-390. Blaise S, Alberto JM, Nedelec E, Ayav A, Pourie G, et al. 2005. Mild neonatal hypoxia exacerbates the effects of vitamin‐ deficient diet on homocysteine metabolism in rats. Pediatr Res 57: 777-782. Bostom AG, Jacques PF, Liaugaudas G, Rogers G, Rosenberg IH, et al. 2002. Total homocysteine lowering treatment among coronary artery disease patients in the era of folic acid‐fortified cereal grain flour. Arterioscler Thromb Vasc Biol 22: 488-491. Bostom AG, Selhub J, Jacques PF, Rosenberg IH. 2001. Power shortage: Clinical trials testing the ‘‘homocysteine hypothesis’’ against a background of folic acid‐fortified cereal grain flour. Ann Intern Med 135: 133-137.

Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. 1995. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: Probable benefits of increasing folic acid intakes. JAMA 274: 1049-1057. Brenton DP, Cusworth DC, Gaull GE. 1965. Homocystinuria: Biochemical studies of tissues including a comparison with cystathioninuria. Pediatrics 35: 50-56. Brown FC, Gordon PH. 1971. A study of L‐[14C] cystathionine metabolism in the brain, kidney, and liver of pyridoxine‐ deficient rats. Biochim Biophys Acta 230: 434-445. Brune GG, Himwich HE. 1962. Effects of methionine loading on the behavior of schizophrenic patients. J Nerv Ment Dis 134: 447-450. Burke G, Robinson K, Refsum H, Stuart B, Drumm J, et al. 1992. Intrauterine growth retardation, perinatal death, and maternal homocysteine levels. N Engl J Med 326: 69-70. Cardo E, Vilaseca MA, Campistol J, Artuch R, Colome C, et al. 1999. Evaluation of hyperhomocysteinaemia in children with stroke. Eur J Paediatr 3: 113-117.

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Stress Response Signal Transduction

Xiaoming Hu . J. R. Perez‐Polo

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 2 Homeostasis: Stress, Inflammation, and Recovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 3 Inflammatory Responses: Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 4 Cytokine Responses and Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 5 Transcription Factors: NF‐kB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 6 Inflammatory Gene Expression: COX‐2 and iNOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 7 Decoy Treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 8 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

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Abstract: Trauma evokes a common response in all organ systems that includes early energy depletion followed by dysfunction of ionic gradients and triggering of stress response mechanisms with outcomes ranging from recovery of function to delayed cell death and further dysfunction. The specific outcome to trauma in terms of this spectrum is determined to some extent by the specific signaling mechanisms triggered. In the central nervous system (CNS), stress response signaling includes changes in transcription of cytokines and chemokines and also inflammatory enzymes. A common regulatory component in all of these is the nuclear factor kappa B (NF-κB) transcription factor. Interventions interfering with NF-κB activation can affect the outcome to CNS trauma. List of Abbreviations: CNS, central nervous system; COX‐2, cyclooxygenase‐2; iNOS, inducible nitric oxide synthase; ICE, IL‐1‐converting enzyme; IL‐1, interleukin‐1; IL‐1R, IL‐1 receptor; IL‐1Ra, IL‐1Rantagonist; IL‐1RAacP, IL‐1R accessory protein; LPS, lipopolysaccharides; MCAO, middle cerebral arterial occlusion; MnSOD, manganese superoxide dismutase; RPA, ribonuclease protection assay; RT‐PCR, real‐ time polymerase chain reaction; TNF, tumor necrosis factor

1 Introduction Classical stress response studies dealt with hormonal changes in the face of abrupt environmental stimuli and their effects on physiological processes: circulation, temperature regulation, motor activity, and so on. This was followed by inclusion of immune responses as part of the physiological stress responses, typically involving assessments of cytokine expression changes in the central nervous system (CNS) and associated growth factors. At about this time, quantitative molecular approaches began to take the place of cellular assays based on migration and inflammatory events at the organismic level. In addition, the role of cytokine expression changes in the CNS in response to more traumatic and acute events, typically related to abrupt physical trauma and ischemia, became more common even as various experimental and animal models of head trauma, stroke, perinatal ischemia, and spinal cord injury were developed and characterized. Here, we focus on some of the more common molecular components of stress in the CNS in the acute and chronic situation. By necessity, this is not a definitive treatment but rather an attempt to integrate some mechanisms of responses to ‘‘trauma’’ in CNS into a cohesive, albeit complex, conceptual framework.

2 Homeostasis: Stress, Inflammation, and Recovery Cellular stress responses are best characterized by an early stage of energy depletion inactivating membrane‐ bound pumps responsible for the maintenance of transmembrane gradients of an assortment of ions and signaling molecules. This departure from cellular homeostasis triggers an inflammatory response that, if sufficient energy stores are available, triggers active secondary cell death with apoptotic features. Cellular apoptosis processes include chromatin condensation and subsequent margination against the nuclear envelope, cytoplasmic and nuclear condensation, overall cell shrinkage, cytoplasmic vacuolization, and convolution of the nuclear and the cytoplasmic membranes followed by the formation of apoptotic bodies. These apoptotic bodies then undergo phagocytosis. Under conditions in which the initial disruption of cellular processes is sufficiently robust, the cell death process displays necrotic features, characterized by overall cell swelling, chromatin clumping, and organelle disruption, with early loss of membrane integrity (Hockenbery, 1995; Majno and Joris, 1995). For example, necrotic cell death is typically an early event at the core of the traumatic insult to the CNS in the case of hypoxia ischemia (HI) (Ray et al., 2003). Delayed cell death with apoptotic features usually starts between 12 and 24 h later at the core site and spreads over time to sites away from the impacted area and includes apoptosis of neurons and glia (Northington et al., 2001). In adult animal models, both apoptosis and necrosis have been reported following focal cerebral ischemia in rats and mice (Charriaut‐ Marlangue et al., 1996; Dux et al., 1996; Chen et al., 1997; Matsushita et al., 1998; Guegan and Sola, 2000) and after global cerebral ischemia (Nitatori et al., 1995; Ni et al., 1998), often emphasizing delayed neuronal death in the selectively vulnerable regions of the hippocampus.

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3 Inflammatory Responses: Cytokines Inflammatory responses have also been implicated in chronic glial activation in the aged rodent brain, in rodent models of adult spinal cord injury and stroke, head injury, as well as hypoxia ischemia (Yang et al., 1995; Hagberg et al., 1996; Bona et al., 1999; Nesic et al., 2001; Rossner et al., 2001; Chu et al., 2002; Hedtjarn et al., 2002; Nesic et al., 2002; Toliver‐Kinsky et al., 2002; Cole and Perez‐Polo, 2004; Hu et al., 2005; Rossner et al., 2005). Typically, CNS trauma increases glial expression of the proinflammatory cytokines: interleukin‐ 1 (IL‐1)b, tumor necrosis factor‐a/b (TNF‐a/b), and IL‐6 (Foster‐Barber et al., 2001; Lau and Yu, 2001; Nesic et al., 2001, 2002; Saliba and Henrot, 2001; Hu et al., 2005). The best‐characterized early response inflammatory cytokine is IL‐1 (Szaflarski et al., 1995). The IL‐1 family includes the agonists IL‐1a and IL‐1b, the endogenous receptor antagonist IL‐1 receptor antagonist (IL‐1Ra), and IL‐18/IL‐1g (Shapiro et al., 1998). The role of IL‐1b in CNS trauma is probably the best understood (Rothwell et al., 1997; Nesic et al., 2001; Hu et al., 2005). There are reports showing that IL‐1b mRNA and protein levels increase as early as 1 h after brain injury and persist during the development of infarction in various adult animal models (Minami et al., 1992; Zhang et al., 1998a, b; Davies et al., 1999). Sources of IL‐1 in CNS are endothelial cells, microglia, and macrophages or monocytes (Buttini et al., 1994) although neurons, astrocytes, and oligodendrocytes can also secrete IL‐1b under traumatic conditions (Sairanen et al., 1997). Levels of IL‐1a in CNS have been shown to also increase promptly and robustly after temporary or permanent occlusion of the middle cerebral artery in mice (Hill et al., 1999; Touzani et al., 1999). Following synthesis, pro‐IL‐1b (31 KD) remains primarily in the cytoplasm until cleaved by the IL‐1b‐ converting enzyme (ICE, caspase‐1), producing mature and active IL‐1b (17 KD). Caspase‐1 also cleaves and activates IL‐18 but not IL‐1a (Dinarello, 1999). There is evidence showing correlation between increased IL‐1b levels and subsequent neurodegeneration. Thus, administration of exogenous IL‐1b markedly exacerbates neuronal or glial damage in rodents exposed to focal cerebral ischemia or excitotoxin administration (Lawrence et al., 1998; Stroemer and Rothwell, 1998). Decreasing effective IL‐1b levels using anti‐IL‐1b antibodies (Touzani et al., 1999) or inhibitors of ICE (Loddick and Rothwell, 1996; Hara et al., 1997) decreases neuronal losses after cerebral ischemia. IL‐1Ra is widely used to study the function of IL‐1 because it is a selective endogenous receptor antagonist that blocks the actions of IL‐1a and IL‐1b (Dinarello et al., 1998). Injection or overexpression of IL‐1Ra significantly inhibits cell death after hypoxia ischemia although the mechanisms involved were not determined (Betz et al., 1995; Stroemer and Rothwell, 1997, 1998; Yang et al., 1998).

4 Cytokine Responses and Cell Survival The responses to IL‐1a and IL‐1b are initiated by binding to IL‐1 type‐I receptor (IL‐1RI), which then associates with an IL‐1 receptor accessory protein (IL‐1RAcP) (Wesche et al., 1997), leading to signal transduction (Cao et al., 1996; Muzio et al., 1997). IL‐1RIs are expressed on both glia and neurons (French et al., 1999; Pinteaux et al., 2002). Ligand binding of IL‐1, in situ hybridization, and immunohistochemical studies have shown the expression of IL‐1RI receptors in specific brain regions in rats, particularly, the hippocampus, the dentate gyrus, the choroid plexus, and the cerebellum. A second receptor IL‐1 type‐II receptor (IL‐1RII) has a much shorter cytoplasmic domain and is thought to act as a decoy (nonsignaling) receptor (McMahan et al., 1991). The activation of IL‐1RI stimulates ubiquitous transcription factors (NF‐kB, AP‐1, AFP) and some key intracellular signaling molecules (JNK, IP3, or PKC kinases; Auron, 1998; O’Neill and Greene, 1998). Based on in vitro studies, it has been suggested that activation of the NF‐kB pathway through IL‐1RI binding after ischemia results in gene transcription with deleterious outcomes (Dunn et al., 2002). For hypoxia ischemia, it has been shown that IL‐1 mRNA and protein levels increase as early as 1 h after the injury and persist during the development of infarction (Minami et al., 1992; Szaflarski et al., 1995; Hagberg et al., 1996; Zhang et al., 1998a, b; Davies et al., 1999; Qiu et al., 2004). Regional differences in IL‐ 1b distribution patterns have been observed in normal rodent brain (Ilyin et al., 1998; Vitkovic et al., 2000);

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for example, there are lower levels of IL‐1b in hippocampus compared with cortex in sham‐treated animals (Hu et al., 2005). This variation might reflect endogenous neuromodulatory activities in an untreated rat. It is reported that the hippocampus is more sensitive to hypoxia ischemia injury than other brain structures (Cervos‐Navarro and Diemer, 1991; Grafe et al., 1994; Yue et al., 1997). The time course of IL‐1b protein levels correlates well with the time course of cell death level and caspase‐3 activity observed after injury. Thus, it would be reasonable to speculate that the cell death‐inducing threshold of IL‐1 is lower in hippocampus than that in the cortex, which explains the lower level of IL‐1b after hypoxia ischemia in hippocampus. This is consistent with there being temporal and spatial differences in IL‐1b protein expression after HI, not surprising given similar reports of spatial temporal differences between hippocampus and basal forebrain cell death mechanisms in an adult rodent hypoxia model (Qiu et al., 2001). Thus, it is likely that there are different mechanisms of stress response signal transduction at the level of IL‐1 signaling in different brain regions. This could also explain some of the particular aspects of long‐term outcomes in the clinical context and the reported fragility of hippocampus, for example, over other brain regions. Using the techniques of in situ hybridization, ribonuclease protection assay (RPA), and real‐time polymerase chain reaction (RT‐PCR), it has been reported that IL‐1RI mRNA was expressed in high levels in cortex (Gayle et al., 1997; Wang et al., 1997) and in low to moderate levels in hippocampus (Wong and Licinio, 1994). At the cellular level, IL‐1RI mRNA has been localized in cerebrovascular endothelium, astrocytic and oligodendroglial (Wong and Licinio, 1994; Hu et al., 2005), as well as neuronal components (Takao et al., 1990; Wong and Licinio, 1994; Hu et al., 2005). Significantly protective effects of IL‐1Ra have been documented in an adult stroke rat model, where cell death after stroke was significantly reduced when IL‐1Ra was administered as a single injection at the time of middle cerebral artery occlusion and hypoxia ischemia (Mulcahy et al., 2003; Hu et al., 2005). The maximal cortical protection occurred after intracerebroventricular injection of IL‐1Ra 2 h post middle cerebral arterial occlusion (MCAO) (Mulcahy et al., 2003). Protective effects of IL‐1Ra can last for at least 7 days after injury (Garcia et al., 1995; Liu et al., 1999; Loddick and Rothwell, 1996). Injections of L‐1Ra 2 h after hypoxia ischemia injury markedly decreased hippocampal cell death levels and caspase‐3 activity when measured 24 h after injury, in support of the hypothesis that IL‐1 contributes to hypoxia ischemia‐induced cell death.

5 Transcription Factors: NF‐kB The transcription factor ‘‘NF‐kB’’ consists of homo‐ or heterodimers of subunits, which constitute a family of related proteins, including p50, p52 (also called p49), p65 (also called Rel‐A), C‐Rel, and Rel‐B (> Figure 5‐1). All of them share a highly conserved
300 residue NH2‐terminal domain for DNA binding or dimerization (Rel homology domain), which enables them to form dimers and bind to an array of homologous decanucleotide sequences with varying affinities. NF‐kB/Rel proteins can be divided into two classes based on their C‐terminal domain. One class includes p65, C‐Rel, and Rel‐B proteins. All of them contain C‐ terminal transactivation domain. Another class includes p50 and p52, which have no transactivation domain at the C terminus (Bours et al., 1990, 1992; Ghosh and Baltimore, 1990; Nolan et al., 1991; Ruben et al., 1991; Schmid et al., 1991; Ryseck et al., 1992). p50 and p52 are first synthesized as precursors p105 and p100, respectively, both of which have ankyrin‐like repeats with close homology to that of IkB family in their C terminus. After degradation of the C‐terminal region, mature p50 and p52 are released. Dimerization of NF‐kB subunits produces species with various intrinsic DNA‐binding specificities, transactivation properties, and subcellular localization (Baeuerle, 1991; Siebenlist et al., 1994). NF‐kB dimeric combinations must contain at least one of the transactivating members (p65, C‐Rel, or Rel‐B) to have the ability to activate DNA transcription (Baeuerle, 1991; Lenardo and Siebenlist, 1994; Siebenlist et al., 1994; Chen et al., 1998). The most commonly described active NF‐kB subunit combinations are the p65/p50 and C‐Rel/p50 heterodimer. Homodimeric combinations of p50 or p52 have no transactivation activity and often behave in vivo as transcriptional inhibitors at high affinity‐binding sites (Franzoso et al., 1992, 1993; Kang et al., 1992).

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. Figure 5‐1 Schematic diagram illustrating possible effects of trauma‐induced IL‐1 increases on NF‐kB activation. IL‐1 stimulates degradation of IkBa and increased nuclear levels of Bcl‐3. IkBa degradation enables nuclear translocation of p65/p50. Nuclear Bcl‐3 binds and dissociates p50 homodimers from cognate NF‐kB‐binding sites enabling p65/p50 dimers to bind and activate target genes

A mechanism underlying IL‐1‐induced cell death involves the activation of the transcription factor NF‐kB and the subsequent induction of multiple genes. NF‐kB is constitutively expressed in CNS at low levels (Kaltschmidt et al., 1994), but is stimulated above basal levels by stimuli associated with ischemic insults. In the rodent brain, NF‐kB activation is stimulated in the forebrain and the hippocampus after hypoxia ischemia (Koong et al., 1994; Schmidt et al., 1995; Yang et al., 1995; Gabriel et al., 1999; Nurmi et al., 2004). In most of these studies, the expression and activity of p65/p50 was measured and used as a representative for NF‐kB activity, which overlooks changes in other NF‐kB proteins. For example, NF‐kB C‐Rel/p50 selectively binds the Bcl‐x gene promoter CS4 sequence while p65/p50 preferentially binds the IgG‐kB promoter sequence (Hu et al., 2005). NF‐kB regulates the expression of antiapoptotic genes such as Bcl‐xL, Bcl‐2, Manganese superoxide dismutase (MnSOD), and inhibitor‐of‐apoptosis proteins, and proinflammatory genes such as IL‐1, TNFa, matrix metalloproteinase‐9, cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS), thereby playing a dual role in neuronal survival (Mattson and Camandola, 2001). Given that the p65/p50 dimer activates genes coding for proteins with proinflammatory properties, while C‐Rel/p50 dimer activates genes coding for Bcl‐xL protein that prevent cell death (Qiu et al., 2001; Pizzi et al., 2002), we believe that some of the present confusions in the field as to the proapoptotic or antiapoptotic nature of NF‐kB activities in in vivo lesion paradigms result from a lack of data addressing activation of specific promoters by different

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NF‐kB dimer species. The activation of different NF‐kB dimers under different stimuli and at different time points may be responsible for the differential beneficial or detrimental outcomes in cell survival. NF‐kB is expressed in the nervous system and shows a low level of constitutive activity in neurons (Kaltschmidt et al., 1994). NF‐kB is induced above constitutive levels in the brain by various stimuli, most of which are stressful or neurotoxic. For example, rodent NF‐kB is induced in the forebrain and the hippocampus following hypoxia ischemia (Koong et al., 1994; Schmidt et al., 1995; Yang et al., 1995; Qiu et al., 2001), in the cortex after traumatic brain injury (Yang et al., 1995), and in the limbic structures following seizures (Rong and Baudry, 1996). NF‐kB is reported to regulate cell death by modulating a diverse array of genes, known to be important for cell death or survival, such as Bcl‐2, Bcl‐xL, MnSOD, iNOS, COX‐2, and some inflammatory cytokines (IL‐1a/b, TNF‐a/b) (Foster‐Barber et al., 2001; Lau and Yu, 2001; Saliba and Henrot, 2001). The extent to which NF‐kB activation contributes to neuropathology versus neuroprotection and recovery remains unresolved. In a number of experimental models, including cell lines and tissues under different stimuli, NF‐kB activation appears to result in both apoptotic and antiapoptotic outcomes (Abbadie et al., 1993; Barger et al., 1995; Jung et al., 1995; Barger and Mattson, 1996; Grilli et al., 1996; Grimm et al., 1996; Baichwal and Baeuerle, 1997; Lipton, 1997; Grilli and Memo, 1999). It is possible that the different compositions of NF‐kB protein dimers determine the outcomes of their activation. For example, the p65/p50 dimer may activate genes coding for proteins with proapoptotic properties whereas C‐Rel/p50 dimer may activate genes coding for proteins that prevent cell death (Qiu et al., 2001; Pizzi et al., 2002; Hu et al., 2005). Therefore, different stimuli might activate different NF‐kB dimers, resulting in beneficial or detrimental outcomes. There is a generic NF‐kB DNA sequence consisting of a 10‐bp consensus sequence that encompasses 128 different decameric sequences, 50 ‐GGGRNNTYCC‐30 (G¼guanine, R¼purine, N¼any nucleotide, Y¼ pyrimidine, C¼cytosine, T¼thymine). Distinct NF‐kB subunit combinations exhibit different binding affinities for the multiple consensus sequence combinations, and this complex variation likely determines the specificity of NF‐kB transcriptional regulation (Perkins et al., 1992; Qiu et al., 2001). For example, p65/p50 heterodimers bind preferentially to the decameric sequence present on the IgG‐kB promoter (50 ‐GGGACTTTCC‐30 ) and the C‐Rel/p50 heterodimer binds preferentially to the decameric sequence 50 ‐ GGGGTCTCC‐30 present on the Bcl‐x promoter (Qiu et al., 2001). Specific combinations of NF‐kB proteins can thus distinguish among the various kB sites to selectively regulate gene expression. Nadjar et al. (2003) documented the activation of NF‐kB by the visualization of p65 translocation in the cells of adult rat brain after intraperitoneal or i.c.v. IL‐1b injection. IL‐1RI‐deficient mice demonstrated impaired NF‐kB activation upon the stimulation of IL‐1b. Huang et al. (2003) documented that decreased NF‐kB activity was observed in ischemic ICE null adult mice and suggested that IL‐1 signaling contributes to NF‐kB activation and subsequent ischemic damage. One of the most important features of NF‐kB proteins is that the function of NF‐kB is strictly regulated by its subcellular localization. Its activation depends on the translocation from the cytoplasm to the nucleus. The movement of NF‐kB proteins into the nucleus and binding to DNA is controlled by a family of inhibitor proteins called IkB (Zabel and Baeuerle, 1990; Haskill et al., 1991; Thompson et al., 1995). There are several inhibitory IkB proteins (IkBa, IkBb, IkBg, and Bcl‐3, > Figure 5‐1) that regulate NF‐kB activity. These proteins have several homologous amino acid stretches known as ankyrin repeats that specifically interact with NF‐kB/Rel proteins (Bours et al., 1992; Grilli et al., 1993). IkBa and Bcl‐3 are the two most important proteins in this family. They contribute to the regulation of NF‐kB activity via different mechanisms. IkBa takes effect in the cytoplasm, where they bind to NF‐kB heterodimers and inhibit their action. In response to stimuli, including IL‐1, TNF‐a, and bacterial lipopolysaccharides (LPS), IkB is phosphorylated by an IkB kinase, ubiquinated and degraded by proteasomes (Ghosh and Baltimore, 1990; Liu et al., 1993; Israel, 1995), uncovering masked nuclear localization signals on the NF‐kB dimers, which are then translocated to the nucleus, bind to DNA consensus sequences on gene promoters, and activate transcription (Ghosh et al., 1998). In particular, it has been shown that IkBa must be phosphorylated on Ser‐32 and Ser‐36 and ubiquitinated at Lys‐21 and Lys‐22 so that it can be targeted for subsequent degradation by the ubiquitin and proteasome system (Traenckner et al., 1995; Baldwin, 1996). Interestingly, only the NF‐kB

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dimers that contain at least one transactivation member (p65, C‐Rel, or Rel‐B) are effectively regulated by IkBa. Thus, the p52 and p50 homodimers are not usually retained in the cytoplasm by IkBa. Their activity is regulated within the nucleus by Bcl‐3. Although structurally homologous, Bcl‐3 and IkBs act differently in regulating NF‐kB activity. Whereas the IkBs are primarily cytoplasmic, Bcl‐3 is predominantly nuclear bound and has a binding specificity for p52 or p50 homodimer (Bours et al., 1993; Franzoso et al., 1993; Fujita et al., 1993; Nolan et al., 1993; Bundy and McKeithan, 1997). There are two mechanisms by which Bcl‐3 can regulate the NF‐kB activity. First, Bcl‐3 can successfully compete with promoter DNA‐bound p50 or p52 homodimers, removing the NF‐kB homodimers away from cognate‐binding sites on target promoter DNA sites (Franzoso et al., 1993; Nolan et al., 1993; Siebenlist et al., 1994; Zhang et al., 1998a, b), allowing p65/p50 or C‐Rel/p65 to bind and activate gene transcription. Second, Bcl‐3, together with p52 or p50 homodimer, can bind to DNA kB site and form a ternary complex. The tethering of Bcl‐3 to DNA via p52 or p50 homodimer allows the activation of gene transcription, whereas Bcl‐3 and p50/p52 homodimers alone cannot since Bcl‐3 has only the transactivation domain and p50/p52 has only the DNA‐binding domain (Bours et al., 1993; Fujita et al., 1993; Rocha et al., 2003). The different regulatory effects of Bcl‐3 may depend on its phosphorylation states and concentration in the nucleus (Fujita et al., 1993; Nolan et al., 1993; Bundy and McKeithan, 1997; Cogswell et al., 2000). NF‐kB transactivation involves IkBa and Bcl‐3 proteins. IkBa phosphorylation at Ser‐32 and Ser‐36 (Traenckner et al., 1995) is a requirement for its ubiquitination and subsequent degradation by the 26 S proteasome, an important step in the translocation of NF‐kB from the cytoplasm to the nucleus. An in vitro study of IL‐1b‐challenged C6 (astrocytoma) cells showed newly synthesized IkBa to be phosphorylated at Ser‐32 (Uehara et al., 1999), which may explain the increase of phosphorylated IkBa 24 h after hypoxia ischemia (Hu et al., 2005). Increased nuclear Bcl‐3 levels also stimulate NF‐kB activation. Western blot analyses have shown an increase in translocation of Bcl‐3 from cytoplasm to nucleus 24 h after hypoxia ischemia in hippocampus and cortex (Hu et al., 2005). A report based on DNA microarray analyses and RT‐ PCR by Elliott et al. (2002) using cultured SW‐1353 cells also shows Bcl‐3 transcription to be stimulated by IL‐1b signaling. It is reported that NF‐kB is stimulated by hypoxia via increased IkBa degradation and NF‐k B nuclear translocation. A small number of researches indicated that an independent pathway involving Bcl‐3 stimulation also might contribute to the NF‐kB activation after hypoxia (Gozal et al., 1998; Zhang et al., 1998a, b; Qiu et al., 2001).

6 Inflammatory Gene Expression: COX‐2 and iNOS There is evidence that nitric oxide (NO) is an important mediator of ischemic and neurodegenerative pathology in the CNS (Hara et al., 1996; Mizushima et al., 2002; Brown and Bal‐Price, 2003). NO is endogenously produced as a byproduct of arginine metabolism by different nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Both nNOS and eNOS are constitutively expressed, whereas iNOS is expressed in response to a variety of stimuli. There are reports documenting the relationship between iNOS induction and ischemic lesions in the brain (Hara et al., 1996; Mizushima et al., 2002). In addition, the administration of IL‐1b to cultured brain endothelial cells and hippocampal neurons induces the expression of iNOS mRNA and increases NO release (Bonmann et al., 1997; Serou et al., 1999). These observations, together with evidence showing that induction of iNOS is regulated by NF‐kB p65/p50 (Teng et al., 2002), also support the hypothesis that activation of p65/p50 by IL‐1 after CNS trauma may further stimulate iNOS synthesis and NO formation, which in turn triggers cell death. COX is an important enzyme in the inflammatory process. COX catalyzes the rate‐limiting step in the conversion of arachidonic acid to prostaglandins. There are two isoforms of COX, designated COX‐1 and COX‐2. COX‐1 is expressed constitutively and appears to be responsible for ongoing physiological function, whereas COX‐2 is present only in certain tissues where it is transiently induced by growth factors, inflammatory cytokines, tumor promoters, and bacterial toxins (Chun et al., 2004). There is evidence to support an involvement of COX‐2 in brain damage (Hara et al., 1998; Nagayama et al., 1999; Sugimoto and Iadecola, 2003). IL‐1b transcriptional control of COX‐2 by NF‐kB p65/p50 has been demonstrated in cell

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culture (Newton et al., 1997). There is a significant increase in iNOS and COX‐2 expression in the hippocampus and the cortex 24 h after hypoxia ischemia (Hu et al., 2003). Treatment with IL‐1Ra reversed the HI‐induced increases in iNOS and COX‐2, a novel finding. Interestingly, sequence analyses of the iNOS and COX‐2 promoters have shown the presence of DNA‐binding consensus sequences specific to the p65/ p50 binding (Hu et al., 2005).

7 Decoy Treatments Sequence‐specific inhibition of NF‐kB can be accomplished with synthetic double‐stranded (ds) phosphothiorate oligonucleotides containing a NF‐kB consensus sequence, which acts in vivo as a ‘‘decoy’’ cis element to bind transcription factors and block the activation of cognate genes (Morishita et al., 1997; Qiu et al., 2004; Tomita et al., 1998, 2000, 2001). The principle of the transcription factor decoy approach is simply the inhibition of promoter activity due to the binding of endogenous transcription factors to saturating amounts of exogenous double‐stranded oligonucleotides displaying specific DNA sequences that are present in the promoter region of the gene of interest. For example, an IgG‐kB decoy, which contains the NF‐kB consensus sequence in the IgG‐kB promoter, has been used to inhibit p65/p50 activity (Blondeau et al., 2001). However, there is no information about decoys targeted to other NF‐kB dimers. The pharmacokinetic profiles of phosphothiorated oligonucleotides of varying base composition in rat are similar (Agrawal and Zhao, 1998). The stereotaxic injection of fluorescent IgG‐kB decoys into brain results in the cellular incorporation of decoys in hippocampi as early as 2 h after injection, with dissipation taking place by 7 h. The ability to selectively intervene in the binding of different NF‐kB‐binding consensus sequences to cognate NF‐kB protein dimers during a fairly short time period should allow for the determination of the role of NF‐kB in the regulation of specific genes at specific times after a traumatic insult to the CNS. It has been proposed that transcriptional injury responses are biphasic and that there are different consequences to delayed transcriptional activation or to persistent versus transient transcriptional factor activation of the NF‐kB transcription factor. The ability to abolish NF‐kB binding to selective promoter sites in a transient fashion should provide a useful tool in establishing the role of different trauma‐induced pathways in cellular commitment to cell death and survival. Since IgG‐kB decoy can specifically inhibit p65/p50‐binding activity, it is not surprising that it can inhibit the transcription of inflammatory cytokines, which are the target genes for NF‐kB p65/p50 (Hu et al., 2005). These results are consistent with recent reports showing antiapoptotic features to the stimulation of C‐Rel and proapoptotic features to the stimulation of p65 DNA binding (Qiu et al., 2001; Pizzi et al., 2002). These results are also consistent with the C‐Rel‐mediated regulation of Bcl‐x gene transcription in response to hypoxia ischemia (Qiu et al., 2001, 2004). Genes that were significantly downregulated by IgG‐kB decoys were not affected by Bcl‐x decoys, and vice versa.

8 Conclusion Following injury to the CNS, there is a cascade of molecular and cellular responses that determine outcome in terms of cell survival and function. One important pathway activated by CNS trauma is via induction of IL‐1, which binds to cognate receptors and activates selective gene expression of genes such as COX‐2 and iNOS via the transcription factor NF‐kB. The aim here was to describe the activation mechanisms by which IL‐1 contributes to cell death. We also discuss the effects of selective interventions in (i) IL‐1 receptor binding or (ii) injury‐induced p65/p50 NF‐kB activation. The absence of side effects of IL‐1Ra and its safety in clinical trials in rheumatoid arthritis would suggest that this intervention may be applicable to perinatal ischemia. Decoys to NF‐kB‐ binding DNA consensus sequences are useful to determine regulatory features affecting individual genes. Use of decoy treatments to alter physiological and pathological outcomes to trauma requires concerted pulsed treatments with ‘‘cocktail’’ of different decoy sequences that take into account the large number of genes displaying NF‐kB‐binding sites in their promoters.

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Aging and Oxidative Stress Response in the CNS

V. Calabrese . D. A. Butterfield . A. M. Giuffrida Stella

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

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The Free‐Radical Hypothesis of Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 The Mitochondrial Theory of Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 Mitochondrial Damage, Reactive Nitrogen Species, and Neurodegenerative Disorders . . . . . . . . 110

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Oxidative Stress and Brain Stress Tolerance: Role of Vitagenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 Neurogasobiology of Nitric Oxide and Carbon Monoxide: Two Molecules That Promote Adaptive Responses in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 3.2 Nitric Oxide Synthase and Its Isoforms in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.3 Nitric Oxide as a Neurotransmitter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.4 Redox Activities Elicited by NO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 3.5 Regulation of Gene Expression by Oxidative and Nitrosative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112 3.6 Carbon Monoxide: A Signaling Molecule Endowed with Antiinflammatory Properties . . . . . . . 113 3.7 The Heat‐Shock Pathway of Brain Stress Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 3.8 HO System: A Putative Vitagene Target for Neuroprotection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 3.9 Regulation of HO Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118 3.10 Glutathione, Thiol Redox State, and RNS: Intracellular Modulators of HO‐1 Expression . . . . . 119 3.11 Heme Oxygenase in Brain Function and Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 3.12 Bilirubin and Biliverdin: An Endogenous Antioxidant System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 4 4.1

Caloric Restriction and Endogenous Oxidative Stress: Relevance to Aging and Cell Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 Therapeutic Potential of Nutritional Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

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Major CNS Disorders Associated with ROS/RNS‐Mediated Damage . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 Multiple Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 Friedreich’s Ataxia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130 Huntington’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Down’s Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132 Ischemia/Reperfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

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HO‐1 and Hsp70 as a Therapeutic Funnel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

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Abstract: Cellular oxidant/antioxidant balance has become the subject of intense study, particularly focused on brain aging and neurodegenerative disorders. There is now evidence to suggest that reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes for both the aging processes and neurodegenerative diseases. However, to survive different types of injuries, brain cells have evolved networks of different responses, which detect and control diverse forms of stress. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so‐called longevity assurance processes, which are composed of several genes termed ‘‘vitagenes.’’ Among these, heat‐shock proteins (Hsps), proteasome, and mitochondrial uncoupling protein systems are highly conserved mechanisms responsible for the preservation and repair of the correct conformation of cellular macromolecules, such as proteins, RNA, and DNA. Recent studies have shown that the heat‐shock response contributes to establishing a cytoprotective state in a wide variety of human diseases, including ischemia and reperfusion damage, inflammation, metabolic disorders, cancer, infection, trauma, and aging. Among the various Hsps, Hsp32 also known as heme oxygenase I (HO‐1), has received considerable attention, as it has been recently demonstrated that HO‐1 induction, by generating the vasoactive molecule carbon monoxide (CO) and the potent antioxidant bilirubin, could represent a protective system potentially active against brain oxidative injury. The major neurodegenerative diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD), and Friedreich’s ataxia (FA), are all associated with the presence of abnormal proteins. Given the broad cytoprotective properties of the heat‐shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat‐shock response. These findings have opened up new perspectives in medicine and pharmacology, as molecules inducing this defense mechanism appear to be possible candidates for novel cytoprotective strategies. Particularly, modulation of endogenous cellular defense mechanisms such as the heat‐shock response, and the proteasomal system, through nutritional antioxidants or pharmacological compounds may represent an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration. Moreover, by maintaining or recovering the activity of vitagenes, it would be possible to delay the aging process and decrease the occurrence of age‐related diseases with resulting prolongation of a healthy life span. List of Abbreviations: AD, Alzheimer’s disease; AP‐1, activator protein‐1; Ab, amyloid beta‐peptide; CNS, central nervous system; GSH, reduced glutathione; GSSG, oxidized glutathione; Hsp, heat‐shock protein; JAK, janus kinase; JNK, c‐jun N‐terminal kinase; MAPK, mitogen‐activated protein kinase; NF‐kB, nuclear factor kappa‐B; NFT, intraneuronal fibrillary tangles; NOS, nitric oxide synthase; PD, Parkinson’s disease; PLA2, phospholipase A2; RNS, reactive nitrogen species; ROS, reactive oxygen species; SAPK, stress‐ activated protein kinase; STAT, signal transducer and transcription activator; TNF, tumor necrosis factor

1

Introduction

Reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes in the aging processes and neurodegenerative diseases (Mattson et al., 2002). Numerous theories have been suggested to explain the aging process (> Table 6‐1). However, current thoughts generally propose that senescence results from various extrinsic events that lead progressively to cell damage and death and/or characteristic intrinsic events related to the genome‐based theory. These general theories have been presented in various ways. It is agreed that aging is a combination of several theories, the free radicals and mitochondrial theories presumably being the most important, while others may play a definite, although less critical, role. However, it should be emphasized that no single theory is entirely satisfactory. Although several lines of evidence suggest that accumulation of oxidative molecular damage is a primary causal factor in senescence, it is increasingly evident that the mitochondrial genome may play a key role in aging and neurodegenerative diseases. Mitochondrial dysfunction is characteristic of several neurodegenerative disorders, and evidence for mitochondria being a site of damage in neurodegenerative

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. Table 6‐1 Theories of aging A. Stochastic (random event) 1. Somatic mutation 2. Error catastrophe 3. Protein glycosylation B. Developmental 1. Immune 2. Neuroendocrine C. Genome‐based 1. Intrinsic mutagenesis 2. Programmed D. Free radical and mitochondrial dysfunction

disorders is partially based on decreases in respiratory chain complex activities in Parkinson’s disease (PD), Alzheimer’s disease (AD), and Huntington’s disease (HD) (Calabrese et al., 2001a). Such defects in respiratory complex activities, possibly associated with oxidant/antioxidant balance perturbation, are thought to underlie defects in energy metabolism and induce cellular degeneration. Among these, chaperones are highly conserved proteins responsible for the preservation and repair of the correct conformation of cellular macromolecules, such as proteins, RNA, and DNA. Chaperone‐buffered silent mutations may be activated during the aging process and lead to the phenotypic exposure of previously hidden features and contribute to the onset of polygenic diseases, such as age‐related disorders, atherosclerosis, and cancer (Soti and Csermely, 2002). Recently, the involvement of the heme oxygenase (HO) pathway in antidegenerative mechanisms operating in AD has received considerable attention, as it has been demonstrated that the expression of HO is closely correlated to that of amyloid precursor protein (APP) (Dore, 2002; Perry et al., 2003). HO induction, which occurs together with the induction of other Hsps during various physiopathological conditions, by generating the vasoactive molecule carbon monoxide (CO) and the potent antioxidant bilirubin, represents a protective system potentially active against brain oxidative injury. HO‐1 gene is redox regulated and this is supported by the fact that HO‐1 gene has a heat‐shock consensus sequence as well as activator proteins (AP)‐1, ‐2, and nuclear factor kappa‐B (NF‐kB) binding sites in its promoter region. In addition, HO‐1 expression is rapidly upregulated by oxidative and nitrosative stresses, as well as by glutathione depletion. Given the broad cytoprotective properties of the heat‐shock response, there is now strong interest in discovering and developing pharmacological agents capable of inducing the heat‐shock response (Butterfield et al., 2002a). In the present chapter, we discuss the role of free radicals and mitochondria in brain aging and neurodegenerative disorders, then review the role of NO and CO gases and that of the heat‐shock system in brain stress tolerance and their relevance to mechanisms of longevity.

2

The Free‐Radical Hypothesis of Aging

As one of the most prominent current theories of aging, the free‐radical theory postulates that free radicals generated through mitochondrial metabolism can act as a causative factor of abnormal function and cell death. Various toxins in the environment can injure mitochondrial enzymes, leading to increased generation of free radicals that over the life span would eventually play a major role in aging (Knight, 2000; Fries, 2002; Anisimov et al., 2003; Sastre et al., 2003). During the last few years, cellular oxidant/antioxidant balance has become the subject of intense study, particularly by those interested in brain aging and in neurodegenerative mechanisms. Several lines of evidence suggest that accumulation of oxidative molecular damage is a causal factor in senescence. The direct evidence for this hypothesis is that overexpression of antioxidative genes for

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Cu/Zn‐superoxide dismutase (Cu/Zn‐SOD) and catalase in transgenic Drosophila melanogaster prolongs the life span, retarding the age‐associated accumulation of oxidative damage (Biesalski, 2002). Among the correlative evidence supporting the involvement of oxidative stress are the following: (1) oxidative damage to DNA and proteins increases exponentially with age, and concomitantly, the rates of mitochondrial O2
 and H2O2 generation as well as the susceptibility of tissues to experimentally induced oxidative stress are increased; (2) experimental regimens that extend life span, such as caloric restriction in mammals and reduction of metabolic rate in insects, decrease the accumulation rates of oxidative damage; and (3) mitochondria make two rather contradictory contributions to cell survival. The classically recognized function is the synthesis of ATP for energizing endergonic reactions and the other is generation of reactive oxygen species (ROS) that may compromise the long‐term survival of cells and constitute a major underlying cause of the aging process. Indeed, these two rather conflicting functions are part of the same process, namely mitochondrial respiration. More than 95% of the O2 taken up by the human body is used by mitochondrial cytochrome oxidase, which adds four electrons to oxygen to generate a molecule of water. Cytochrome oxidase normally does not release ROS into its surroundings. However, a number of investigations have indicated that brain mitochondria undergo oxidative stress damage and a decrease of cytochrome c oxidase activity during aging (Harris et al., 2003). It has been postulated that this complex may act as a bottleneck, creating a situation of ‘‘electron traffic jam’’ upstream, which would alter the redox state of oxidoreductases in the electron transfer chain and increase their autoxidizability and rate of superoxide generation. A finding that lends credibility to this hypothesis is that cytochrome c oxidase activity is directly correlated with the average life span in different species (Sharman and Bondy, 2001). Oxidative damage to key intracellular targets such as DNA or proteins is an important feature of the normal cellular aging process in the brain, and several studies have shown that oxidative damage to DNA or protein extracted from brain tissue increases with age (Kalyuzhny, 2002). Oxidative damage to DNA has been shown to be extensive and could be a major cause of the degenerative diseases related to aging such as cancer. With respect to this, it has been proposed that DNA damage is a major factor underlying neuronal degeneration in normal aging and that accelerated damage to DNA may be the basis of neurodegenerative conditions such as AD, and it has been demonstrated that DNA damage distribution in the human brain, as shown by in situ end labeling, shows area‐specific differences in aging and in AD (Mecocci et al., 1998). Levels of the oxidized nucleotide 8‐hydroxy‐deoxyguanosine (8‐OH‐dG), a biomarker of DNA damage, have also been shown to accumulate with aging. In several tissues, including brain and muscle, levels of 8‐OH‐dG in mitochondrial DNA (mtDNA) exceed that of nuclear DNA (nDNA) some 16‐fold, although as yet there have been no studies performed using absolutely pure mtDNA (Halliwell, 1999). It has been demonstrated that 8‐OH‐dG most frequently base pairs with cytosine, but also mispairs with adenine approximately 1% of the time, causing misreading of adjacent residues. Mecocci and coworkers (1997) found that 8‐OH‐dG significantly correlates with increases in levels of a 7.4‐kb deletion in the human brain. The major DNA product formed by methylating agents in vitro and in vivo is 7‐methylguanine, and it has been shown that in nDNA of normal mouse brains steady‐state levels of 7‐methylguanine increased approximately twofold between 11 and 28 months of age and that following treatment in vivo with methylnitrosourea, a fraction of DNA damage in brain tissue was refractory to repair and was lost from DNA much more slowly (Gaubatz and Tan, 1993). This repair‐resistant fraction of damage was greater in DNA from old tissues, and it was suggested that although DNA repair enzymes are present and active in senescent postmitotic tissues such as the brain, changes in the structure and function of ‘‘old’’ chromatin somehow decrease the capacity of the DNA repair enzymes present in the nucleus to repair oxidatively damaged DNA (Gaubatz and Tan, 1994). In addition, single‐strand and double‐strand breaks in DNA accumulate in the aging brain, and on exposure of neurons isolated from young and aged rats to an excitotoxic insult, more extensive DNA breaks were measured in neurons isolated from older rats (Mandavilli and Rao, 1996). During the course of normal metabolism in the brain there is production of ROS such as superoxide and hydroxyl radicals, as well as the production of reactive nitrogen species (RNS) such as nitric oxide (NO) and peroxynitrite. Therefore, the ability of DNA repair mechanisms within the nuclei of brain cells to repair damage caused by a diverse range of oxidizing species is important to maintain normal brain functions.

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There are several enzymes systems that have been found to repair damage to DNA caused by oxidizing species (Demple and Harrison, 1994). These include endonucleases, exonucleases, thymine glycol glycolases, and DNA polymerases. DNA polymerases so far detected in the mammalian brain (a, b, d, and E) undergo age‐ dependent changes in activity, but it is not known which cell types contain which polymerases, and the ability of nuclei from different brain regions to repair specific types of oxidative DNA damage is unknown. In addition, recent evidence indicates that genetic instability, such as telomere loss and somatic and mtDNA mutations, increases with age (Aviv et al., 2003). Levels of oxidative damage in mtDNA isolated from various brain regions appear to be at least tenfold higher than those of nDNA (Bohr and Dianov, 1999), although owing to technical difficulties there has as yet been no definitive study of oxidative damage to mtDNA (Beckman and Ames, 1998). This increase correlates with the 17‐fold higher evolutionary mutation rate in mtDNA compared with nDNA. These higher levels of oxidative damage and mutations in mtDNA recognize different causal factors, including location of the DNA near the inner mitochondrial membrane sites (where oxidants are generated), lack of protective histones, mitochondrial polymerase errors, and activations of genes involved in error‐prone DNA repair (Schapira, 1998). The age‐associated accumulation of oxidative damage to mtDNA correlates with the level of mtDNA deletions found in various tissues composed of postmitotic cells (Floyd and Hensley, 2002). It is possible that this damage leads to mutations that result in mitochondrial dysfunction, which has been suggested to be involved in the pathogenesis of neurodegenerative disorders (Calabrese et al., 2003a). An increase in protein oxidative damage, as indicated by the loss of protein sulfhydryl groups and by a decline in the activity of enzymes such as glutamine synthetase (GS) and glucose‐6‐phosphate dehydrogenase (G‐6‐PDH), has been demonstrated to occur in the brain during aging (Stadtman, 2001). A number of experimental evidence indicates that increased rate of free‐radical generation and decreased efficiency of the reparative/degradative mechanisms, such as proteolysis, are the two factors that primarily contribute to age‐ related elevation in the level of oxidative stress and brain damage. With respect to this, it has been suggested that decreases in levels of enzymes which ordinarily protect neuronal cells against oxidative stress with age may be responsible for increased levels of free‐radical damage in the brain, or that these enzymes themselves are susceptible to inactivation by free‐radical molecules which increase with age in the brain (Calabrese et al., 2000a). During aging a number of enzymes accumulate as catalytically inactive or less active forms. The age‐related changes in catalytic activity are due in part to reactions of proteins with oxygen and/or nitrogen free‐radical species produced during exposure to ionizing radiation or to metal ion‐catalyzed oxidation systems. The levels of oxidized proteins in brain extracts of rats of different ages increase progressively with age, and in old rats can represent 30%–50% of the total cellular protein (Calabrese et al., 2001a). The age‐related increase in oxidized protein is accompanied by a loss of GS and G‐6‐PDH activities, and by a decrease in the level of cytosolic neutral protease activity, which is responsible for the degradation of oxidized (denatured) protein. Of particular significance are the results of experiments showing that similar age‐related changes occur in the gerbil brain and that these changes are accompanied by a loss of short‐term memory. Chronic treatment of old animals with the free‐radical spin‐trap reagent N‐tert‐butyl‐a‐phenylnitrone (PBN) resulted in normalization of the several biochemical parameters to those characteristic of the young animals; coincidentally, the short‐term memory index was restored to the values seen in young animals (Carney et al., 1991). These results provide strong evidence that there is a linkage between the age‐dependent accumulation of oxidized proteins and the loss in brain physiological functions. It has recently been proposed that a primary mechanism leading to neuronal cell death in aging and common neurodegenerative disorders is interference with proteasome function (Hyun et al., 2003). Proteasomal dysfunction can involve genetic defects, direct inactivation of proteasome by reactive oxygen and nitrogen species, or overloading with proteins. The latter can be caused by excessive production of normal proteins or by the formation of poorly degradable proteins as a result of genetic mutations, faulty posttranslational modification, or protein modification by free‐radical damage. The major neurodegenerative diseases, Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Huntington’s disease (HD) and Friedreich’s ataxia (FA), are all associated with the presence of abnormal proteins (Hyun et al., 2002). The origin of HD and FA involve specific genetic defects that lead to production of abnormal proteins, whereas AD, ALS, and PD have been described mostly as sporadic,

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although familial types of AD, ALS, and PD are recognized. Examples are the rare mutations in synuclein or parkin that cause familial PD and the 2% of patients with ALS associated with mutation in the gene encoding Cu/Zn‐SOD (Halliwell, 2002). Even in the more common sporadic version of PD, ALS, and AD abnormal proteins are present to a significant extent. Thus, the senile plaques typical of AD contain not only b‐amyloid but also a wide range of other proteins. Most of them are oxidized and nitrated. Similar damage has been described for proteins in Lewy bodies in sporadic PD. Oxidative as well as nitrosative protein damage are also elevated in ALS. In nondividing cells, such as the great majority of neurons in the adult brain, the protein content of cells is approximately constant. Since protein synthesis is continuous, there must be an equilibrium between synthesis and degradation. Cellular protein can be degraded by the lysosomal system, but a system of equal or greater importance is the proteasome. The 20S proteasome (according to its sedimentation coefficient) is a cylindrical structure comprised of multiple protein subunits and containing a narrow channel where proteolysis occurs. The 20S proteasome can degrade a wide range of proteins including oxidatively damaged proteins, but most or all of the 20S proteasome in the cell is associated with a 19S ‘‘cap complex,’’ which binds in an ATP‐dependent manner and confers specificity for the degradation of polyubiquitinated proteins (Halliwell, 2001). Several other proteins are known that can associate with proteasomes and increase (or in some case decrease) rates of protein clearance. Although it is usually assumed that increased levels of oxidative damage are due to the increased generation of free‐radical species, however, increased levels of oxidative damage can equally ensue a decreased clearance of oxidatively modified biomolecules. Oxidized protein levels in the central nervous system (CNS) tend to increase with age (Friguet, 2002), consistent with several reports that proteasome activity decreases with age and in neurodegenerative disorders (Mc Naught et al., 2001). The age‐dependent accumulation of oxidized dysfunctional proteins with reactive carbonyl groups leads to inter‐and intramolecular cross‐links with protein amino groups, thus altering the efficiency of the electron transport. Imbalances in the stoichiometry of functional electron transport proteins is proposed to lead to a leakage in the flow of electrons to the terminal electron acceptor, cytochrome oxidase (Ojaimi et al., 1999), and increased likelihood of superoxide generation. Studies on isoprostanes, the end product of lipid peroxidation that can be measured in the CSF and urine in various neurodegenerative disorders, suggest that lipid peroxidation is an early stage in these disease processes. Similarly, another end product of lipid peroxidation, the aldehyde 4‐hydroxy‐trans‐nonenal (HNE), which is highly neurotoxic, avidly binds to proteins, and HNE–protein adducts are demonstrable in senile plaques and tangles in AD, tissues from ALS patients, and Lewy bodies in PD. Protein carbonyls can be generated by direct oxidative damage to proteins, by binding of cytotoxic aldehyde such as HNE to proteins, and by glycosidation of proteins (Drake et al., 2003a). The content of protein carbonyls in Alzheimer’s brain samples is greater than in age‐ matched controls (Butterfield, 2002), and this provides the clearest indication of greater accumulation of oxidized proteins in this disease. Brain regions show specific changes in this regard, and carbonyl levels correlate well with tangles (Butterfield, 2002). Importantly, the accumulation of oxidation products in particular regions of the brain seems to be related to specific cognitive defects (Forster et al., 1996). In support of this, administration of the spin‐trap PBN to gerbils retards both protein oxidation and such neurological defects (Carney et al., 1991).

2.1 The Mitochondrial Theory of Aging Harman in 1972 first proposed that mitochondria may have a central role in the process of aging (Harman, 1981). According to this theory, free radicals generated through mitochondrial metabolism can act as a causative factor of abnormal function and cell death. Mitochondria are the cell’s most significant source of oxidants and in vitro studies have indicated that approximately 1%–2% of electron flow through the electron transport chain (ETC) results in the univalent generation of superoxide (Calabrese et al., 2000a). Moreover, various toxins in the environment can injure mitochondrial enzymes, leading to increased generation of free radicals that over the life span would eventually play a major role in aging (Calabrese et al., 2004a). Ultrastructural changes have been also reported to occur in mitochondria with age. They become larger and less numerous with vacuolization, cristae rupture, and accumulation of paracrystalline

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inclusions. Cardiolipin, an acidic phospholipid that occurs only in mitochondria, has been shown to decrease with age (Paradies et al., 2002). This inner membrane lipid is known to have optimal electrical insulating properties, thereby contributing significantly to the transmembrane potential that drives the formation of ATP via ATP synthase. Indeed, a decrease in membrane potential in mitochondria from older animals has been demonstrated (Calabrese et al., 2001a). It has been proposed that accumulation of mtDNA during life is a major cause of age‐related disease, and this is because of its high mutagenic propensity. As discussed before, the lack of introns and protective histones, limited nucleotide excision and recombination DNA repair mechanisms, and location in proximity of the inner mitochondrial membrane which expose it to an enriched free‐radical milieu are all factors contributing to a tenfold higher mutation rate occurring in the mtDNA than in the nDNA. Moreover, a large body of evidence indicates that mtDNA mutations increase as a function of age, reaching the highest levels in the brain and muscle. More than 20 different types of deletions have been documented to accumulate in aging human tissues. The first report on an age‐related increase in mtDNA deletion was found in brains from elderly subjects and in PD (Calabrese et al., 2001a). This deletion has been described to occur between 13‐bp sequence repeats beginning at nucleotides 8470 and 13447, removing almost a 5‐kb region of mtDNA between the ATPase 8 and the ND5 genes. The deletion is thought to occur during replication of the mtDNA, the absent sequence encoding for six essential polypeptides of the respiratory chain and 5 tRNAs. It has been associated with several clinical diseases, such as chronic progressive external ophthalmoplegia and Kearns Sayre syndrome. Several age‐related disorders have been shown to be linked to higher levels of mtDNA mutations than age‐matched controls. In the CNS, 17 times higher levels of the common deletion in the striatum of patients with PD have been demonstrated, compared with age‐ matched controls. Evidence also exists indicating higher levels of this deletion in patients with AD, which parallel increased levels in the oxidized nucleotide 8‐OH‐dG (Bohr and Dianov, 1999). A major feature of mtDNA disease in humans is the presence of cells with low cytochrome c oxidase activity, and evidence exists that indicates that the mechanism for these changes is likely to be clonal expansion of individual mtDNA deletions within single cells (Schapira, 1998). Complex IV‐deficient cells, which occurred only sporadically earlier than the sixth decade of life, were present regularly after this age, with the loss of enzyme activity being always confined to single, randomly distributed cells. Similarly, cytochrome c oxidase‐negative neurons have been demonstrated to exist in abundance in the CNS of patients with mitochondrial disorders (Cottrell et al., 2001). These findings establish the relationship between age‐associated accumulation of mtDNA mutations and bioenergy dysfunction as a key feature of the aging process, at least in tissues predominantly composed of postmitotic cells, such as the CNS and skeletal muscle. Relevant to mitochondrial bioenergetics, in fact, is the finding of a significant decrease in state 3/state 4 ratio, which has been observed to occur in brain during aging (Calabrese et al., 2001a). Since this ratio relates to the coupling efficiency between electron flux through the ETC and ATP production, an increase in state 4 would result in a more reductive state of mitochondrial complexes and, consequently, to an increase in free‐radical species production. A decrease in state 3/state 4 respiration during aging has been found associated with a significant decrease in cardiolipin content in brain mitochondria (Cottrell and Turnbull, 2000). This loss could play a critically important role in the age‐related decrements in mitochondrial function and appears to be associated with both quantitative and qualitative region‐specific protein changes, which are parallel to structural changes, such as decrease of the inner membrane surface, smaller as well as sparser cristae, decreased fluidity, and increased fragility. Modifications in cardiolipin composition is recognized to accompany functional changes in brain mitochondria, which include all proteins of the inner mitochondrial membrane that generally require interaction with cardiolipin for optimal catalytic activity (Portero‐Otin et al., 2001; Quiles et al., 2002). Acetylcarnitine fed to old rats increased cardiolipin levels to that of young rats and also restored protein synthesis in the inner mitochondrial membrane, as well as cellular oxidant/antioxidant balance (Paradies et al., 1999), suggesting that administration of this compound may improve cellular bioenergetics in aged rats (Hagen et al., 1998a, b). Interestingly, caloric restriction, a dietary regimen that extends life span in rodents, maintains the levels of 18:2 acyl side chains and inhibits the cardiolipin composition changes (Selman et al., 2003). In addition, caloric restriction was shown to retard the aging‐associated changes in oxidative damage, mitochondrial oxidant generation, and antioxidant defenses (Mattson, 2003).

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2.2 Mitochondrial Damage, Reactive Nitrogen Species, and Neurodegenerative Disorders Increasing evidence sustains the hypothesis that mitochondrial energy metabolism underlies the pathogenesis of neurodegenerative diseases (Papa and Skulachev, 1997; Genova et al., 2004). Decreased complex I activity is reported in the substantia nigra of postmortem samples obtained from patients with PD (Beal, 1998). Similarly, impaired complex IV activity has been demonstrated in AD (Heales et al., 1999). Increased free‐radical‐induced oxidative stress has been associated with the development of such disorders and a large body of evidence suggests that NO
plays a central role (Stamler and Hausladen, 1998). Cytokines (INF‐g) that are present in the normal brain are elevated in numerous pathological states, including PD (Mayer, 2003), AD (Moore et al., 2003), multiple sclerosis (MS) (Calabrese et al., 1994, 1998, 2002a, 2003b; Bagasra et al., 1995), ischemia, encephalitis, and viral infections of the CNS (Calabrese et al., 2001a). Accordingly, as cytokines promote the induction of nitric oxide synthase (NOS) in the brain, a possible role for a glial‐ derived NO
in the pathogenesis of these diseases has been suggested (Stamler and Hausladen, 1998). Excessive formation of NO
from glial origin has been evidenced in some study in which NADPH diaphorase (a cytochemical marker of NOS activity)‐positive glial cells have been identified in the substantia nigra of postmortem brains obtained from individuals with PD (Hyun et al., 2003). Loss of nigral GSH is considered an early and crucial event in the pathogenesis of PD (Beal, 1998, 2003) and as a consequence decreased peroxynitrite scavenging may also occur. Therefore, such perturbations in thiol homeostasis may constitute the starting point for a vicious cycle leading to excessive ONOO generation in PD. In support of this it has been reported that the selective inhibition of neuronal NOS (nNOS) prevents 1‐methyl‐4‐phenyl‐ 1,2,3,6‐tetrahydropyridine (MPTP)‐induced parkinsonism in experimental animals (Dawson and Dawson, 2002; Moore et al., 2003).

3

Oxidative Stress and Brain Stress Tolerance: Role of Vitagenes

There is now evidence to suggest that reduction of cellular expression and activity of antioxidant proteins and the resulting increase of oxidative stress are fundamental causes for both aging process and neurodegenerative diseases (Ferri et al., 2003; Genova et al., 2003). However, to survive different types of injuries, brain cells have evolved networks of different responses, which detect and control diverse forms of stress. Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so‐called longevity assurance processes, which are composed of several genes termed vitagenes. Signaling mechanisms adopted by regulatory proteins to control gene expression in response to alterations in the intracellular redox status are very common in prokaryotes. Gene activation by oxidative stress was first described in bacteria where regulatory proteins such as OxyR were discovered as an activator of antioxidant‐and stress‐responsive genes. As the cytoprotective mechanism triggered by SoxR in E. coli includes the expression of critical antioxidant defensive proteins, such as superoxide dismutase, the emerging concept is that an analogous system might operate in mammalian cells (Calabrese et al., 2001a, 2003a, 2004a). In eukaryotes, typical examples are the HO and Hsp genes, thioredoxin, detoxificant enzymes (Mn‐SOD, glutathione‐S‐transferase, NADPH/quinone reductase), cytokines, immunoreceptors and growth factors (Calabrese et al., 2003c; Colombrita et al., 2003).

3.1 Neurogasobiology of Nitric Oxide and Carbon Monoxide: Two Molecules That Promote Adaptive Responses in the CNS CO is the second gas discovered in the last 25 years to have salutary effects, the first being NO. Certain findings raise the conceivable possibility that HO‐1 and/or CO and NOS2 and/or NO are functionally interrelated in mediating their protective effects. In some situations, CO can activate the expression of

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NOS2 and, in others, inhibits the expression of NOS2 and consequently of NO (Motterlini et al., 2002a). NO upregulates HO‐1 with production of CO (Maines, 1997). We have recently found evidence for a functional relationship between CO and NO. In endotoxic shock, the salutary action of CO in the rat brain appears to depend sequentially on the activation of NF‐kB, which triggers transcription of NOS2 with production of NO, and subsequently in the upregulation of HO‐1. In the absence of any of these steps, the beneficial effect of CO is lost (Scapagnini et al., 2002a). This has been also demonstrated in mice treated for hepatitis induced by TNF‐a and D‐galactosamine (Otterbein et al., 2003a). To what extent CO and NO act interdependently in other physiopathological conditions that are responsive to CO and/or NO is unknown.

3.2 Nitric Oxide Synthase and Its Isoforms in the CNS The enzyme responsible for NO synthesis is the NOS family of enzymes, which catalyze the conversion of arginine to citrulline and NO
. NOS, localized in the CNS and in the periphery (Calabrese et al., 2000a), is present in three well‐characterized isoforms: (1) neuronal NOS (nNOS; type I), (2) endothelial NOS (eNOS; type III), and (3) inducible NOS (iNOS; type II). Activation of different isoforms of NOS requires various factors and cofactors. In addition to a supply of arginine and oxygen, an increase in intracellular calcium leads to activation of eNOS and nNOS, and formation of calcium/calmodulin complexes is a prerequisite before the functionally active dimer exhibits NOS activity, which depends also on cofactors such as tetrahydrobiopterin (BH4), FAD, FMN, and NADPH (Dawson and Dawson, 1995). nNOS has a predominant cytosolic localization whereas the eNOS is bound to the plasma membrane by N‐terminal myristaglation (Calabrese et al., 2000a). In contrast to nNOS and eNOS, iNOS can bind to calmodulin even at very low concentrations of intracellular calcium; thus, iNOS can exert its activity in a calcium‐independent manner. iNOS, usually present only in the cytosol, also requires NADPH, FAD, FMN, and BH4 for full activity. eNOS, expressed in cerebral endothelial cells, critically regulates cerebral blood flow. However, a small population of neurons in the pyramidal cells of CA1, CA2, and CA3 subfields of the hippocampus and granule cells of the dentate gyrus express eNOS. nNOS, which is expressed in neurons, is critically involved in synaptic plasticity, neuronal signaling, and neurotoxicity. Activation of nNOS forms part of the cascade pathway triggered by glutamate receptor activation that leads to intracellular cGMP elevation. The levels of iNOS in the CNS are generally fairly low. However, an increased expression of iNOS in astrocytes and microglia occurs following viral infection and trauma (Bredt, 1999). Activation of iNOS requires gene transcription, and the induction can be influenced by endotoxin and cytokines (Calabrese et al., 2000a) (IL‐1, IL‐2, lipopolysaccharide, IFN‐g, TNF). This activation can be blocked by antiinflammatory drugs (dexamethasone), inhibitory cytokines (IL‐4, IL‐10), prostaglandins (PGA2), and tissue growth factors or inhibitors of protein synthesis, e.g., cycloheximide.

3.3 Nitric Oxide as a Neurotransmitter The discovery of the role of NO as a messenger molecule has revolutionized the concept of neuronal communication in the CNS. NO is a gas freely permeable to the plasma membrane, thus, NO does not need a biological receptor to influence the intracellular communication or signaling transduction mechanisms (Stamler and Hausladen, 1998). Once generated, the cell cannot regulate the local concentration of NO, therefore the other way to influence NO activity is to control its synthesis. The activity of NO also terminates when it chemically reacts with a target substrate. NO when produced in small quantities can regulate cerebral blood flow, local brain metabolism (Calabrese et al., 2001a), and neurotransmitter release and gene expression, and play a key role in morphogenesis and synaptic plasticity. It is also generally accepted that NO is a major component in signaling transduction pathways controlling smooth muscle tone, platelet aggregation, host response to infection, and a wide array of other physiological and pathophysiological processes. Under conditions of excessive formation, NO is emerging as an important mediator of neurotoxicity in a variety of disorders of the nervous system (Heales et al., 1999).

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3.4 Redox Activities Elicited by NO In the last several years, a number of studies have shown a protective effect of NO in a variety of paradigms of cell injury and cell death. These include (1) direct scavenging of free radicals, such as superoxide with effects on intracellular iron metabolism, including interaction with iron to prevent, through formation of nitrosyl‐iron complexes, release of iron from ferritin (Sergent et al., 1977); (2) interaction of NO
(through its congener NOþ) with thiol group on the NMDA receptor with consequent downregulation and inhibition of calcium influx (Ignarro, 2002); (3) inactivation of caspases (Ignarro, 2002); (4) activation of a cGMP‐dependent survival pathway, as demonstrated in PC12 cells (Motterlini et al., 2002a); (5) inducing expression of cytoprotective proteins, such as heat‐shock proteins (Hsps) (Motterlini et al., 2000a); and (6) inhibition of NF‐kB activation or GADPH, whose activity appears to be required in one paradigm of neuronal apoptosis (Piantadosi et al., 1997). In general, the current opinion holds that the intracellular redox state is the critical factor determining whether in brain cells NO is toxic or protective (Rosenberg et al., 1999). In addition, it has been proposed that NO might inhibit T‐cell activation and cell trafficking across the blood–brain barrier, and hence limiting the setting of the autoimmune cascade associated with degenerative damage (Dawson and Dawson, 1995). The difficulty in delineating a mechanistic involvement of NO as proinflammatory or antiinflammatory agent and the controversy arising on whether excessive NO elicits cytoprotective or cytotoxic actions are better appreciated by recognizing the complexity of NO chemistry when applied to biological systems (Motterlini et al., 2002a). As minutely detailed by Stamler and colleagues, the reactivity of the NO groups is dictated by the oxidation state of the nitrogen atom, which enables the molecule to exist in different redox‐activated forms (Stamler and Hausladen, 1998). In contrast to NO, which contains one unpaired electron in the outer orbital, nitrosonium cation (NOþ) and nitroxyl anion (NO) are charged molecules being, respectively, the one‐electron oxidation and reduction products of NO. Whereas NOþ can be transferred reversibly between cysteine residues (transnitrosation), NO can be formed by hemoglobin, nNOS, and S‐nitrosothiols (RSNO). A fundamental aspect of NO biochemistry is the attachment of NO groups to sulfhydryl centers to form S‐nitrosyl derivatives or RSNO (Ignarro, 2002). This chemical process, known as S‐nitrosation, has been suggested to represent a refined endogenous tool to stabilize and preserve NO biological activity (Rosenberg et al., 1999; Motterlini et al., 2002a). It has been speculated that low‐molecular weight RSNO, such as S‐nitrosoglutathione or nitrosocysteine, may also represent a mechanism for storage of NO in vivo (Stamler et al., 1992; Rosenberg et al., 1999). In this regard, glutathione becomes an important determinant of the reactivity and fate of NO because this cysteine‐containing tripeptide is very abundant in most tissues and biological fluids. In addition, S‐nitrosation is also an important process in modulating the activity and function of several enzymes and proteins. However, deleterious and oxidative modification in protein structure and function may occur when RNS reach a critical threshold, and hence nitrosative stress may ensue (Hausladen et al., 1996). At the cellular level, nitrosative stress has been linked to inhibition of cell growth and apoptosis, and implicated in NO pathogenesis (Sergent et al., 1977). The intriguing aspect in the parallelism between the effects mediated by increased RNS and ROS is the ability of cells to respond to these two types of stress and, depending on the severity of the nitrosative/oxidative insult, this response may result in both adaptation and resistance to toxicity (Calabrese et al., 2002b).

3.5 Regulation of Gene Expression by Oxidative and Nitrosative Stress Signaling mechanisms adopted by regulatory proteins to control gene expression in response to alterations in the intracellular redox status are very common in prokaryotes. Gene activation by oxidative stress was first described in bacteria where regulatory proteins such as OxyR was discovered as an activator of antioxidant‐ and stress‐responsive genes. OxyR is a homotetramer that is activated by hydrogen peroxide and S‐nitrosothiols. The protein contains six cysteine residues, one of each is absolutely necessary for activity and two are required for maximal activation. Recent studies suggested that oxidation of a single thiol to a sulfenic acid may represent a sensor mechanism, whereas the activation mechanism can be ascribed to formation of an intramolecular disulfide, or alternatively to S‐nitrosylation of a single cysteine residue, with

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Cys 199 being a likely candidate site of posttranslational modification (Rosenberg et al., 1999; Motterlini et al., 2002a). The expression of these protective genes renders bacteria more resistant to oxidant damage (Motterlini et al., 2002b). As the cytoprotective mechanism triggered by SoxR in E. coli includes the expression of critical antioxidant defensive proteins, such as superoxide dismutase (Motterlini et al., 2003), the emerging concept is that analogous systems might operate in mammalian cells. In eukaryotes, typical examples are genes such as the HO gene, thioredoxin and detoxificant enzymes (Mn‐SOD, glutathione‐S‐transferase, NADPH/quinone reductase), cytokines, immunoreceptors, and growth factors. That the antioxidant protein HO could ‘‘sense’’ NO, and thus protecting against ROS and RNS insults, is supported by the following findings: (1) NO and NO‐related species induce HO‐1 expression and increase HO activity in human glioblastoma cells, hepatocytes, and aortic vascular cells; (2) cells pretreated with various NO‐releasing molecules acquire increased resistance to H2O2‐mediated cytotoxicity when HO is maximally activated; and (3) bilirubin, one of the end products of heme degradation by HO, protects against the cytotoxic effects caused by strong oxidants such as H2O2 and ONOO (Rosenberg et al., 1999; Motterlini et al., 2002a). The conception that NO and RNS can be directly involved in the modulation of HO‐1 expression in eukaryotes is based on the evidence that different NO‐releasing agents can markedly increase HO‐1 mRNA and protein, as well as HO activity, in a variety of tissues, including brain cells (Scapagnini et al., 2002a). In rat glial cells, treatment with lipopolysaccharide (LPS) and interferon‐g (IFN‐g) results in a rapid increase in both iNOS expression and nitrite levels followed by enhancement of HO‐1 protein (Calabrese et al., 2000b). In the same study, the presence of NOS inhibitors suppressed both nitrite accumulation and HO‐1 mRNA expression. Modulation of HO‐1 mRNA expression by iNOS‐ derived NO following stimulation with LPS has also been reported in different brain regions, particularly in the hippocampus and substantia nigra in an in vivo rat model of septic shock (Scapagnini et al., 2002a). Moreover, the early increase in iNOS protein levels observed in endothelial cells exposed to low oxygen tension seems to precede the stimulation of HO‐1 expression and activity, an effect that appears to be finely regulated by redox reactions involving glutathione (Motterlini et al., 2000a, 2002a). Taken together, these findings point to the central role of NO as a signaling molecule which, by triggering expression of cytoprotective genes such as HO‐1, may lead to adaptation and resistance of brain cells to subsequent, eventually more severe, nitrosative and oxidative stress insults (Butterfield et al., 2002b). Thus, a direct interaction of NO groups with selective chemical sites localized in transcription proteins that can be activated through nitrosative reactions could effectively contribute to the enhancement of both HO‐1 gene expression and stress tolerance. Recent knowledge concerning the modulation by thiol redox state of the activity of several transcription factors that recognize specific binding sites within the promoter and distal enhancer regions of the HO‐1 gene include Fos/Jun (AP‐1), NF‐kB, and the more recently identified Nrf2 proteins (Balogun et al., 2003; Poon et al., 2004a). Importantly, both AP‐1 and NF‐kB contain cysteine residues whose interaction with oxidant or nitrosant species might be crucial for determining the DNA‐ binding activity (Rosenberg et al., 1999; Motterlini et al., 2002a). Data in the literature show that NO can either activate or inhibit these transcription factors, and that in many circumstances activation depends on the reversibility of the posttranslational modification elicited by the various RNS (Butterfield et al., 2002a; Poon et al., 2004b). We have demonstrated in astroglial cell cultures that cytokine‐induced nitrosative stress is associated with an increased synthesis of Hsp70 stress proteins, which was also found after treatment of cells with the NO‐generating compound sodium nitroprusside (SNP), thus suggesting a role for NO in inducing Hsp70 protein expression (Calabrese et al., 2000b). In vivo experiments performed in our laboratory have also demonstrated that the redox glutathione status is a critical factor for induction of cytoprotective Hsp70 (Calabrese et al., 2000c, 2002c, 2004a; Balogun et al., 2003).

3.6 Carbon Monoxide: A Signaling Molecule Endowed with Antiinflammatory Properties Grehan first detected a combustible gas in the blood in 1894 (Grehan, 1894). This gas was supposed by de Saint Martin and Nicloux to be CO. However, it was not until 1949 that Siorstrand discovered that

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endogenously produced CO arose from the degradation of hemoglobin released from senescing erythrocytes (Siorstrand, 1949). Greater than 75% of CO produced in humans arises from erythrocyte turnover generated as a by‐product of heme metabolism. In 1969, the source of endogenous CO was discovered, as Tenhunen and collaborators (1969) described and characterized HO as the enzyme responsible for breaking down heme in the body, demonstrating that heme catalysis resulted in the subsequent release of CO and free iron as by‐products (Tenhunen et al., 1969). Since then, supported by a large body of experimental evidence, CO is proving to be an extraordinary signaling molecule generated by the cell, which is vital in the regulation of cellular homeostasis. In the brain, CO is emerging as a chemical messenger molecule which can influence physiological and pathological processes in the central and peripheral nervous systems. This gaseous molecule is now considered a putative neurotransmitter, owing to its capability to diffuse freely from one cell to another, thereby influencing intracellular signal transduction mechanisms. However, unlike conventional neurotransmitters, CO is not stored in synaptic vesicles and is not released by membrane depolarization and exocytosis. It seems likely that CO is involved in the mechanism of cell injury (Turcanu et al., 1998). This is evidenced by the fact that CO binds to iron of heme of the enzyme guanylyl cyclase to activate cGMP (Piantadosi et al., 1997). Indeed, it has been found that CO is responsible for maintaining endogenous levels of cGMP. This effect is blocked by potent HO inhibitors but not by NO inhibitors (Maines, 1997). On the basis of endogenous distribution of HO in the CNS, it has been suggested that CO can influence neurotransmission like NO (Verma et al., 1993). CO appears to be involved as a retrograde messenger in LTP and also in mediating glutamate action at metabotropic receptors (Graser et al., 1990). This is evident from the fact that metabotropic receptor activation in the brain regulates the conductance of specific ions channels via a cGMP‐dependent mechanism, which is blocked by HO inhibitors (Glaum and Miller, 1993). Experimental evidence suggests that CO plays a similar role like NO in the signal transduction mechanism in regulating cell function and cell‐to‐cell communication (Maines, 1997). HO resembles NOS in that the electrons for CO synthesis are donated by cytochrome P450 reductase, which is 60% homologous at the amino acid level to the half carboxyterminal of NOS (Calabrese et al., 2001a). CO like NO binds to iron in the heme moiety of guanylyl cyclase. However, there are some differences in function between CO and NO. Thus, NO mainly mediates glutamate effect at NMDA receptors while CO is primarily responsible for glutamate action at metabotropic receptors. Taken together, it appears that CO and NO play an important role in the regulation of CNS function, therefore impairment of CO and NO metabolism results in abnormal brain function (Calabrese et al., 2000a). A number of evidence suggest a possible role of CO in regulating nitrergic transmission. Endogenous CO has been suggested to control constitutive NOS activity. Moreover, CO may interfere with NO binding to guanylyl cyclase, and this in addition to the important role of HO in regulating NO generation, owing to its function in the control of heme intracellular levels, as part of the normal protein turnover (Calabrese et al., 2003a). This hypothesis is sustained by recent findings showing that HO inhibition increases NO production in mouse macrophages exposed to endotoxin (Turcanu et al., 1998). CO may also act as a signaling effector molecule, by interacting with targets other than guanylate cyclase. Notably, it has been recently demonstrated that K(Ca) channels are activated by CO in a cGMP‐independent manner (Wang and Wu, 2003) and also that CO‐induced vascular relaxation results from the inhibition of the synthesis of the vasoconstrictor endothelin‐1 (Coceani et al., 1997). Little, however, is known about how CO is sensed on a biological ground. Interestingly, the photosynthetic bacterium Rhodospirillum rubrum has the ability to respond to CO through the heme protein CooA that, upon exposure to CO, acquires DNA‐binding transcriptional activity for the CO dehydrogenase gene, thereby encoding for CO dehydrogenase, which is the key enzyme involved in the oxidative conversion of CO to CO2. Remarkably, heart cytochrome c oxidase possesses CO‐oxygenase activity, thus metabolizing CO to CO2 (Calabrese et al., 2001a). Whether this occurs also in brain mitochondria remains to be elucidated. Aside from the CNS, the protective effects of CO were initially demonstrated in a model of acute lung injury and endotoxic shock, and subsequently in a mouse cardiac xenotransplantation model (Otterbein et al., 2003a). Mouse heart transplanted to immunosuppressed rats survive indefinitely. However, if HO‐1 activity cannot be expressed in the mouse heart, either as a consequence of absent phenotypical expression of the HO‐1 gene (mice hmox/) or as a consequence of HO‐1 activity being inhibited with a selective inhibitor Tin protoporphyrin (SnPPIX), the hearts are rejected rapidly. HO‐1 expression in the transplanted heart is essential to prevent rejection in this

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model. Surprisingly, if the donor and recipient were both treated with 250 ppm CO, even a heart that cannot express HO‐1 activity still survives indefinitely (Otterbein, 2002). In this scenario CO appears to be able to substitute for HO‐1 in suppressing the proinflammatory response, which is the leading cause of graft rejection. CO emerges as a powerful antiinflammatory promoting agent acting at the level of the macrophage cell line, a cell that probably controls the balance of inflammation in many conditions. Macrophages stimulated with bacterial LPS produce proinflammatory cytokines such as TNFa and an inflammatory cytokine interleukin‐10 (IL‐10) is also produced (Otterbein et al., 2003b). If macrophages overexpress HO‐1 or are exposed to CO in vitro before stimulation with LPS, the proinflammatory response, and consequently TNFa, is markedly diminished, whereas the antiinflammatory response, characterized by IL‐10 production, is enhanced. At least, three important actions of CO contribute to its antiinflammatory effects: (1) CO prevents platelet aggregation and the consequent thrombosis (Otterbein et al., 2003a); (2) CO downmodulates the expression of plasminogen activator inhibitor type 1 (PAI‐1); and (3) CO prevents apoptosis in several cell types, including endothelial cells, fibroblasts, hepatocytes, and pancreatic b‐cells (Otterbein et al., 2003a). In addition, CO suppresses the proliferative response of smooth muscle cells, which contribute to neointimal proliferation associated with inflammatory lesions in vivo. Many of the observed effects of CO have been obtained by exposing cells or animals to gaseous CO and its subsequent inhalation. Interestingly, the recently discovered CO‐releasing molecules (CORMS) appear to afford similar protective action, thereby providing an alternative therapeutic approach for those pathophysiological conditions where CO administration is warranted (Motterlini et al., 2002b, 2003).

3.7 The Heat‐Shock Pathway of Brain Stress Tolerance It is well known that living cells are continually challenged by conditions that cause acute or chronic stress. To adapt to environmental changes and survive different types of injuries, eukaryotic cells have evolved networks of different responses, which detect and control diverse forms of stress. One of these responses, known as the heat‐shock response, has attracted a great deal of attention as a universal fundamental mechanism necessary for cell survival under a wide variety of toxic conditions. In mammalian cells, Hsp synthesis is induced not only after hyperthermia but also following alterations in the intracellular redox environment and exposure to heavy metals, amino acid analogs, or cytotoxic drugs. While prolonged exposure to conditions of extreme stress is harmful and can lead to cell death, induction of Hsp synthesis can result in stress tolerance and cytoprotection against stress‐induced molecular damage. Furthermore, transient exposure to elevated temperatures has a cross‐protective effect against sustained, normally lethal exposures to other pathogenic stimuli. Hence, the heat‐shock response contributes to establish a cytoprotective state in a variety of metabolic disturbances and injuries, including stroke, epilepsy, cell and tissue trauma, neurodegenerative disease, and aging (Calabrese et al., 2001a; Mattson et al., 2002). This has opened new perspectives in medicine and pharmacology, as molecules activating this defense mechanism appear as possible candidates for novel cytoprotective strategies (Scapagnini et al., 2002b; Soti and Csermely, 2002; Colombrita et al., 2003). In mammalian cells, the induction of the heat‐shock response requires the activation and translocation to the nucleus of one or more heat‐shock transcription factors, which control the expression of a specific set of genes encoding cytoprotective Hsps. Some of the known Hsps include ubiquitin, Hsp10, Hsp27, Hsp32 (or HO‐1), Hsp47, Hsp60, Hsc70, Hsp70 (or Hsp72), Hsp90, and Hsp100/105. Most of the proteins are named according to their molecular weight. HSP70. The 70‐kDa family of stress proteins is one of the most extensively studied. Included in this family are Hsc70 (heat‐shock cognate, the constitutive form), Hsp70 (the inducible form, also referred to as Hsp72), and GRP75 (a constitutively expressed glucose‐regulated protein found in the endoplasmic reticulum). After a variety of CNS insults, Hsp70 is synthesized at high levels and is present in the cytosol, nucleus, and endoplasmic reticulum. Denatured proteins are thought to serve as stimuli for induction. These denatured proteins activate heat‐shock factors (HSFs) within the cytosol by dissociating other Hsps that are normally bound to HSF (Balogun et al., 2003; Calabrese et al., 2003a; Poon et al., 2004a, b). Freed HSF is phosphorylated and forms trimers, which enter the nucleus and bind to heat‐shock elements (HSEs) within the promoters of different heat‐shock genes, leading to transcription and synthesis of Hsps.

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After heat shock, for instance the synthesis of Hsp70 increases to a point where it becomes the most abundant single protein in a cell. Once synthesized, Hsp70 binds to denatured proteins in an ATP‐ dependent manner. The N‐terminal end contains an ATP‐binding domain, whereas the C‐terminal region contains a substrate‐binding domain. Hsps serve as chaperones that bind to other proteins and regulate their conformation, regulate protein movement across membranes or through organelles, or regulate the availability of a receptor or activity of an enzyme. In the nervous system, Hsps are induced in a variety of pathological conditions, including cerebral ischemia, neurodegenerative disorders, epilepsy, and trauma. Expression of the gene encoding Hsps has been found in various cell populations within the nervous system, including neurons, glia, and endothelial cells (Kelly et al., 2002). Hsps consist of both stress‐inducible and constitutive family members. Whether stress proteins are neuroprotective has been the subject of much debate, as it has been speculated that these proteins might be merely an epiphenomenon unrelated to cell survival. Only recently, however, with the availability of transgenic animals and gene transfer, it has become possible to overexpress the gene encoding Hsp70 to test directly the hypothesis that stress proteins protect cells from injury, and it has been demonstrated that overproduction of Hsp70 leads to protection in several different models of nervous system injury (Wang and Wu, 2003). Following focal cerebral ischemia, mRNA encoding Hsp70 is synthesized in most ischemic cells except in areas of very low blood flow, because of limited ATP levels. Hsp70 proteins are produced mainly in endothelial cells, in the core of infarcts in cells that are most resistant to ischemia, in glial cells at the edges of infarcts, and in neurons outside the areas of infarction. It has been suggested that this neuronal expression of Hsp70 outside an infarct can be used to define the ischemic penumbras, which is the zone of protein denaturation in the ischemic areas (Balogun et al., 2003). A number of in vitro studies show that both heat shock and Hsp overproduction protect CNS cells against both necrosis and apoptosis. Mild heat shock protects neurons against glutamate‐mediated toxicity and protects astrocytes against injury produced by lethal acidosis (Narasimhan et al., 1996). Transfection of cultured astrocytes with Hsp70 protects them from ischemia or glucose deprivation (Fink et al., 1997). Hsp70 has been demonstrated to inhibit caspase‐3 activation caused by ceramide, and also affects JUN kinase and p38‐kinase activation (Mosser et al., 1997). In addition, Hsp70 binds to and modulates the function of BAG‐1, the bcl‐2 binding protein (McLaughlin et al., 2003), thus modulating some type of apoptosis‐related cell death. A large body of evidence now suggests a correlation between mechanisms of oxidative and/or nitrosative stress and Hsp induction. Current opinion holds also the possibility that the heat‐shock response can exert its protective effects through inhibition of NF‐kB‐signaling pathway (Calabrese et al., 2001a). We have demonstrated in astroglial cell cultures that cytokine‐induced nitrosative stress is associated with an increased synthesis of Hsp70. Increase in Hsp70 protein expression was also found after treatment of cells with the NO‐generating compound SNP, thus suggesting a role for NO in inducing Hsp70 proteins. The molecular mechanisms regulating the NO‐induced activation of the heat‐shock signal seems to involve cellular oxidant/antioxidant balance, mainly represented by the glutathione status and the antioxidant enzymes (Calabrese et al., 2000a, 2003a; Motterlini et al., 2000a). Ubiquitin. Ubiquitin is one of the smallest Hsps and is expressed throughout brain in response to ischemia. It is involved in targeting and chaperoning of proteins degraded in proteasomes, which include NF‐kB, cyclins, HSFs, hypoxia‐inducible factor, some apoptosis‐related proteins, tumor necrosis factor, and erythropoietin receptors (Mayer, 2003). Hsp27. Hsp 27 is synthesized mainly in astrocytes in response to ischemic situations or to kainic acid administration. It chaperones cytoskeletal proteins such as intermediate filaments, actin, or glial fibrillary acidic protein following stress in astrocytes. It also protects against Fas‐Apo‐1, staurosporine, TNF, and etoposide‐induced apoptotic cell death as well as H2O2‐induced necrosis (Bechtold and Brown, 2003). Hsp47. Hsp47 is synthesized mainly in microglia following cerebral ischemia and subarachnoid hemorrhage (Valentim et al., 2003). Hsp60, glucose‐regulated protein 75 (GRP75), and Hsp10. Hsp60, glucose‐regulated protein 75 (GRP75), and Hsp10 chaperone proteins within mitochondria. GRP75 and GRP78, also called oxygen‐regulated proteins (ORPs), are produced by low levels of oxygen and glucose. These protect brain cells against ischemia and seizures in vivo, after viral‐induced overexpression (Turner et al., 1999). Hsp60 is encoded in

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the nucleus and resides mainly in the mitochondria (Calabrese et al., 2003c, 2004a, 2005). Hsp60 forms the chaperonin complex, which is implicated in protein folding and assembly within the mitochondria under normal conditions (Izaki et al., 2001). Most mitochondrial proteins are synthesized in the cytosol and must be imported into the organelles in an unfolded state (Izaki et al., 2001). During translocation, the proteins interact with Hsp70. ATP‐dependent binding and release of Hsp70 provide the major driving force for complete transport of polypeptides into the matrix. Most imported polypeptides are released from soluble Hsp70; however, a subset of aggregation‐sensitive polypeptides must be transferred from Hsp70 to Hsp60 for folding (Okubo et al., 2000). Owing to the close functional interaction between this chaperonin system and the Hsp70 system, it is likely that upregulation of Hsp60 may be a fundamental mechanism targeted by nutritional interventions leading to restoration of mitochondrial respiratory complex function compromised by oxidative stress (Calabrese et al., 2004a, 2005; Perluigi et al., 2005; Poon et al., 2005; Sultana et al., 2005). Hsp32. Hsp32 or HO is the rate‐limiting enzyme in the production of bilirubin. There are three isoforms of HO, HO‐1 or inducible isoform, HO‐2 or constitutive isoform, and the recently discovered HO‐3 (Scapagnini et al., 2002c).

3.8 HO System: A Putative Vitagene Target for Neuroprotection In the last decade the HO system has been strongly highlighted for its potential significance in maintaining cellular homeostasis. It is found in the endoplasmic reticulum in a complex with NADPH cytochrome c P450 reductase. It catalyzes the degradation of heme in a multistep, energy‐requiring system. The reaction catalyzed by HO is the a‐specific oxidative cleavage of the heme molecule to form equimolar amounts of biliverdin and CO. Iron is reduced to its ferrous state through the action of NADPH cytochrome c P450 reductase. CO is released by elimination of the a‐methene bridge of the porphyrin ring. Further degradation of biliverdin to bilirubin occurs through the action of biliverdin reductase. Biliverdin complexes with iron until its final release (Calabrese et al., 2003a). HO is present in various tissues with the highest activity in the brain, liver, spleen, and testes. There are three isoforms of HO, HO‐1 or inducible isoform (Calabrese et al., 2004b), HO‐2 or constitutive isoform (Ewing and Maines, 1992; Hon et al., 2000), and the recently discovered HO‐3, cloned only in rat to date (Scapagnini et al., 2002c). They are all products of different genes and, unlike HO‐3, which is a poor heme degrading catalyst, both HO‐1 and HO‐2 catalyze the same reaction (i.e., degradation of heme) but differ in many respects and are regulated under separate mechanisms. The similarity between HO‐1 and HO‐2 consists of a common 24 amino acid domain (differing in just one residue) called the ‘‘HO signature,’’ which renders both proteins extremely active in their ability to catabolize heme (Calabrese et al., 2004b). They have different localization, similar substrate and cofactor requirements, while presenting different molecular weights. They also display different antigenicity, electrophoretic mobility, inducibility, as well as susceptibility to degradation. The proteins for HO‐1 and HO‐2 are immunologically distinct and, in humans, the two genes are located on different chromosome arms i.e., 22q12 for HO‐1 and 16q13.3 for HO‐2 (Ewing and Maines, 1992). Various tissues have different amounts of HO‐1 and HO‐2. Brain and testes have a predominance of HO‐2, whereas HO‐1 predominates in the spleen. In the lung not subjected to oxidative stress more than 70% of HO activity is accounted for by HO‐2, whereas in the testes the pattern of HO isoenzyme expression differs according to the cell type, although HO‐1 expression predominates after heat shock. This also occurs in the brain tissue, where HO isoforms appear to be distributed in a cell‐specific manner, and HO‐1 distribution is widely apparent after heat shock or oxidative stress. Although previous reports from us and other groups have not found detectable levels of HO‐1 protein in the normal brain (Ewing and Maines, 1992), we have recently demonstrated that HO‐1 mRNA expression is physiologically detectable in the brain and shows a characteristic regional distribution, with high level of expression in the hippocampus and cerebellum (Scapagnini et al., 2002c). This evidence may suggest the possible existence of a cellular reserve of HO‐1 transcripts quickly available for protein synthesis and a posttranscriptional regulation of its expression.

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HO isoenzymes are also seen to colocalize with different enzymes depending on the cell type. In the kidney, HO‐1 colocalizes with erythropoietin, whereas in smooth muscle cells HO‐1 colocalizes with NOS. In neurons, HO‐2 colocalizes with NOS, whereas the endothelium exhibits the same isoform that colocalizes with NOS III. The cellular specificity of this pattern of colocalization lends further support to the concept that CO may serve a function similar to that of NO. Furthermore, the brain expression pattern shown by HO‐2 protein and HO‐1 mRNA overlaps with the distribution of guanylate cyclase, the main CO functional target (Coceani et al., 1997). HO‐3, the third isoform of HO, shares a high homology with HO‐2, both at the nucleotide (88%) and protein (81%) levels. Both HO‐2 and HO‐3, but not HO‐1, are endowed with two Cys‐Pro residues considered the core of the heme‐responsive motif (HRM), a domain critical for heme binding but not for its catalysis (Hon et al., 2000). Although the biological properties of this isoenzyme still remains to be elucidated, the presence of two HRM motifs in its amino acidic sequence might suggest a role in cellular heme regulation (Maines, 2000). Studying the HO‐3 mRNA sequence (GenBank accession no.: AF058787), we have observed that its 50 ‐ portion corresponds to an L‐1 retrotransposon sequence, a member of a family of retrotransposons recently found to be involved in evolutionary mechanisms (Kazazian, 2000). On the basis of the close similarity to a paralogous gene (HO‐2) and on the preliminary data from our group demonstrating the absence of introns in the HO‐3 gene (Scapagnini et al., 2002c), it is possible that this 50 -portion could have originated from the retrotransposition of the HO‐2 gene. In addition, this genetic mutation in the rat may represent a species‐ specific event since no other sequence in the public databases match that of the rat HO‐3. Induction of HO‐1 gene could be used for clinical diagnosis. However, the length of the GT polymorphism in the promoter of the gene encoding HO‐1 that regulates the magnitude of the HO‐1 response to a given stress signal can render this approach difficult for those individuals with long GT repeats, which are associated with low HO‐1 responsiveness. This polymorphism appears to be of functional significance in the short repeats, which are associated with high responsiveness and seem to be also associated with lesser likelihood of re‐stenosis after angioplasty (Otterbein et al., 2003a).

3.9 Regulation of HO Genes Coupling of metabolic activity and gene expression is fundamental to maintain homeostasis. Heme is an essential molecule that plays a central role as the prosthetic group of many heme proteins in reactions involving molecular oxygen, electron transfer, and diatomic gases. Although heme is integral to life, it is toxic because of its ability to catalyze the formation of ROS, and consequently oxidative damage to cellular macromolecules. In higher eukaryotes, toxic effects of heme are counteracted by the inducible HO‐1 system (Maines, 2000). As in the classic theory of metabolic control, expression of HO‐1 is induced by the substrate heme (Kanakiriya et al., 2003). In addition, expression of HO‐1 is robustly induced in mammalian cells by various proinflammatory stimuli, such as cytokines, heavy metals, heat shock, and oxidants that induce inflammatory damage (Keyse and Tyrrell, 1989). Thus, HO‐1 is an essential antioxidant defense enzyme that converts toxic heme into antioxidants and is fundamental for coping with various aspects of cellular stress and for regulating iron metabolism (Poon et al., 2004a). In clinical conditions, HO‐1 expression has been associated with increased resistance to tissue injury, thus leading to a gene therapy approach employing HO‐1 (Otterbein, 2002). HO‐2 gene consists of five exons and four introns. HO‐2 has a molecular weight of 34 kDa and exhibits 40% homology in amino acid sequence with HO‐1. It is generally considered a constitutive isoenzyme; however, in situ hybridization studies have shown increases in HO‐2 mRNA synthesis, associated with increased HO‐2 protein and enzyme activity in the neonatal rat brain after treatment with corticosterone (Raju et al., 1997). The organization of the HO‐2 gene needs to be fully elucidated, although a consensus sequence of the glucocorticoid response element (GRE) has been demonstrated in the promoter region of the HO‐2 gene (Liu et al., 2000). In addition, endothelial cells treated with the NOS inhibitor L‐NAME and HO inhibitor zinc mesoporphyrin exhibited a significant upregulation of HO‐2 mRNA.

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HO‐1 gene is induced by a variety of factors, including metalloporphyrins and hemin, as well as ultraviolet A (UVA) irradiation, hydrogen peroxide, prooxidant states, or inflammation (Tyrrell, 1999). This characteristic inducibility of HO‐1 gene strictly relies on its configuration: the 6.8‐kb gene is organized into four introns and five exons. A promoter sequence is located approximately 28 base pairs upstream from the transcriptional site of initiation. In addition, different transcriptional enhancer elements such as the HSE and metal regulatory element reside in the flanking 50 ‐region. Also, inducer‐responsive sequences have been identified in the proximal enhancer located upstream the promoter and, more distally, in two enhancers located 4 kb and 10 kb upstream the initiation site (Hill‐Kapturczak et al., 2003). The molecular mechanism that confers inducible expression of HO‐1 in response to numerous and diverse conditions has remained elusive. One important clue has recently emerged from a detailed analysis of the transcriptional regulatory mechanisms controlling the mouse and human HO‐1 genes. The induction of HO‐1 is regulated principally by two upstream enhancers, E1 and E2 (Sun et al., 2002). Both enhancer regions contain multiple stress (or antioxidant)‐responsive elements (StRE, also called ARE) that also conform to the sequence of the Maf recognition element (MARE) (Martin et al., 2004) with a consensus sequence (GCnnnGTA) similar to that of other antioxidant enzymes (Balogun et al., 2003). There is now evidence to suggest that heterodimers of NF‐E2‐related factors 2 (Nrf2) and one or another of the small Maf proteins (i.e., MafK, MafF and MafG) are directly involved in induction of HO‐1 through these MAREs (Gong et al., 2002). A possible model, centered on Nrf2 activity, suggests that the HO‐1 locus is situated in a chromatin environment that is permissive for activation. Since the MARE can be bound by various heterodimeric basic leucine zipper (bZip) factors including NF‐E2, as well as several other NF‐E2‐related factors (Nrf1, Nrf2, and Nrf3), Bach, Maf, and AP‐1 families (Sun et al., 2002), random interaction of activators with the HO‐1 enhancers would be expected to cause spurious expression. This raises a paradox as to how cells reduce transcriptional noise from the HO‐1 locus in the absence of metabolic or environmental stimulation. This problem could be reconciled by the activity of repressors that prevent nonspecific activation. One possible candidate is the heme protein Bach1, a transcriptional repressor endowed with DNA‐binding activity, which is negatively regulated upon binding with heme. Bach1–heme interaction is mediated by evolutionarily conserved heme regulatory motifs (HRM), including the cysteine–proline dipeptide sequence in Bach1. Hence, a plausible model accounting for the regulation of HO‐1 expression by Bach1 and heme is that expression of HO‐1 gene is regulated through antagonism between transcription activators and the repressor Bach1. While under normal physiological conditions expression of HO‐1 is repressed by Bach1/Maf complex, increased levels of heme displace Bach1 from the enhancers and allow activators, such as heterodimer of Maf with Nrf2, to promote the transcription of HO‐1 gene (Sun et al., 2002). To our knowledge, the Bach1/HO‐1 system is the first example in higher eukaryotes that involves a direct regulation of a transcription factor for an enzyme gene by its substrate. Thus, regulation of HO‐1 involves a direct sensing of heme levels by Bach1 (by analogy to lac repressor sensitivity to lactose), generating a simple feedback loop whereby the substrate effects repressor–activator antagonism. The promoter region also contains two metal‐responsive elements, similar to those found in the metallothionein‐1 gene, which respond to heavy metals (cadmium and zinc) only after recruitment of another fragment located upstream, between 3.5 and 12 kbp (CdRE). In addition, a 163‐bp fragment containing two binding sites for HSF‐1, which mediates HO‐1 transcription, are located 9.5 kb upstream of the initiation site (Balogun et al., 2003). The distal enhancer regions are important in regulating HO‐1 in inflammation, as they have been demonstrated to be responsive to endotoxin. In the promoter region, there also resides a 56 bp fragment that responds to the STAT‐3 acute‐phase response factor, involved in the downregulation of HO‐1 gene induced by glucocorticoid (Raju et al., 1997).

3.10 Glutathione, Thiol Redox State, and RNS: Intracellular Modulators of HO‐1 Expression The major regulator of intracellular redox state is glutathione, a cysteine‐containing tripeptide with reducing and nucleophilic properties. This tripeptide (GSH) is essential for the cellular detoxification of

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ROS in brain cells (Butterfield et al., 2002b). A compromised GSH system in the brain has been associated with the oxidative stress occurring in neurological diseases (Butterfield et al., 2002c). Recent data demonstrate that, besides intracellular functions, GSH has also important extracellular functions in the brain. In this respect astrocytes appear to play a key role in the GSH metabolism of the brain, since astroglial GSH export is essential for providing GSH precursors to neurons (Dringen and Hirrlinger, 2003). Of the different brain cell types studied in vitro only astrocytes release substantial amounts of GSH. In addition, during oxidative stress astrocytes efficiently export glutathione disulfide (GSSG). The multidrug‐resistance protein 1 participates in both the export of GSH and GSSG from astrocytes (Dringen and Hirrlinger, 2003). Glutathione exists in either a reduced (GSH) or oxidized (GSSG) form and participates in redox reactions through the reversible oxidation of its active thiol. In addition, GSH acts as a coenzyme of numerous enzymes involved in cell defense. In unstressed cells the majority (99%) of this redox regulator is in the reduced form, and its intracellular concentration is between 0.5 and 10 mM depending on the cell type (Drake et al., 2003b). Depletion of glutathione has been shown to occur in conditions of moderate or severe oxidative stress and has been associated with increased susceptibility to cell damage (Calabrese et al., 2000b; Balogun et al., 2003; Catania et al., 2003). There is now evidence to suggest that a direct link between a decrease in glutathione levels by oxidant stress and rapid upregulation of HO‐1 mRNA and protein exist in a variety of cells, including the rat brain, human fibroblasts, endothelial cells, and rat cardiomyocytes (Foresti and Motterlini, 1999). This finding is supported by the fact that N‐acetyl‐cysteine (a precursor of glutathione) abolishes oxidative stress‐mediated induction of HO‐1 gene (Foresti et al., 1997, 2001). In addition, increased production of NO and RSNO can also lead to changes in intracellular glutathione. In astroglial cell cultures, stimulation of iNOS by exposure to LPS and IFN‐g decreases total glutathione, while increasing GSSG, and this effect was abolished by pretreatment of glial cells with NOS inhibitors (Calabrese et al., 2000b). Moreover, elevation of intracellular glutathione prior to exposure of endothelial cells to NO donors almost completely abolishes activation of the HO pathway, which suggests that thiols can antagonize the effect of NO and NO‐related species on HO‐1 induction (Calabrese et al., 2000b). We have recently demonstrated in endothelial cells subjected to hypoxia that induction of HO‐1 is associated with a decrease in the GSH/GSSG ratio and with an increase in RSNO levels resulting from early induction of iNOS (Motterlini et al., 2000a). This implies that in conditions of low oxygen availability both oxidative and nitrosative reactions may serve as a trigger for induction of the HO‐1 gene (Motterlini et al., 2000b). All these evidence corroborate the notion that generation of ROS and RNS are important signal transduction mechanisms linking HO‐1 activation to cell stress tolerance (Mancuso et al., 2003; Pocernich et al., 2005).

3.11 Heme Oxygenase in Brain Function and Dysfunction In the brain, the HO system has been reported to be very active and its modulation seems to play a crucial role in the pathogenesis of neurodegenerative disorders. The HO pathway, in fact, has been shown to act as a fundamental defensive mechanism for neurons exposed to an oxidant challenge (Chen et al., 2000). Induction of HO occurs together with the induction of other Hsps in the brain during various experimental conditions including ischemia (Dore, 2002). Injection of blood or hemoglobin results in increased expression of the gene encoding HO‐1, which has been shown to occur mainly in microglia throughout the brain (Calabrese et al., 2001a). This suggests that microglia take up extracellular heme protein following cell lysis or hemorrhage. Once in the microglia, heme induces the transcription of HO‐1. In human brains following traumatic brain injury, accumulation of HO‐1þ microglia/macrophages at the hemorrhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months (Beschorner et al., 2000). There is now evidence that oxidative stress contributes to secondary injury after spinal cord trauma. Induction of HO‐1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter, was found in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO‐1 was induced in both the grey and the white matter. Thereafter, HO‐1 expression was limited to degenerating fiber tracts. Interestingly, Hsp70 was consistently colocalized with HO‐1 in the microglia and macrophages, indicating that long‐term induction of HO‐1 and Hsp70 in

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microglia and macrophages occur long after traumatic injury and are correlated with Wallerian degeneration and remodeling of surviving tissue (Mautes et al., 2000). Since the expression of Hsps is closely related to that of APP, Hsps proteins have been studied in the brain of patients with AD. Significant increases in the levels of HO‐1 have been observed in AD brains in association with neurofibrillary tangles (NFTs) (Takeda et al., 2000), and also HO‐1 mRNA was found increased in AD neocortex and cerebral vessels (Premkumar et al., 1995). HO‐1 increase was not only in association with NFTs but also colocalized with senile plaques and glial fibrillary acidic protein‐positive astrocytes in AD brains (Schipper et al., 2000). It is conceivable that the dramatic increase in HO‐1 in AD may be a direct response to increased free heme associated with neurodegeneration and an attempt to convert the highly damaging heme into the antioxidants biliverdin and bilirubin (Calabrese et al., 2004b). Upregulation of HO‐1 in the substantia nigra of patients with PD has been demonstrated. In these patients, nigral neurons containing cytoplasmic Lewy bodies exhibited in their proximity maximum HO‐1 immunoreactivity (Ewing and Maines, 1992). New evidence showed a specific upregulation of HO‐1 by oxidative stress in the nigral dopaminergic neurons (Poon et al., 2004a). Multiple sclerosis (MS) is a common, often disabling disease of the CNS. It has been suggested that inappropriate stress response within the CNS could influence both the permeability of the blood–brain barrier and the expression of Hsps, thereby initiating the MS lesion (Aquino et al., 1977; Maines, 2000). However, cytokines, immunoglobulins, and complement complexes may elicit a survival response in the oligodendrocytes, involving the induction of endogenous Hsps and other protective molecules, which indicates that redox systems and therefore the oxidant/antioxidant balance in these cells are of great importance in MS (Calabrese et al., 1994, 1998, 2002a, 2003b; Bagasra et al., 1995). The expression of HO‐1 is increased in the CNS of mice and rats with experimental allergic encephalomyelitis (EAE), an animal model of MS (Catania et al., 2003). To investigate the role of HO‐1 in EAE, SnPPIX was administered to SJL mice during active disease. SnPPIX (200 mmol/kg) attenuated clinical scores, weight loss, and some signs of pathology in comparison to vehicle treatment. Glutathione levels were greater in treated EAE mice than in those receiving vehicle, indicating lower oxidative stress in the former group. These data suggest that inhibition of HO‐1 attenuated disease and suppressed free‐radical production (Chakrabarty et al., 2003). On the contrary, in another study, high expression of HO‐1 in lesions of EAE was enhanced by hemin treatment, a procedure that resulted in the attenuation of clinical signs of pathology, whereas tin mesoporphyrin, an inhibitor of HO‐1, markedly exacerbated EAE (Liu et al., 2001). These results strongly suggest that endogenous HO‐1 plays an important protective role in EAE, and that targeted induction of HO‐1 overexpression may represent a new therapy for the treatment of MS. We have recently shown that thiol disruption and nitrosative stress are associated in active MS with induction of Hsp70 and HO‐1 in central and peripheral tissues of MS patients and that acetylcarnitine was able to counteract nitrosative stress‐mediated damage, an effect associated with enhancement of Hsp stress signaling (Poon et al., 2004a). All these findings can open up new therapeutic perspectives, as molecules activating these defense mechanisms appear to be possible candidates for novel neuroprotective strategies (Calabrese et al., 2000c).

3.12 Bilirubin and Biliverdin: An Endogenous Antioxidant System Supraphysiological levels (>300 mM) of nonconjugated bilirubin, as in the case of neonatal jaundice, are associated with severe brain damage. This is a plausible reason whereby bilirubin has generally been recognized as a cytotoxic waste product. However, only in recent years its emerging role as a powerful antioxidant has received wide sustain. The specific role of endogenously derived bilirubin as a potent antioxidant has been demonstrated in hippocampal and cortical neurons, where accumulation of this metabolite owing to phosphorylative‐dependent enhancement of HO‐2 activity protected against hydrogen peroxide‐induced cytotoxicity (Stocker et al., 1987; Mancuso et al., 2003). Moreover, nanomolar concentrations of bilirubin resulted in a significant protection against hydrogen peroxide‐induced toxicity in cultured neurons as well as in glial cells following experimental subarachnoid hemorrhage. In addition, neuronal damage following middle cerebral artery occlusion was substantially worsened in HO‐2 lacking mice (Dore et al., 2000). Bilirubin can become particularly important as a cytoprotective agent for tissues

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with relatively weak endogenous antioxidant defenses such as the CNS and the myocardium. Interestingly, increased levels of bilirubin have been found in the cerebrospinal fluid in AD, which may reflect the increase of degraded bilirubin metabolites in the AD brain, derived from the scavenging reaction against chronic oxidative stress (Kimpara et al., 2000). Similarly, a decreased risk for coronary artery disease is associated with mildly elevated serum bilirubin, with a protective effect comparable to that of HDL cholesterol (Dore et al., 2000). The most likely explanation for the potent neuroprotective effect of bilirubin is that a redox cycle exists between bilirubin and biliverdin, the major oxidation product of bilirubin. In mediating the antioxidant actions, bilirubin would be transformed to biliverdin, then rapidly converted back to bilirubin by biliverdin reductase, which in the brain is present in large functional excess, suggesting a mechanism to amplify the antioxidant effect (Poon et al., 2004b). Remarkably, the rapid activation of HO‐2 by protein kinase C (PKC) phosphorylation parallels the availability of nNOS. Both are constitutive enzymes localized in neurons, and nNOS is activated by calcium entry into cells binding to calmodulin. Similarly, PKC phosphorylation of HO‐2 and the transient increase in intracellular bilirubin would provide a way for a rapid response to calcium entry, this being a major activator of PKC. Recent evidence has demonstrated that bilirubin and biliverdin possess strong antioxidant activities toward peroxyl radical, hydroxyl radical, and hydrogen peroxide. Exposure of bilirubin and biliverdin to agents that release NO or nitroxyl resulted in a concentration‐and time‐dependent loss of bilirubin and biliverdin. Increasing concentrations of thiols prevented bilirubin and biliverdin consumption by nitroxyl, indicating that bile pigments and thiol groups can compete and/or synergize the cellular defence against NO‐related species. In view of the high inducibility of heme oxygenase‐1 by NO‐releasing agents in different cell types, these findings highlight novel antinitrosative characteristics of bilirubin and biliverdin, suggesting a potential function for bile pigments against the damaging effects of uncontrolled NO production (Kaur et al., 2003).

4

Caloric Restriction and Endogenous Oxidative Stress: Relevance to Aging and Cell Survival

Caloric restriction in mammals has been recognized as the best characterized and most reproducible strategy for extending maximum life span, retarding physiological aging, and delaying the onset of age‐ related pathological situations. The overwhelming majority of studies using caloric restriction have used short‐lived rodent species, although current work using monkeys should reveal whether this paradigm is also relevant for manipulating the rate of primate aging. The mechanisms by which restricted calorie intake modifies the rate of aging and cellular pathology have been the subject of much controversy, although an attenuation of accumulating oxidative damage appears to be a central feature (Hursting et al., 2003). A major effect of calorie‐restricted feeding now appears to be on the rate of production or leak of free radicals from mitochondrial sites, although the details of the adaptation and the signaling pathway that induce this effect are currently unknown. General consensus, however, has been achieved that caloric restriction feeding regimes reduce the rate of accrual of oxidative damage as measured by lipid peroxidation, nuclear and mtDNA damage, and protein carbonyl formation. An analysis of published studies that used a degree of food restriction in the range of 40%–50% ad libitum intake revealed a significant positive correlation between survival parameters, such as mean, maximum, and average survival time, and duration of caloric restriction. The longer the animals are maintained on low calorie intake during the postweaning period of the life span, the greater is the survival (Mattson et al., 2002). It is unclear whether caloric restriction protects against random oxidative damage per se or is protective for those vulnerable proteins of key pathways, such as those containing iron‐sulfur centers of the ETC or DNA‐binding signaling proteins. This is directly related to the question whether oxidative damage in genomic and mtDNA is primarily random as a function of age or whether there is a specific pattern of distribution of ROS which may vary depending on the tissue or the state of the cell cycle within any particular cell. It is generally accepted that age‐related accrual of ROS‐induced damage represents a balance between generation and defences, such as antioxidant enzymes, repair systems, and turnover. It has been demonstrated that caloric restriction reduces cellular injury and improves heat tolerance of old animals by lowering radical production and preserving cellular ability to adapt to stress through antioxidant enzyme induction and translocation of these proteins to the

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nucleus (Calabrese et al., 2001a). It has been also demonstrated that mitochondria from calorie‐restricted animals produce less ROS per nanomole of O2 during state 4 respiration, and recent work on ETC complexes suggests a modification in the Km for complex III associated with a retention of high‐affinity binding sites for complex IV as a possible mechanism operating in reducing superoxide generation (Mattson et al., 2003). It is conceivable that low calorie‐induced changes in unsaturated fatty acid composition of the mitochondrial membranes not only may protect against ROS‐induced lipid peroxidation but also may influence the binding properties of ETC proteins embedded in the membrane and the related transport processes. However, several questions need to be addressed such as the signaling pathway underlying the adaptive responses triggered by caloric restriction, or the effect of chronic caloric restriction on either the bioenergetics of individual mitochondria or the mitochondrial number and turnover rate. High‐density oligonucleotide array studies have recently provided compelling evidence that aging results in a differential gene expression pattern indicative of a marked stress response associated with lower expression of metabolic and biosynthetic genes, and also, these alterations are either completely or partially prevented by caloric restriction. In addition, the transcriptional patterns of calorie‐restricted animals suggest that caloric restriction retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage (Martin et al., 2003; Strauss, 2003; Calabrese et al., 2004b).

4.1 Therapeutic Potential of Nutritional Antioxidants Recently, considerable attention has been focused on identifying dietary and medicinal phytochemicals that can inhibit, retard, or reverse the multistage pathophysiological events underlying AD pathology (Butterfield et al., 2002a). Spices and herbs contain phenolic substances with potent antioxidative and chemopreventive properties (Scapagnini et al., 2002d). The active antioxidant principle in Curcuma longa, a coloring agent and food additive used in Indian culinary preparations, has been identified as curcumin (diferuloylmethane). Because of the presence in its structure of two electrophilic a, b‐unsaturated carbonyl groups which, by virtue of the Michael reaction, can react with nucleophiles such as glutathione, curcumin has the potential to inhibit lipid peroxidation and effectively to intercept and neutralize reactive oxygen and NO‐based free radicals (Poon et al., 2004a). This agent is a potent inhibitor of tumor initiation in vivo and possesses antiproliferative activities against tumor cells in vitro (Butterfield et al., 2002b). Recent epidemiological studies (Ganguli et al., 2000) have raised the possibility that this molecule, as one of the most prevalent nutritional and medicinal compounds used by the Indian population, is responsible for the significantly reduced (4.4‐fold) prevalence of AD in India compared with the United States. On the basis of these findings, compelling evidence has been provided that dietary curcumin given to an Alzheimer transgenic APPSw mouse model (Tg2576) for 6 months resulted in a suppression of indices of inflammation and oxidative damage in the brain of these mice (Lim et al., 2001). Furthermore, in a human neuroblastoma cell line it has recently been shown that curcumin inhibits NF‐kB activation, effectively preventing neuronal cell death (Poon et al., 2004a). Remarkably, recent evidence has demonstrated that curcumin is a potent inducer of HO‐1 in vascular endothelial cells (Balogun et al., 2003). We have also recently demonstrated in astroglial cells the role of caffeic acid phenethyl ester (CAPE), an active component of propolis, as a novel HO‐1 inducer (Scapagnini et al., 2002d). The similarity of CAPE to curcumin is striking because CAPE is also a Michael reaction acceptor, endowed with antiinflammatory, antioxidant, and anticancer effects (Butterfield et al., 2002a). These agents all appear capable of transcriptionally activating a gene battery that includes antioxidant enzymes and HO (Dinkova‐Kostova et al., 2001). Gene induction occurs through the antioxidant‐responsive element (ARE) (Alam, 2002; Alam and Cook, 2003). Thus, increased expression of genes regulated by the ARE in cells of the CNS may provide protection against oxidative stress.

5

Major CNS Disorders Associated with ROS/RNS‐Mediated Damage

The major CNS disorders associated with ROS/RNS‐mediated damage are listed in > Table 6‐2.

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. Table 6‐2 Neurodegenerative disorders associated with free‐radical damage 1. Amyotrophic lateral sclerosis 2. Alzheimer’s disease 3. Parkinson’s disease 4. Multiple sclerosis 5. Friedreich’s ataxia 6. Down’s syndrome 7. Huntington’s disease 8. Ischemia/reperfusion

5.1 Amyotrophic Lateral Sclerosis Amyotrophic lateral sclerosis (ALS) is a remarkably debilitating disease with inevitable lethal consequences. It typically affects adults in midlife with progressive paralysis and causes death generally within 5 years. It is characterized by degeneration of the motor neurons. These neural components include the anterior horn cells of the spinal cord, motor nuclei of the brain stem, particularly the hypoglossal nuclei, and the upper motor neurons of the cerebral cortex. ALS is currently untreatable and the pathogenesis is unknown, although numerous possible etiologies have been studied including viral, immunologic, and metabolic. However, none of these is considered a serious etiological candidate. On the other hand, the pathogenetic role of oxidative stress has emerged as a distinct possibility. Recent data from a multicenter study indicate that some but not all cases of inherited ALS arise because of mutations in the gene encoding the cytosolic form of Cu/Zn‐SOD (Calabrese et al., 2004b). Familial ALS patients heterozygous for SOD mutations have less than 50% of normal SOD activity in their erythrocytes and brains. This defective SOD gene is on chromosome 21 (the gene for mtSOD is on chromosome 6). Thus, the implication is that the degeneration of motoneurons in ALS may be initiated by oxidative free‐radical damage. An alternative hypothesis is that the mutation might impart harmful properties to the enzyme. This gain‐of‐function theory is based primarily on the fact that familial ALS is dominantly inherited, and thus only one copy of the enzyme is required to cause the disease, which arise from the toxic influence exerted by the abnormal protein. In this regard, transgenic mice containing extra copies of human Cu/Zn‐SOD and ALS mutations showed a disorder closely resembling human ALS, whereas overexpression of normal Cu/Zn‐SOD alone did not produce the disorder (Brwon, 1994). These studies suggest that the enzyme is endowed with neurotoxic properties. What the neurotoxic function might be remains to be elucidated. With regard to the familial form of the disease, one line of evidence suggests that interaction between NO and superoxide, by yielding the powerful oxidant ONOO, might constitute the primary pathogenic event leading to protein nitration, which slowly injures the motor neurons (Beckman et al., 1993). It is possible that the active site of SOD is altered, allowing greater access of ONOO to the copper center, and so favoring the subsequent formation of a nitronium‐like species which nitrosylates tyrosine residues (Heales et al., 1999). Immunocytochemistry studies have also revealed, in the neurofilament aggregates associated with ALS, a close association between SOD‐1 and NOS activity (Chou et al., 1996). Since light neurofilaments are rich in tyrosine, it is proposed that nitrotyrosine formation occurs, which impairs neurofilament assembly and ultimately leads to motoneuron death. Recently, increased nitrotyrosine immunoreactivity has been demonstrated in motor neurons of both sporadic and familial ALS, suggesting that ONOO‐mediated oxidative damage may play a role in the pathogenesis of both forms of the disease (Beal et al., 1997). Some evidence is now available to suggest that mitochondrial dysfunction is a central event in the disease process. Thus, a significant decrease in complex IV activity is reported in the spinal cord (ventral, lateral, and dorsal regions) of patients with sporadic ALS (Fujita et al., 1996). In addition, studies with a transgenic mouse model of ALS also suggest that axonal transport of organelles, in particular mitochondrial transport, is impaired and may be an important factor in ALS (Collard et al., 1995).

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5.2 Alzheimer’s Disease AD affects over two million Americans and is the major cause of admission to nursing homes. AD, which rarely occurs before the age of 50 years, usually becomes clinically apparent as a subtly impaired cognitive function or as affectivity disturbance. With time there is a progressive memory loss and disorientation, which eventually progresses into dementia. Although most cases are sporadic, 5%–10% or more are familial. Gross examination of the brain in AD shows a variable degree of cortical atrophy with narrowed gyri and widened sulci most apparent in the frontal, parietal, and temporal lobes. Microscopically, the features include NFTs, neurite (senile) plaques, amyloid angiopathy, granulovacuolar degeneration, and Hirano bodies. Importantly, all of these changes are present in the brains of nondemented older individuals but to a much lesser extent. The finding that choline acetyltransferase is decreased by 40%–90% in the cerebral cortex and hippocampus of patients with AD has led to the hypothesis that AD is consequence of a deficit in the cholinergic system (Calabrese et al., 2001a). Several lines of evidence now support an important role for free‐radical‐mediated event in the pathogenesis of the disease. Advanced glycosylation end products (AGEs) are a family of complex posttranslationally modified proteins that are initiated by condensation of reducing sugars with proteins amino groups via the Maillard reaction. It has become evident that glycation of proteins occurs in vivo in aged individuals (Christen, 2000). Oxidative stress increases the frequency of hydroxyl radical‐induced autoxidation of unsaturated membrane lipids. Reactive aldehydes, resulting by metal ion‐mediated fragmentation of lipid hydroperoxides, can modify proteins through alteration of protein–protein interactions and intermolecular crosslinking. Age modifications and oxidative stress mechanisms can synergistically accelerate protein damage (Butterfield, 2004). Several potential sources of oxidative stress should be considered in the pathogenesis of AD. First, the concentration of iron, a potent catalyst of oxyradical generation, is increased in NFT‐bearing neurons (Calabrese et al., 2000a). Second, increased concentrations of iron would result in increased protein modifications, which are catalyzed by metal ions and reducing sugars (Calabrese et al., 2000a). Third, microglia are activated and increased in number in AD and represent a major source of free radicals (El Khoury et al., 1998). Fourth, the increased lipid peroxidation and the resulting membrane disturbances, which are observed in degenerating neurons and neurites, are expected to lead to an influx of calcium, which causes destabilization of the cytoskeleton and activation of specific degradative enzymes (Markesbery, 1997; Drake et al., 2004). A decrease of complex IV activity has been reported in the cerebral cortex of individuals who died of AD (Kish et al., 1992). While the exact mechanism for this loss of activity is not clear, it is known that this enzyme complex is particularly susceptible to oxidative damage (Butterfield, 2002). In addition, there is now evidence to suggest that NO metabolism is affected in AD. The glial‐derived factor, S‐100‐b, which is overexpressed in this condition, causes induction of iNOS in astrocytes associated with NO‐mediated neuronal cell death in a co‐culture system (Hu et al., 1997). Furthermore, b‐amyloid is reported to activate NOS in a substantia nigra/neuroblastoma hybrid cell line (Heales et al., 1999). Analysis of postmortem material has revealed in AD brain the presence of tyrosine, as result of the reaction of ONOO and nitrotyrosine residues in protein, which was not detectable in age‐matched control brains (Smith et al., 1997). In addition, using antibodies specifically directed against iNOS, the presence of this isoform in neurofibrillary tangle‐bearing neurons was demonstrated (Sayre et al., 2000). Despite evidence for activation of NO metabolism in AD, analysis of the CSF nitrite þ nitrate (stable end products of ONOO degradation) concentration revealed levels in AD patients comparable to controls (Corregidor and De Pasamonte, 1996). While this observation does not dismiss a role for NO/ONOO in the etiology of AD, it implies that formation of RNS occurs at a level that not necessarily leads to a rise in CSF RNS concentration. Amyloid beta‐peptide (Ab), the principal component of senile plaques and the major neuropathological hallmark of AD, is considered to be central to the pathogenesis of AD. Ab is a 40–42 amino acid peptide that accumulates in the neuritic plaques in AD. The AD brain is under extensive oxidative stress (Butterfield et al., 2002a). These two observations were joined by a model to potentially account for neurodegeneration in AD brain: the Ab‐associated free‐radical oxidative stress hypothesis of brain cell death in AD (Castegna et al., 2004; Drake et al., 2004). In this model, Ab‐associated free radicals initiate lipid peroxidation, protein oxidation, ROS formation, intracellular and mitochondrial Ca2þ accumulation, and eventual death of

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neurons. A prediction of this model is that the antioxidant vitamin E should prevent or modulate these Ab‐ induced effects to neurons (Butterfield et al., 1997). Consistent with this model, this free‐radical scavenger was shown to block Ab‐initiated lipid peroxidation in cortical synaptosomes (Butterfield et al., 1999). Further, protein oxidation induced by Ab in astrocyte cultures and assessed by increased protein carbonyl content was abrogated by the more soluble form of vitamin E, trolox (Koppal et al., 1998). Vitamin E also blocked Ab‐induced inhibition of transmembrane protein function, including ion‐motive ATPases, glucose and glutamate transporters, G‐protein coupled signal transduction, and the energy‐related enzyme creatine kinase, and the methionine residue 35 of Ab1–42 and Ab1–40 was shown to be critical to the oxidative stress properties of these peptides (Butterfield and Kanski, 2002). Human Ab1–42, expressed in vivo in transgenic Caenorhabditis elegans nematodes, led to protein oxidation in the living animal, and methionine was important in this process as well (Yatin et al., 1999). A risk factor for AD is the presence of allele 4 of apolipoprotein E (apoE) (Roses, 1996). Synaptosomes from apoE‐knockout mice, containing no gene for apoE, show increased susceptibility to oxidative stress induced by Ab, (Lauderback et al., 2001), while synaptosomes from knockout mice containing human apoE4 with no mouse background show significantly increased Ab‐induced oxidative stress compared with synaptosomes from human apoE2 or apoE3 knockin mice (Lauderback et al., 2002). Thus, apoE may serve an antioxidant function, but apoE4 may be less able than apoE2 or apoE3 to do so (Lauderback et al., 2002). This notion was tested using 1‐month‐old control and apoE‐deficient mice. Both received dietary vitamin E for 12 months. Vitamin E‐fed animals had better behavioral outcomes of spatial motor activity and decreased levels of lipid peroxidation relative to apoE‐deficient mice fed a normal diet (Veinbergs et al., 2000). The sum of these studies suggests a decreased risk for and diminished oxidative stress in AD in persons taking high dose dietary, or perhaps supplemental, vitamin E (and vitamin C to regenerate vitamin E from the tocopherol radical). Brains of AD patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat‐shock response, which is one form of stress response. Hsps are proteins serving as molecular chaperones involved in the protection of cells from various forms of stress. Increasing interest has focused on identifying dietary compounds that can inhibit, retard, or reverse the multistage pathophysiological events underlying AD pathology. AD, in fact, involves a chronic inflammatory response associated with both brain injury and Ab‐associated pathology. All of the above evidence suggests that stimulation of various repair pathways by mild stress has significant effects on delaying the onset of various age‐associated alterations in cells, tissues, and organisms. Spice and herbs contain phenolic substances with potent antioxidative and chemopreventive properties, and it is generally assumed that the phenol moiety is responsible for the antioxidant activity. In particular, curcumin, a powerful antioxidant derived from the curry spice turmeric, has emerged as a strong inducer of the heat‐shock response. In light of this finding, curcumin supplementation has been recently considered as an alternative, nutritional approach to reduce oxidative damage and amyloid pathology associated with AD (Butterfield et al., 2002a, c; Calabrese et al., 2004b). Conceivably, dietary supplementation with vitamin E or with polyphenolic agents, such as curcumin and its derivatives, can forestall the development of AD, consistent with a major ‘‘metabolic’’ component to this disorder. Nutritional biochemical research is providing optimism that this devastating brain disorder of aging may be significantly delayed and/or modulated.

5.3 Parkinson’s Disease PD is a progressive neurodegenerative disorder that increases in frequency after the age of about 50 years. The major clinical disturbances in PD result from dopamine depletion in the striatum, because of nigral neuronal loss. Although, a number of hypothesis, including defective DNA repair mechanisms, specific genetic defects, viral disorder, lack of a neurotrophic hormones, or toxic compounds present in the environment, have been proposed, none completely explains the cascade of events responsible for the cause and the course of the disease. A large body of evidence supports the role of free radicals in the pathogenesis of the disease (Hyun et al., 2003). Levels of lipid hydroperoxides are increased tenfold in the substantia nigra in PD (Hyun et al., 2002).

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Decreased glutathione peroxidase and catalase activities associated to increased SOD activity leads to increased levels of hydrogen peroxide (Dexter et al., 1991). This, in dopaminergic cells, is primarily produced by MAO via deamination of dopamine and also nonenzymatically by autoxidation of dopamine. Hydrogen peroxide, by reacting with reduced forms of transition metals, e.g., iron (II) or copper (I), gives rise to the powerful oxidant hydroxyl radical, and oxidative damage to nigral membrane lipids, proteins, and DNA ensues. The role of iron in brain oxidative injury has been extensively considered (Mattson et al., 2002). Dexter and coworkers (1991) reported a 31%–35% increase in the total iron content in parkinsonian SN compared with control tissue, which was associated with decreased levels of the iron storage protein ferritin, contrasting with a significant decrease of the levels of iron‐binding protein in the CSF. A shift of iron II/iron III ratio in the substantia nigra from almost 2:1 in the normal brain to 1:2 in the parkinsonian brain is also served (Li and Dryhurst, 1997). Hence, a distinct possibility exists that excessive free‐radical generation occurs in this region, leading to the death of nigral neurons. In addition, substantia nigra is a dopamine‐rich brain area, and catechols, including DOPA and dopamine, have been demonstrated to be cytotoxic in vitro, presumably by formation of covalent bonds between their quinone forms and macromolecules of vital importance, primarily represented by thiol groups (Spencer et al., 2002). In fact, an intermediate in the autoxidation of catechols to quinone is the free radical semiquinone. Both autoxidation steps generate reduced forms of molecular oxygen such as superoxide anion and hydrogen peroxide which, in addition to hydrogen peroxide produced by the MAO‐dependent catabolism of dopamine, contribute to maintain considerable levels of the highly reactive hydroxyl radical, which on reacting with free thiol groups may contribute to the decreased levels of GSH and a corresponding increase in GSSG found in the SN (Calabrese et al., 2002b, 2004a). This is of special importance considering that nigral cells also contain neuromelanin, a pigmented substance related to lipofuscin and derived from dopamine. Neuromelanin has been demonstrated to have high affinity for iron III, and this iron–melanin interaction might have pathogenetic implications. In fact, the synthesis of neuromelanins from dopamine is known to produce more oxidative damage than the synthesis from other catecholamines (Spencer et al., 1994) and, in addition, neuromelanins polymerize from pheomelanin in a process that requires cysteine for synthesis, thus competing with g‐glutamyl cysteine synthetase which utilizes cysteine for GSH synthesis. Under these circumstances the GSH system in the substantia nigra could result in a position of increased demand and decreased synthetic capability, and hence contribute to the high vulnerability of this region to peroxidative injury (Calabrese et al., 2001a, 2002b, 2004a). This is confirmed by the study of Perry and coworkers (1982), which showed that GSH levels in the SN were significantly lower than in other brain regions. Moreover, a 40% decrease in GSH in the SN of PD, associated with significant increase in oxidized glutathione, has been also reported (Sian et al., 1994). Recently, it has been demonstrated in PD patients that the proportion of dopaminergic neurons with immunoreactive NF‐kB in their nuclei was more than 70‐fold than that in control subjects (Hunot et al., 1997). A possible relationship between the nuclear localization of NF‐kB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF‐kB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin‐dependent signaling pathway with C2‐ ceramide (France‐Lanord et al., 1997). Data suggest that this oxidant‐mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD (Schapira et al., 1990; Mc Naught et al., 2001; Dawson and Dawson, 2002; Moore et al., 2003). Moreover, a potential role for excitotoxic processes in PD has been strengthened by the observation that there appears to be a mitochondrially encoded defect in complex I activity of the ETC (Schapira et al., 1990). An impairment of oxidative phosphorylation will enhance vulnerability to excitotoxicity (Xin et al., 2000). Substantia nigra neurons possess N‐methyl‐D‐aspartate receptors, and there are glutamatergic inputs into the substantia nigra from both the cerebral cortex and the subthalamic nucleus. After activation of excitatory amino acid receptors, it has been suggested that there is an influx of calcium followed by activation of nNOS, which can then lead to the generation of peroxynitrite (Bechtold and Brown, 2003). Consistent with such a mechanism, studies of MPTP neurotoxicity in both mice and primates have shown that inhibition of nNOS exerts neuroprotective effects, raising the prospect that excitatory amino acid antagonists for nNOS inhibitors might be useful in the treatment of PD (Dawson and Dawson, 1995, 2002).

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5.4 Multiple Sclerosis MS is a common often disabling disease of the CNS. Although evidence indicates that MS is a complex trait caused by interaction of genetic and environmental factors, little is known about its cause or the factors that contribute to its unpredictable course (Risch and Merikangas, 1996). It is generally accepted that vascular factors, metabolic alterations, virus infections of the CNS, or disturbed immune mechanisms are responsible for the cause and course of MS. The clinical symptoms of MS result from inflammatory damage to the insulating myelin sheath of axons in the CNS and at later stages to axons themselves. A local autoimmune process involving activation of TH cells against CNS protein components is likely to be crucial for this development. Once triggered, the immune system attacks and destroys myelin and the myelin‐forming cells (Calabrese et al., 1998). Evidence exists which indicate that oligodendrocytes and their secreted products respond to the attack by immune cells through modulation of its metabolism and gene expression (Lindsey et al., 1997). It has been also suggested that inappropriate stress response within the CNS could influence both the permeability of the blood–brain barrier and the expression of Hsps, thereby initiating the MS lesion (Calabrese et al., 2002a). In addition, cytokines, immunoglobulins, and complement complexes may elicit a survival response in the oligodendrocytes, involving the induction of endogenous Hsps and other protective molecules, which indicates that redox systems and therefore the oxidant/antioxidant balance in these cells are of great importance in MS (Calabrese et al., 2003b). A variety of studies support a role for oxidative stress in MS. These include studies on increased serum peroxide levels in MS relative to control (Toshniwal and Zarling, 1992; Calabrese et al., 1994). Patients with MS in acute exacerbation exhibit significantly higher levels of pentane and exane (products of lipid peroxidation) in expired breath compared with either MS patients in remission or control subjects (Toshniwal and Zarling, 1992). Moreover, recent clinical and animal studies suggest that NO and its reactive derivative peroxynitrite are implicated in the pathogenesis of MS (Bagasra et al., 1995). Patients dying with MS demonstrate increased astrocytic iNOS activity as well as increased levels of iNOS mRNA and nitrotyrosine residues (Bagasra et al., 1995; Cross et al., 1998). In EAE, both astrocytes and microglia express iNOS (Tran et al., 1997). All this is consistent with the demonstration that NO‐derivative species are cytotoxic to oligodendrocytes and neurons by inhibiting the mitochondrial respiratory chain (complex II–III and IV) and certain key intracellular enzymes (Stamler and Hausladen, 1998; Heales et al., 1999), thereby representing a critical determinant in the etiology of the disease. MS is a relatively common disease of the CNS, the course of which is often of a progressive but relapsing/remitting nature. The clinical symptoms of MS during relapse (numbness, paralysis, blindness, and a variety of others) are mainly due to conduction block of axonal electrical impulses, caused by a variety of different molecular pathologies, including inflammation and demyelination (Calabrese et al., 2001a). A local autoimmune process involving activation of glia is likely to be crucial in the development of this damage (Tran et al., 1997). Activated glia secrete RNS products of NO metabolism with superoxide radicals (O2
) to form peroxynitrite anion (ONOO). At physiological pH, it protonates to its conjugate acid peroxynitrous acid, which decomposes with a t1/2 of less than 1 s. One of the fastest reactions of ONOO is with (3 to 5.8  104 M1 s1 at 37 C). Together with the high concentrations of CO2 (1.3 mM) and HCO 3 (25 mM) this reaction is the most probable pathway of ONOO decomposition in vivo (Calabrese et al., 2003b). RNS can cause nitrosative stress, which results in the destruction of myelin and (myelin‐forming) oligodendrocyte cells (Calabrese et al., 2002a). A direct link between NO and the conduction block that occurs in MS has been suggested, as NO donors cause reversible conduction block in both normal and demyelinated axons of the central and peripheral nervous systems (Calabrese et al., 2002a). In addition, conduction in demyelinated and early remyelinated axons is particularly sensitive to block by NO (Heales et al., 1999). This may be due to the direct effects of NO on glutamatergic neurotransmission, as it has been shown that NMDA receptor is inactivated by nitrosylation (Heales et al., 1999). Furthermore, the formation of S‐nitrosoglutathione (GSNO) can cause GSH depletion, and hence trigger redox‐dependent changes in cellular signaling as well as modification of key intracellular enzymes, such as chain respiratory complex activities (Gegg et al., 2003).

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Recent clinical and animal studies also indicate that NO and ONOO play a central role in the pathogenesis of MS (reviewed in Calabrese et al., 2001a). It has also been shown that in CSF and plasma nitrite þ nitrate (stable end products of NO metabolism) levels are elevated in patients with MS (Giovannoni, 1998). ROS and RNS have a major role in the mediation of cell damage, and free sulfhydryl groups are vital in cellular defence against endogenous or exogenous oxidants (Calabrese et al., 2000a, 2001b). The possible links between MS and oxidant/antioxidant balance in cell perturbation may be suggested by several factors, including increased incidence of MS in populations consuming high proportions of animal fat, (Calabrese et al., 1994), increased malonaldehyde levels in blood, and decreased glutathione peroxidase activity in MS erythrocytes, lymphocytes and granulocytes (Calabrese et al., 1998) and, in addition, an inappropriate expression of Hsps on oligodendrocytes (Calabrese et al., 2002a). This last event could represent a possible initiating factor at the level of MS lesions, capable of modulating the subsequent susceptibility or resistance of cells to oxidative stress. Moreover, a decrease in sulfhydryl groups and increased amounts of lipid peroxidation products have also been measured in the CSF and plasma of MS patients (Calabrese et al., 2002a). Nitrosative stress in isolated astrocytes in vitro causes modifications in the endogenous thiol pool associated with induction of Hsp32 or HO‐1, which is prevented by antioxidants, suggesting a biochemical link between nitrosative stress, sulfhydryl function, and the heat‐shock pathway (Scapagnini et al., 2002d; Calabrese et al., 2004b). In addition, this evidence suggests that redox‐active compounds such as glutathione and the overall oxidant/antioxidant balance in the CNS are potentially of great importance in MS, although as yet there has been few studies addressing the relationships between NO, ONOO, and glutathione in MS. The chemical composition of human CSF is considered to reflect brain metabolism (Thompson, 1988), and we have recently demonstrated in MS patients decreased levels of protein sulfhydryl groups associated with an increase in RNS and peroxidative products (Calabrese et al., 2002a, 2003b). More recently, we have provided experimental evidence that increased levels of RNS are present in the CSF of MS patients, and this is associated with increased nitrosylation of sulfhydryl moieties. Our results are consistent with evidence indicating increased protein nitrosylation in MS patients (Cross et al., 1997) and pose intriguing implications regarding clinical manifestations in MS, which are potentially linked to a failure of action potentials to propagate along damaged axons and involve inflammatory processes as primary causative factors in addition to demyelination. In favor of this possibility is the evidence that NO donors are capable of blocking conduction in rat demyelinated axons (Garthwaite et al., 2002). All this would suggest a broader potential role for NO in the symptomatic manifestations of MS. Whether or not NO is central to the pathogenesis of MS remains to be clarified, owing to its role of being a double‐edged sword.. Consistently, a recent study (Sellebjerg et al., 2002) has demonstrated an association between high CSF levels of NO metabolites with severe disease activity in relapsing/remitting MS, and high concentrations of NO metabolites were associated with more pronounced treatment responses after methylprednisolone treatment. However, other studies have shown no significant correlation between NO metabolites and disability score, disease progression index, MRI activity, and development of cortical atrophy on MRI. (Yuceyar et al., 2001). We have also demonstrated in MS patients an increase in nitrosative stress, which was associated with a significant decrease of both protein SH groups and GSH, with increased levels of GSSG and nitrosothiols (Calabrese et al., 2002a). Interestingly, treatment of MS patients with acetylcarnitine resulted in decreased CSF levels of NO‐reactive metabolites and protein nitration and in a significantly higher content of both GSH and GSH/GSSG ratio. In addition, urinary nitrites, which were higher in MS patients than in controls, decreased significantly after treatment with acetylcarnitine. Several studies have shown the capability of carnitines to interfere with changes in oxidant/antioxidant balance and metabolism induced by oxidants (Hagen et al., 1998a, b). Although, so far, the exact mechanisms of action of acetylcarnitine are still unknown, current research points to its ability to enhance neuronal mitochondrial bioenergetics (Calabrese and Rizza, 1999), which in turn may influence cellular oxidant/antioxidant balance (Calabrese et al., 2002d). We have recently shown in astrocytes exposed to LPS and INFg‐induced nitrosative stress that acetylcarnitine protects against cytokine‐mediated mitochondrial chain respiratory complex impairment and the associated increase in protein and lipid peroxidation. The increase in astroglial antioxidative potential observed after acetylcarnitine treatment involves a secondary line of antioxidant defenses, represented by stress‐responsive genes, such as HO‐1 and the mitochondrial Hsp60

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and SOD (Calabrese et al., 2005). Furthermore, as a brain energy enhancer, acetylcarnitine could improve survival of damaged neurons (Scapagnini et al., 2002b), and metabolic studies conducted noninvasively in humans with NMR indicate that acetylcarnitine helps the brain to maintain the constant supply of energy needed for effective homeostasis. MS is a progressive inflammatory neurodegenerative disease. However, despite increasing research efforts and although several explanations have been proposed for destruction of myelin and oligodendrocytes in MS, there is still no proven mechanism of injury. The possibility of manipulating these complex glial cell functions and controlling their pathologic interactions with immune cells probably will illuminate how myelin damage can be contained and how the injured tissue can be repaired.

5.5 Friedreich’s Ataxia FA is an autosomal recessive neurodegenerative disorder involving both the central and peripheral nervous systems. Patients also show a systemic clinical picture presenting heart disease and diabetes mellitus or glucose intolerance. The disease is caused by mutations in the FA gene mapped on chromosome arm 9q13. The product of the gene is frataxin, an 18‐kDa soluble mitochondrial protein with 210 amino acids. Crystal structure suggests a new, not previously reported, protein fold (Durr et al., 1996). The most frequent mutation is the expansion of a GAA trinucleotide repeat located within the first intron of the gene, and represents 98% of the mutations. This triplet motif can adopt a triple‐helical DNA structure that inhibits transcription (Harding, 1981). The severity of the disease correlates directly with the number of triplet units and consequent decrease in protein levels, with patients having frataxin levels ranging from 6% to 30% of that of normal subjects (Campuzano et al., 1996). The primary tissues affected in the disease include the large sensory neurons in the dorsal root ganglia and the nucleus dentatus, as well as cardiac and pancreatic cells. The progressive gait and limb ataxia, hypertrophic cardiomyopathy, and diabetes mellitus found in FA patients are attributed to low levels of ATP produced in these energy‐intensive tissues (Durr et al., 1996). Point mutations are described in compound heterozygous subjects with one expanded allele. A two‐step model of GAA normal alleles toward premutation alleles, which might generate further full expanded mutations in the population with Indo‐European ancestry, has been postulated. Clinical phenotype is variable and an inverse correlation with the GAA expansion size has been observed. Analysis of the GAA triplet is a strong molecular tool for clinical diagnosis, genetic counseling, and prenatal diagnosis. Many approaches have been undertaken to understand FA, but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, increasing evidence indicates that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to FA pathogenesis. Brains of FA patients undergo many changes, such as disruption of protein synthesis and degradation, classically associated with the heat‐shock response, which is the most important form of stress response. The precise sequence of events in FA pathogenesis is uncertain. The impaired intramitochondrial metabolism with increased free iron levels and a defective mitochondrial respiratory chain will result in increased free‐radical generation, causing oxidative damage, which may be considered a possible mechanism that compromises cell viability. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols, and in addition, decreased expression of frataxin protein is associated with FA. Recent studies have shown that frataxin acts as a chaperone for Fe(II) and a storage compartment for excess iron (Babcock et al., 1997). This is consistent with the roles played by frataxin in iron export, Fe–S cluster assembly, heme biosynthesis, and prevention of oxidative stress. Also, frataxin plays a direct role in the mitochondrial energy activation and oxidative phosphorylation. Several model systems have been developed in an effort to understand the disease. In mouse models, deletion of the frataxin gene results in embryonic lethality (Radisky et al., 1999) while its selective inactivation in neuronal and cardiac tissues leads to neurological symptoms and cardiomyopathy associated with mitochondrial iron–sulfur cluster‐containing

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enzyme deficiencies and time‐dependent mitochondrial iron accumulation. In contrast, a model expressing 25%–35% of wild‐type frataxin levels, by virtue of a (GAA)230 expansion inserted in the first intron of the mouse gene, has no obvious phenotype (Bradley et al., 2000). Over the last 5 years, it has become clear that mitochondrial iron accumulation generates oxidative stress and results in damage to critical biological molecules. Studies using the budding yeast Saccharomyces cerevisiae have provided a further understanding of the consequences of frataxin loss (Radisky et al., 1999). Deletion of the yeast frataxin homolog YFH1 results in a tenfold increase in iron within the mitochondria along with increased ROS production (Lodi et al., 2002). This leads to loss of mitochondrial function and the appearance of a petite phenotype in nearly all strains that have been examined (Radisky et al., 1999). Bradley and colleagues (2000) demonstrated an impaired oxidative phosphorylation system with severe and significant deficiencies of mitochondrial respiratory chain complexes I and II/III and aconitase activity in cardiac muscle from patients with FA; mtDNA levels were reduced in FA heart and skeletal muscle and increased iron deposition was present in FA heart, liver, and spleen in a pattern consistent with a mitochondrial location. In addition, there is the appearance of nDNA damage (Bradley et al., 2000). Moreover, aconitase deficiency is suggestive that oxidative stress may induce a self‐ amplifying cycle of oxidative damage associated with mitochondrial dysfunction, which may also contribute to cellular toxicity. Iron deposition and enzyme deficiencies have been reported in postmortem heart and brain tissues (Foury and Talibi, 2000) of FA patients. The role of oxidative damage in the pathogenesis of FA is also supported by the finding that idebenone, an antioxidant similar to ubiquinone, can reduce myocardial hypertrophy and also decrease markers of oxidative stress in FA patients (Lodi et al., 2002). Upregulation of protein manganese superoxide dismutase (MnSOD) fails to occur in FA fibroblasts exposed to iron. This finding, together with the absence of activation of the redox‐sensitive factor NF‐kB, suggests that NF‐kB‐independent pathway which may not require free‐radical signaling is responsible for the reduced induction of MnSOD. This impairment could constitute both a novel defense mechanism against iron‐mediated oxidative stress in cells with mitochondrial iron overload and, conversely, an alternative source of free radicals that could contribute to the disease pathology. Iron chelator drugs and antioxidant drugs have therefore been proposed for the treatment of FA. Drugs that reduce oxidative stress have a limited effect on the progression of the disease pathology, probably because these cannot properly remove iron accumulation. The potential role of iron chelator analogues (e.g., 2‐pyridylcarboxaldehyde isonicotinoyl hydrazone (PCIH)) as agents to remove mitochondrial iron deposits have been recently under investigation (Jauslin et al., 2003). These ligands have been specifically designed to enter and target mitochondrial iron pools, which is a property lacking in desferrioxamine, the only chelator in widespread clinical use. This latter drug may not have any beneficial effect in FA patients, probably because of its hydrophilicity that prevents mitochondrial access. Indeed, standard chelation regimens will probably not work in FA, as these patients do not exhibit gross iron loading. Considering that there is no effective treatment for FA, it is essential that the therapeutic potential of iron chelators focusses on the mitochondrial iron pools as their primary target. Remarkably, in an in vitro model of regulated human frataxin overexpression, it was shown that downregulation of the expression of mitogen‐activated protein kinase kinase 4 was associated with a decreased phosphorylation of c‐Jun N‐terminal kinase. In addition, to understand whether this alteration might result in cell death, the caspase pathway was investigated in FA cells, revealing in FA patients a significantly higher activation of caspase‐9 after serum withdrawal compared with controls. These findings suggest the presence, in FA patient cells, of a ‘‘hyperactive’’ stress‐signaling pathway. The role of frataxin in FA pathogenesis could be explained, at least in part, by this hyperactivity. Pilot studies have shown the potential effect of antioxidant therapy using idebenone or coenzyme Q10 with vitamin E administration and provide a strong rationale for designing larger randomized clinical trials (Lodi et al., 2003). There is now strong evidence to suggest that mitochondrially localized antioxidant ameliorates cardiomyopathy in FA patients, as well other lipophilic antioxidants can protect FA cells from cell death, indicating novel treatment strategies for FA and presumably for other neurodegenerative diseases with mitochondrial impairment. Antioxidants targeted to mitochondria appear a promising approach to effectively slow disease progression.

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5.6 Huntington’s Disease HD is an autosomal dominant, completely penetrant inherited neurodegenerative disorder, characterized by the insidious progression of severe neuropsychological and motor disturbances. The clinical manifestations of HD primarily involve psychiatric abnormalities, most commonly mood disturbances, followed by the development of involuntary choreiform movements and dementia. Onset of the disease is typically apparent in the fourth or fifth decade of life, and its long duration of up to 15–20 years results in death as consequence of complicating immobility. The principal neuropathological features of the disease are marked atrophy, neuronal loss, and astrogliosis in the neostriatum. The genetic defect in HD has been recognized in an abnormal expanded trinucleotide (CAG) repeat in a gene located on the short arm of chromosome 4 that encodes a protein termed ‘‘huntingtin,’’ whose function, so far, remains to be elucidated. Several lines of evidence indicate that a defect in mitochondrial energy metabolism might underlie the pathogenesis of the selective neuronal death occurring in HD. Evidence of bioenergetic defects in HD comes from in vivo imaging studies showing a marked hypometabolism, as revealed by PET analysis of [18F]fluorodeoxyglucose (FDG) utilization, in the caudate and putamen of symptomatic HD patients (Calabrese et al., 2004b). Recent studies have also identified cortical hypometabolism in symptomatic HD. Alterations in cerebral glucose utilization predominantly reflect changes in neuronal terminal activity, the principal site of energy consumption. Most of the ATP produced in the brain is used by energy‐ dependent pumps to restore transmembrane potential following synaptic transmission (Calabrese et al., 2004b). Consequently, the marked hypometabolism observed in specific brain regions in HD can be related to loss of synaptic density due to the marked atrophy occurring in these regions. This hypothesis has gained further sustain from NMR studies indicating increased lactate concentrations in the basal ganglia of HD patients (Jenkins et al., 1993). This increase well correlates with the duration of the disease, implying that normal energy metabolism is progressively impaired by the disease process. This might arise by the fact that when oxidative phosphorylation is no longer sufficient in supplying cellular energy demands, cells resort to reducing pyruvate by NADH in order to recycle NAD for ATP production via the glycolytic pathway. A pathogenic role for mitochondrial dysfunction in HD arises from in vivo biochemical studies in postmortem brain tissue, which have evidenced defects in succinate dehydrogenase as well as pyruvate dehydrogenase activities in the striatum of HD patients, and also these defects have been found as a function of illness duration. Further evidence supporting a mitochondrial defect in HD has been provided by an NMR spectroscopy study demonstrating 60% increase in pyruvate levels in the CSF, and 60% reduction in the activities of complex II and III in the caudate of HD patients, compared with controls (Browne, 1997).

5.7 Down’s Syndrome The most important pathological features of Down’s syndrome (DS) are mental retardation and accelerated aging. Numerous studies link both of these disturbances to free‐radical‐induced damage (Calabrese et al., 2000a). DS patients have an extra chromosome 21 (trisomy 21) and the recent assignment to chromosome 21 of the gene for Cu/Zn‐SOD together with the observation of increased SOD activity in red blood cells in DS patients has directed the interest on the role played by free‐radical species in the pathogenesis of the disease (Calabrese et al., 2000a). DS patients have a very high predisposition to develop the characteristics of AD. Therefore, DS patients provide a genetic model for investigating the role of oxidative stress in AD. In this regard, transgenic mice expressing the human SOD gene preferentially localize SOD in the hippocampus, which is the most vulnerable region in AD. However, these changes are not compensated by corresponding increase in catalase or glutathione peroxidase activities. This provides one possible explanation why increase in SOD activity might be detrimental. In addition, increased SOD activity results in decreased steady‐state levels of superoxide anion, which also plays a role in terminating the chain reactions of lipid peroxidation. All these evidence highlight the importance of oxidant/antioxidant balance as a critical determinant, and with this the conceivable possibility that the use of exogenous antioxidants can slow the progression of the disease (Halliwell, 2002).

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5.8 Ischemia/Reperfusion Ischemic brain damage is accompanied by an energy deficiency state and selective neuronal loss (Heales et al., 1999). Under such conditions, there is an increase in the extracellular concentration of glutamate, which may be neurotoxic due to activation of nNOS. Excess NO generation, causing impairment of energy metabolism and other metabolic processes, may also downregulate glutamate (NMDA) receptors, thereby minimizing the effect of glutamate. In addition, NO can cause vasodilation, and hence increase cerebral blood flow to the infarcted area (Heales et al., 1999). These effects may provide an explanation for the contradictory results that have been obtained when nonspecific NOS inhibitors have been evaluated in various models of ischemia. Reperfusion, following ischemia, may exacerbate the generation of oxidizing species, particularly superoxide. In a model of graded ischemia, loss of brain mitochondrial function, at the levels of complexes I, II, II–III, and ATP synthetase, has been reported (Powell and Jackson, 2003). Reperfusion was associated with restoration of activity of these mitochondrial components, followed after 2 h by a dramatic loss of complex IV activity (Brooks et al., 2002). The exact mechanism for this loss of complex IV activity is not known, but could involve the oxidative and/or nitrosative stress‐mediated reactions (Brooks et al., 2002). In fact, ischemia is accompanied by the formation of gliotic scar, principally comprised of reactive astrocytes, which in large amount express iNOS (Heales et al., 1999). Thus, excessive generation of glial‐derived ONOO may constitute an important contributing factor to the mitochondrial damage associated with ischemia. Loss of brain ATP levels and mitochondrial complex II–III and IV activity has been demonstrated in a rat model of perinatal asphyxia (Bolanos et al., 1998); in addition, administration of an NOS inhibitor to the mothers prevented impairment of brain energy metabolism in the hypoxic pups (Bolanos et al., 1998). Notably, ischemic preconditioning, which has been demonstrated to increase Hsp expression, preserves brain mitochondrial functions during middle cerebral artery occlusion (Zhang et al., 2003).

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Genetics of Human Longevity: Role of Vitagenes in Prolongation of Healthy Life Span

The first half of the twentieth century has seen a rapid increase in the life expectancy of individuals in industrialized nations because of improved sanitation, public health, housing, nutrition, medical technology, and pharmaceuticals. The second half of this century has been characterized by a growing concern with the challenge produced by the increasing prevalence of old people in the society. Aging is a very common feature in living organisms and can be described as the total effect of those intrinsic changes in an organism that adversely affect its vitality and render it more susceptible to the many factors that can cause death. Typically, mortality rate accelerates with time, but it is not clear whether this effect is the result of external or internal causes of death. The full extent of aging in a population becomes apparent when most important external hazards are removed, such as captive or laboratory conditions, and average longevity is usually greatly extended (Calabrese et al., 2001a). Even if an organism is immortal it has nonzero probability of dying because of extrinsic causes such as starvation, predation, and accidents. The probability of survival decreases in the course of life and, since natural selection is effective only through the reproductive output of individuals, the strength of natural selection decreases with age (Calabrese et al., 2001a). The first genetic theories on the evolution of aging were proposed in 1957 by Medawar and Williams almost simultaneously to the mechanistic theories of aging, such as the free‐radical and the somatic mutation theory, suggested by Harman (1956) and Szilard (1977), respectively. A synthesis of evolutionary and mechanistic theories occurred in 1977 within the framework of the soma theory of aging postulated by Kirkwood (1977). This theory provides a direct connection between evolutionary and physiological aspects of aging, by recognizing the primary importance of the allocation of metabolic energy resources between growth, somatic maintenance, and reproduction. It is suggested that longevity is determined through the setting of longevity assurance mechanisms so as to provide an optimal compromise between investments in somatic maintenance (including stress resistance) and in reproduction. As a corollary, increasing maintenance promotes the survival and longevity of the organism only at the expense of significant metabolic

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investments that could otherwise be used to accelerate processes such as growth and reproduction. The ‘‘disposable soma’’ theory of the evolution of aging also proposes that a high level of accuracy is maintained in immortal germ line cells, or alternatively, defective germ cells, if any, are eliminated. The evolution of an increase in longevity in mammals may be due to a concomitant reduction in the rates of growth and reproduction, the so‐called ‘‘essential life’’ and an increase in the accuracy of synthesis of macromolecules. The theory can be tested by measuring accuracy in germ line and somatic cells and also by comparing somatic cells from mammals with different longevities. Notably, the HO gene is evolutionarily different in birds and mammals, with the biliverdin reductase–bilirubin step present in the latter but absent in the former.. Consistently, the organism sacrifices the potential for indefinite survival in favor of earlier and more prolific fecundity. From an evolutionary perspective, aging is a nonadaptive phenomenon, since it limits the reproductive potential of an individual. For this reason aging should be opposed by natural selection, and hence the argument that it evolved to provide offspring with living space is now receiving little credence. A clear prediction is that the actual mechanisms of senescence are stochastic, involving most likely processes such as random accumulation of somatic mutations or oxidative damage to macromolecules. In the words of an anonymous poet, ‘‘we are born as copies, but we die as originals.’’ It is becoming increasingly clear that genetic factors are prominently involved in aging, the major lines of empirical evidence being: (1) the life span which in human populations shows significant heritability; (2) different species have different intrinsic life spans due to genomic differences; (3) human populations possess inherited progeroid disorders, such as Werner’s syndrome, a disease characterized by premature age‐related disorders, including atherosclerosis, type II diabetes, osteoporosis, and cancers; and (4) clear evidence of genetic effects on life span have been demonstrated in invertebrate model systems, such as D. melanogaster and C. elegans. In this organism, five different genomic regions appear to be associated with longevity, as assessed by quantitative genetic analysis (Rothschild and Jazwinski, 1988). Also, in S. cerevisiae 13 longevity genes have been identified and cloned. Of these 13 genes, 11 have human homologues (Rothschild and Jazwinski, 1988). At least, three categories of genes are predicted to affect aging and longevity. They are: (1) genes that regulate levels of somatic maintenance and repair; (2) pleiotropic genes, whose expression involves trade‐offs between early‐life fitness benefits and late‐life fitness disadvantages, which do not encompass somatic maintenance; and (3) late‐acting deleterious mutations that have escaped elimination as consequence of the decline in the force of natural selection at old ages (Calabrese et al., 2001a). Efficient functioning of maintenance and repair process seems to be crucial for both survival and physical quality of life. This complex network of the so‐called longevity assurance processes is composed of several genes, termed vitagenes (> Table 6‐2). The homeodynamic property of living systems is a function of such a vitagene network. Because aging is characterized by the failure of homeodynamics, a decreased efficiency and accuracy of the vitagene network can influence gerontogenic processes. It is not clear how various components of the vitagene network operate and influence each other in a concordant or a discordant manner. Since aging is characterized by a progressive failure of maintenance and repair, it is reasoned that genes involved in homeodynamic repair pathways, such as the HO‐1 or Hsp70 genes, are the most likely candidate vitagenes.

7

HO‐1 and Hsp70 as a Therapeutic Funnel

A promising approach for the identification of critical vitagene‐related processes is represented by the hormesis‐like positive effect of stress, including regular muscle exercise (Butterfield et al., 2002a, b; Calabrese et al., 2004b) caloric restriction, which can result in activation of the Hsp signal pathway and, consequently, in stress tolerance. In particular, there is strong evidence that the HO/CO and biliverdin– bilirubin redox system might work critically as a ‘‘therapeutic funnel’’ in a number of physiopathological situations where the sensing of redox‐active events is coupled to acquiring major resistance to the effects of stressful and pathogenic conditions (> Figure 6‐1). HO‐1 activity seems to be required for the action of several other therapeutic molecules. In each case, the expression of HO‐1 or administration of one of its metabolic products substitutes for the actions of the other protective molecule (Otterbein et al., 2003a).

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. Figure 6‐1 Redox regulation of gene expression involving the vitagene system. Proposed role for the vitagene member heat‐shock proteins (Hsps) in modulating cellular redox state and cell stress tolerance. Various proteotoxic (or genotoxic) conditions cause depletion of free Hsps that lead to activation of stress kinase and proinflammatory‐ and apoptotic signaling pathways. Hsp70 prevents stress‐induced apoptosis by interfering with the SAPK/JNK signaling and by blocking caspase proteolytic cascade. Nitrosative‐dependent thiol depletion triggers HO‐1 induction, and increased HO‐1 activity is translated into augmented production of carbon monoxide (CO) and the antioxidant bilirubin. These molecules may counteract increased NOS activity and NO‐mediated cytotoxicity. In addition, HO‐1 may directly decrease NO synthase protein levels by degrading the cofactor heme. Abbreviations: PLA2, phospholipase A2; IL‐6, interleukin‐6; AP‐1, activator protein‐1; SAPK, stress‐activated protein kinase; JNK, c‐jun N‐terminal kinase; NF‐kB, nuclear factor kappa‐B; GSNO: S‐nitrosoglutathione; HO‐1, heme oxygenase‐1

In many inflammatory situations, the ability of IL‐10 to suppress TNF‐a expression in macrophages requires the presence of HO‐1 and the generation of CO; HO‐1 expression or CO administration has the same effects as IL‐10 (Lee and Chau, 2002; Soares et al., 2004). In concert with this conceivable possibility, the protective effect of IL‐10 in a lethal endotoxic shock mice model is strongly dependent on the expression of HO‐1 and the generation of CO (Lee and Chau, 2002; Soares et al., 2004). Moreover, rapamycin appears not to exert its antiproliferative effects on smooth muscle cells unless HO‐1 is present (Lee and Chau, 2002; Akamatsu et al., 2004), and it has been proven that, in order for NO to protect mice livers from hepatitis induced by TNF‐a and galactosamine, upregulation of HO‐1 seems to be essential (Otterbein et al., 2003b). Also, alcohol has antiinflammatory effects as TNF‐a is suppressed and IL‐10 is increased (Otterbein et al., 2003b; Yamashita et al., 2004). However, protection is lost when HO‐1 is blocked (Foresti et al., 1997). In addition, the antiinflammatory effect of 15‐deoxy‐D‐12,14‐prostaglandin J2 has been shown to require the activity of HO‐1 (Lee and Chau, 2002; Otterbein et al., 2003b). Notably, during heat shock, which leads to upregulation of several Hsps endowed with cytoprotective actions, entire cytoprotection is lost if HO‐1 is blocked with SnPPIX. Last, relevant to brain physiopathology, dietary and medicinal phytochemicals that can inhibit, retard, or reverse the multistage pathogenic events associated with degenerative damage,

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particularly polyphenols such as curcumin, caffeic acid, and ferulic acid, all capable of exerting powerful antiinflammatory actions, have been shown to function by upregulating HO‐1 (Scapagnini et al., 2002d, 2004; Poon et al., 2004a). The fact that in all these situations specific molecules or biological phenomena appear to lose most, if not all, of their effects when HO‐1 is absent represents a compelling evidence that the HO‐1 system may represent a final common mediator of many biological events associated to cell stress response and, as such, working as a critical vitagene, which links redox‐dependent pathways of stress tolerance to a versatile biological program of cell life.

8

Conclusion

Modulation of endogenous cellular defense mechanisms via the stress response signaling represents an innovative approach to therapeutic intervention in diseases causing tissue damage, such as neurodegeneration (Calabrese et al., 2005; Sultana et al., 2005). Efficient functioning of maintenance and repair processes seems to be crucial for both survival and physical quality of life. This is accomplished by a complex network of the so‐called longevity assurance processes, which are composed of several genes termed vitagenes. Consistently, by maintaining or recovering the activity of vitagenes it can be possible to delay the aging process and decrease the occurrence of age‐related diseases with resulting prolongation of a healthy life span. As one of the most important neurodegenerative disorders, AD is a progressive disorder with cognitive and memory decline, speech loss, personality changes, and synapse loss. With the increasingly aging population of the United States, the number of AD patients is predicted to reach 14 million in the mid‐twent‐first century in the absence of effective interventions (Butterfield et al., 2002a). This will pose an immense economic and personal burden on the people of this country. Similar considerations apply worldwide, except in sub‐Saharan Africa where HIV infection rates seem to be leading to decreased incidence of AD (Butterfield et al., 2002b). There is now strong evidence to suggest that factors such as oxidative stress and disturbed protein metabolism and their interaction in a vicious cycle are central to AD pathogenesis. Brain‐accessible antioxidants, potentially, may provide the means of implementing this therapeutic strategy of delaying the onset of AD, and more in general all degenerative diseases associated with oxidative stress. As one potentially successful approach, potentiation of endogenous secondary antioxidants systems can be achieved by interventions which target the HO‐1/CO and/or Hsp70 systems. In this chapter, the importance of the stress response signaling and, in particular, the central role of HO‐1 together with the redox‐dependent mechanisms involved in cytoprotection are outlined. The beneficial effects of HO‐1 induction result from heme degradation and cytoprotective regulatory functions of biliverdin/bilirubin redox cycling. Thus, HO‐1 can amplify intracellular cytoprotective mechanisms against a variety of insults. Consequently, induction of HO‐ 1 by increasing CO and/or biliverdin availability can be of clinical relevance. CO has been studied for >100 years and, until the last few years, has been touted as a molecule to avoid, owing to its toxic effects exerted mostly on hemoglobin and cytochrome oxidase functions (Otterbein, 2002). However, these toxic effects are seen at concentrations of CO well above concentrations used experimentally. Beneficial effects are obtained with relative low doses of CO (250 ppm for one to few hours) in rodents (Otterbein et al., 2003b). Carboxyhemoglobin levels generated in such a model are not too high from those of heavy smokers. If this beneficial effect is confirmed also in humans, limited exposure of patients to CO might be considered as therapy for various syndromes, particularly to prevent re‐stenosis after angioplasty or treatment of an organ donor and/or the organ to suppress ischemia–reperfusion injury and to prolong allograft survival. Very importantly, HO‐1 and CO can suppress the development of atherosclerotic lesions associated with chronic rejection of transplanted organs (Otterbein et al., 2003b; Akamatsu et al., 2004). Interestingly, the recently discovered CORMS appears to afford similar protective action, thereby providing an alternative therapeutic approach for those pathophysiological conditions where CO administration may prove to be beneficial (Motterlini et al., 2002b, 2003). Furthermore, administration of biliverdin or bilirubin after the first few weeks of life is proven not to have toxicity and doses as much as 2.5 mg/dl used in experimental paradigms are only slightly above normal levels, yet endowed with cytoprotective effects (Yamashita et al., 2004). Although clinical application of the HO

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6

system should be fully considered, a better understanding of how HO mediates its action will guide therapeutic strategies to enhance or suppress HO effects. Remarkably, the recent envisioned role of Hsp70 as a vehicle for intracytoplasmic and intranuclear delivery of fusion proteins or DNA to modulate gene expression (Wheeler et al., 2003), along with the evidence that binding of HO protein to HO‐1 DNA modifies HO expression via nonenzymatic signaling events (Weng et al., 2003) associated to CO and P‐38‐ dependent induction of Hsp70, opens intriguing perspectives, as it is possible to speculate that synergy between these two systems might impact cell proliferation and apoptotic processes during oxidative stress, hence contributing to programmed cell life or programmed cell death (> Figure 6‐1), depending on the relative extent of activation. Presented here is strong evidence that a cross talk between stress response genes is critical for cell stress tolerance, highlighting a compelling reason for a renewed effort to understand the central role of this most extraordinary defense system in biology and medicine. All of the above evidence supports also the notion that stimulation of various maintenance and repair pathways through exogenous intervention, such as mild stress or nutritional compounds targeting the heat‐shock signal pathway, may have biological significance as a novel approach to delay the onset of various age‐associated alterations in cells, tissues, and organisms (Poon et al., 2004a, b). Hence, by maintaining or recovering the activity of vitagenes (Calabrese et al., 2001a, 2004b, 2005) it can be possible to delay the aging process and decrease the occurrence of age‐related diseases with resulting prolongation of a healthy life span.

Acknowledgments This work was supported in part by grants from the Wellcome Trust, MIUR‐Cluster Biomedicine, and FIRB RBNE01ZK8F, and by grants from the National Institute of Health (D.A.B.). The authors acknowledge helpful discussions with John Clark (Institute of Neurology, UCL, London, UK) and with Roberto Motterlini (Northwick Park Institute for Medical Research, Harrow, UK); Enrico Rizzarelli and Eduardo Puleo (Department of Chemistry, University of Catania).

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Parallels Between Neurodevelopment and Neurodegeneration: A Case Study of Alzheimer’s Disease

X. Zhu . G. Casadesus . K. M. Webber . C. S. Atwood . R. L. Bowen . G. Perry . M. A. Smith

1 1.1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 Review Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

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Alzheimer’s Disease‐Associated Proteins: Critical Roles in Both Neurodevelopment and Neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 2.1 Genetic Associations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 2.1.1 Amyloid‐b Protein Precursor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148 2.1.2 Presenilins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 2.1.3 Apolipoprotein E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 3 3.1 3.2 3.3 3.4

Mitotic Alterations in Alzheimer’s Disease: Similarities to Neurogenesis . . . . . . . . . . . . . . . . . . . . 149 Cell‐Cycle Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 Cyclins and Cyclin‐Dependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 A Mitotic Catastrophe in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150 An Early, Not a Late, Change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

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Possible Mitotic Factors: Clues Provided by Gender Difference in Alzheimer’s Disease and Hints on Therapeutic Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4.1 Cell Cycle and Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4.2 Mitogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4.3 Gender Differences and Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 4.3.1 The HPG Axis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 4.3.2 Luteinizing Hormone/Therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 5

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152

2008 Springer ScienceþBusiness Media, LLC.

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Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimer’s disease

Abstract: An accumulating body of evidence shows significant similarities between cellular processes involved in neurodegeneration and those involved in neurodevelopment. Most striking, in Alzheimer’s disease, cell cycle re-entry appears to be a pathological signature of the disease that also occur in brain development. Such inappropriate reactivation of a fetal program is likely to play a key role in both the etiology and pathogenesis of disease. List of Abbreviations: AD, Alzheimer’s disease; ApoE, apolipoprotein E; AβPP, amyloid-β protein precursor; Aβ, amyloid-β; Cdk, cyclin-dependent kinase; HPG axis, hypothalamic-pituitary-gonadal axis; HRT, hormone replacement therapy; LH, luteinizing hormone; PS, presenilin; ROS, reactive oxygen species

1

Introduction

1.1 Review Summary Alzheimer’s disease (AD) is the most common senile dementia in the elderly, which is characterized by three main structural changes in the brain: neurofibrillary tangles, senile plaques, and extensive neuronal degeneration. Quite surprisingly, a number of recent findings demonstrate similarities between neurogenesis during development and neurodegeneration during AD in that neuronal populations that are known to degenerate in AD display phenotypic changes characteristic of dividing cells (Zhu et al., 2004a). Such a notion of a direct relation between neurodevelopment and neurodegeneration is further strengthened by studies of AD‐associated proteins (Selkoe and Kopan, 2003). In this chapter, we highlight the parallels between early neural development and late‐life neurodegeneration and, thereafter, discuss possible therapeutic strategies that this might afford.

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Alzheimer’s Disease‐Associated Proteins: Critical Roles in Both Neurodevelopment and Neurodegeneration

2.1 Genetic Associations Linkage studies show that mutations in at least three genes, the amyloid‐b protein precursor (AbPP) gene located on chromosome 21, and the two homologous genes, presenilin 1 and 2 (PS1 and PS2) located on chromosome 14 and 1, respectively, are associated with early‐onset AD (Hardy, 1997). AbPP is a type 1 membrane protein with a large extracellular domain and a short cytoplasmic domain and belongs to a larger AbPP family that also includes APLP1 and APLP2 (Coulson et al., 2000). Presenilins are polytopic proteins with six to eight transmembrane domains that play a central role in intramembrane proteolysis (g‐cleavage) of a number of cell surface proteins including AbPP, which gives rise to amyloid‐b (Ab) peptide, the major component of senile plaques (Selkoe and Kopan, 2003). The penetrance of the mutations is almost 100%, suggesting that these proteins play critical roles in the neurodegeneration of AD. Unfortunately, however, their mechanistic roles in the pathogenesis of AD are still elusive.

2.1.1 Amyloid‐b Protein Precursor Interestingly, it appears that these AD‐associated proteins also play central roles in early neural development (Bothwell and Giniger, 2000). For example, the overexpression of human AbPP in Drosophila leads to development of blistered‐wing phenotype (Fossgreen et al., 1998). Mice genetically deficient in AbPP, APLP1, or APLP2 are viable with only subtle neurological deficits, suggesting redundancy in function among the AbPP family members (Heber et al., 2000). More recently, mutant mice lacking all three AbPP genes displayed early lethality as well as high incidence of cortical dysplasia

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suggesting a critical role in neural development (Koo, 2002). However, the actual function of AbPP remains unresolved. Interestingly, in vitro studies suggest that AbPP may function as a cell‐cycle regulator because AbPP and its proteolytic fragments (i.e., Ab peptide and AbPP) are mitogenic (Schubert et al., 1989; Milward et al., 1992; Copani et al., 1999; Hoffmann et al., 2000; Schmitz et al., 2002). Further, its adaptor proteins including Fe65 and AbPP‐BP1 have been shown to function as regulators of the cell cycle. AbPP‐BP1 is a cell‐cycle protein that normally negatively regulates the progression of cells into the S phase and positively regulates the progression into mitosis (Chen et al., 2000), whereas Fe65 is a nuclear protein that negatively regulates G1 to S‐phase progression by inhibiting the key S‐phase enzymes (Bruni et al., 2002). Therefore, it is likely that AbPP may act as a cell surface receptor to relay cell cycle‐related signals.

2.1.2 Presenilins The role of PS1 in neurodevelopment is more firmly established at least partially because of its involvement in the g‐cleavage of the Notch receptor, which plays critical roles in determining cell fate during early development (Selkoe and Kopan, 2003). It is shown that the PS1‐deficient mouse displays profound developmental abnormalities, including failed somite formation and altered neurogenesis (Shen et al., 1997; Wong et al., 1997). Additionally, the loop domain of PS1 serves as a scaffold for b‐catenin degradation (Willert and Nusse, 1998; Kang et al., 2002). It is known that b‐catenin can migrate to the nucleus and interact with transcription factors like TCF/LEF‐1 to induce the expression of genes such as Cyclin D1 and c-myc, which are crucial in the G1/S‐phase transition (Willert and Nusse, 1998). In fact, transgenic mice that express a constitutively active b‐catenin in neuroepithelial precursor cells develop enlarged brains because a greater proportion of neural precursors reenter the cell cycle after mitosis (Chenn and Walsh, 2002). Therefore, PS1 may also be involved in cell‐cycle regulation. Indeed, the association of presenilins with centrosome and centromeres suggests that they play a role in cell division and segregation of chromosomes (Li et al., 1997). In addition, it has been shown that the overexpression of both PS1 and PS2 proteins resulted in G1‐phase arrest of the cell cycle (Janicki and Monteiro, 1999; Janicki et al., 2000), whereas PS1 deficiency results in accelerated entry into (Soriano et al., 2001) and significantly prolonged length (Yuasa et al., 2002) of the S phase of the cell cycle. This evidence suggests that PS1 and PS2 play important roles in cell‐cycle control.

2.1.3 Apolipoprotein E Finally, the apolipoprotein E4 (ApoE4) alleles, risk factor of late‐onset AD, which confer increased susceptibility both to AD and prostate cancer, implicate an association between the ApoE4 allele and a propensity toward developing a dysregulated cell cycle (Yuasa et al., 2002). In support of such a notion, ApoE is itself associated with the amyloid deposits found in pituitary adenomas.

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Mitotic Alterations in Alzheimer’s Disease: Similarities to Neurogenesis

3.1 Cell‐Cycle Control The cell cycle is a highly regulated process and it is the sequential expression and activation of cyclin/Cdk complexes, the main regulators of cell‐cycle progression, which orchestrate the orderly transition from one phase to another. Cyclin dependent kinases (Cdks) are controlled through sophisticated mechanisms, including binding to their appropriate cyclins, phosphorylation modifications, and binding to their specific inhibitors. Adult neurons are generally thought of as being postmitotic and terminally differentiated and therefore, it is somewhat surprising that vulnerable neurons in AD have certain phenotypic markers that resemble a cyclin rather than a quiescent nondividing, cell, which highlights the similarities between

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neurogenesis during development and neurodegeneration during AD. Indeed, this may well reflect the fact that AD‐associated proteins are involved in cell‐cycle regulation, which may be disrupted in the pathogenesis or progression of the disease.

3.2 Cyclins and Cyclin‐Dependent Kinases Neurogenesis involves proliferation of the precursor cells and Cdks are crucial regulators of proliferation in the precursor stage. For example, Cdk2 and Cdk4/6 are both critical in cell‐cycle regulation in neural precursor cells (Ferguson et al., 2000). The activities of Cdks are blocked once the neural cells start differentiation and these Cdks are normally absent in differentiated cells. However, various cell‐cycle markers including Cdks are found in susceptible neurons in AD reminiscent of proliferating precursor cells during neurogenesis. Indeed, the upregulation of Cdk7 and reappearance of Cyclin D, Cdk4, and/or Ki67 suggest that the susceptible neurons are no longer quiescent (Smith and Lippa, 1995; McShea et al., 1997; Nagy et al., 1997a, b; Zhu et al., 2000). The expression of Cdk2/Cyclin E complex reflects that neurons may have emerged from phase G1 (Nagy et al., 1997a), whereas the aberrant expression of Cdc2/ Cyclin B1 complex and CARB indicates that the degenerating neurons may have reached phase G2 at least in some cases (Nagy et al., 1997b; Vincent et al., 1997; Zhu et al., 2004b). The reported presence of coordinated DNA replication suggests that the susceptible neurons may complete a nearly full S phase (Yang et al., 2001), and this is supported by the fact that mitochondrial DNA is also increased (Hirai et al., 2001).

3.3 A Mitotic Catastrophe in Alzheimer’s Disease Despite the increasing evidence of reactivation of cell‐cycle machinery in the susceptible neurons in AD, there is, as yet, no evidence suggesting a successful nuclear division or chromosome condensation in AD. This implies that neurons do not complete mitosis, leading us to speculate that there is a ‘‘mitotic catastrophe.’’ Indeed, the highly unorganized nature of the cell‐cycle reentry in AD neurons as evidenced by the concurrent expression and aberrant localization of Cdk4 and p16 (McShea et al., 1997), and the presence of Cyclin E and B1 but absence of Cyclin D and A (Nagy et al., 1997b), point to an inadequate or a failed control of cell cycle in these neurons which may be due to the inherent incompetence of terminally differentiated neurons to complete cell cycle. Therefore, it is very likely that an unscheduled activation of a mitotic division cycle in postmitotic neurons leads to an abortive reactivation of cell‐cycle machinery and the eventual demise of the cell.

3.4 An Early, Not a Late, Change However, this does not necessarily mean that mitotic proteins are exclusively associated with end stage of neuropathology. Rather, it was shown that cell‐cycle markers are associated with the earliest neuronal change to occur in the disease (Nagy et al., 1997a; Busser et al., 1998; Yang et al., 2003). For example, cell‐ cycle markers have been shown in patients with mild cognitive impairment (MCI) (Yang et al., 2003). It has also been shown that they occur prior to the appearance of gross cytopathological changes (Vincent et al., 1998), which suggests that mitotic alterations may lead to those cytopathological changes. For example, the expression, phosphorylation, and metabolism of AbPP, have been shown to be cell cycle‐ related, thus any perturbation of cell cycle, as apparently seen in AD, could lead to misregulated metabolism and expression of AbPP, which may give rise to Ab production and amyloid pathology. Further, increased phosphorylation and altered microtubule stability are coincident during progression through the cell cycle. Therefore, it is not surprising that microtubule abnormities and tau phosphorylation are also associated with AD, because cell‐cycle reentrance is an early feature in AD and candidate tau kinases such as Cdk2, Cdk5, Cdc2, and MAPK that are involved in cell‐cycle control are all increased in

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AD in a topographical manner that overlaps with phospho‐tau. Moreover, free radicals produced during oxidative stress are speculated to be pathologically important in AD and other neurodegenerative diseases (Cross et al., 1987; Perry et al., 1998). Dysregulated cell‐cycle control may also lead to increased cytoplasmic oxidative damage observed in susceptible neurons in AD, as it may adversely affect mitochondria function. It is reported that mitochondrial DNA and protein are increased (Hirai et al., 2001), which may be due to the same mitogenic signal that triggers cell‐cycle reentry and nuclear DNA duplication. However, the incompetence of postmitotic neurons to complete the cell‐division cycle may result in the failure of organelle kinesis after mitochondrial DNA duplication that probably leads to mitochondrial abnormalities. It is clear that dysregulated mitochondria are major sources of ROS for surrounding cytoplasm.

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Possible Mitotic Factors: Clues Provided by Gender Difference in Alzheimer’s Disease and Hints on Therapeutic Considerations

4.1 Cell Cycle and Pathology As discussed earlier, such mitotic alterations are not only some of the earliest neuronal abnormalities in the disease (McShea et al., 1997; Vincent et al., 1998), but would also lead to all of the other pathological changes reported in the disease, including tau phosphorylation, mitochondria dysfunction, and cytoplasmic oxidative damage, alterations in Ab and the ultimate neuronal dysfunction and death (Raina et al., 2000). This strongly suggests that therapeutic interventions targeted toward normalizing these abnormal mitotic changes may result in the slowing or prevention of disease progression.

4.2 Mitogenic Factors In order to study this possibility, one must first examine possible candidates that can stimulate these neurons to reenter the cell cycle. Among these, probable candidates are growth or cellular differentiation factors. In this regard, aberrant and elevated levels of a variety of molecules with potential neurotrophic and mitogenic activities are found in AD, including nerve growth factor (Allen et al., 1991; Crutcher et al., 1993; Connor et al., 1996), luteinizing hormone (LH) (Bowen et al., 2002), epithelial growth factor (Birecree et al., 1988; Styren et al., 1990), transforming growth factor‐b1, platelet‐derived growth factor (Masliah et al., 1995), hepatocyte growth factor/SF (Fenton et al., 1998), insulin‐like growth factor‐I (Connor et al., 1997), insulin‐like growth factor‐II (Tham et al., 1993), and basic fibroblast growth factor (Gomez‐ Pinilla et al., 1990; Stopa et al., 1990). However, although the precise effects of aberrant release of growth factors in AD remains elusive, the gender bias present in AD has provided valuable information in our attempt to determine the identity of this unknown mitogen.

4.3 Gender Differences and Hormones Extensive epidemiological data suggest a gender‐based predisposition that is specific to AD such that higher prevalence (Jorm et al., 1987; Breitner et al., 1988; Rocca et al., 1991; McGonigal et al., 1993) and incidence (Jorm and Jolley, 1998) of AD is present in women. This has led investigators to focus on the roles of the sex steroids, estrogen and to a lesser extent testosterone, in the pathogenesis of the disease resulting in several lines of evidence suggesting that estrogen deficiency following menopause may contribute to the etiology of AD in women. In this regard, a positive correlation has been shown to exist between AD and decreased estrogen levels following menopause (Manly et al., 2000) and earlier studies also demonstrate a decreased incidence (Henderson et al., 1994) and a delay in the onset (Tang et al., 1996) of AD among women on hormone replacement therapy (HRT) following menopause (Kawas et al., 1997). Nevertheless, HRT protection against AD is restricted to administration during a ‘‘critical period’’ that constitutes the

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climacteric years and is ineffective or even detrimental when administered after such time (Rapp et al., 2003) or during the latent preclinical stage of AD, which usually occurs much later in life (Resnick and Henderson, 2002).

4.3.1 The HPG Axis Although falling levels of estrogen might explain the higher incidence of AD in females than in males, a decline in sex steroids does not account for why males with Down’s syndrome have a higher risk of developing AD‐type changes than females, as the levels of sex steroids in both males and females with Down’s syndrome are comparable to those found in the general population (Schupf et al., 1998). This reversal in gender‐based predisposition of AD combined with questions regarding a ‘‘critical period’’ of HRT‐based protection against dementia has prompted us to investigate the potential role of other hypothalamic–pituitary–gonadal (HPG) axis hormones in the etiology of AD (Bowen et al., 2000, 2002; Bowen 2001; Smith et al., 2003), which, along with sex steroids, regulate reproductive function, and also show expression in many nonreproductive tissues including, most notably, the brain. In this regard, previous reports demonstrate a twofold increase in the gonadotropin LH in AD patients when compared to age‐matched control subjects (Bowen et al., 2000; Short et al., 2001). More important, the fact that the highest density of LH receptors in the brain is found within the hippocampus (Lei et al., 1993; Al‐Hader et al., 1997a, b), the most vulnerable region in AD, and gonadotropins are known to cross the blood–brain barrier (Lukacs et al., 1995), we speculate that elevated LH levels may be a driving pathogenic force in mitogenic dysfunction seen in AD (Bowen et al., 2002).

4.3.2 Luteinizing Hormone/Therapeutics As LH is a powerful mitogen (Harris et al., 2002), and given the temporal and spatial overlap with mitotic changes in AD (Bowen et al., 2002; unpublished observations), it is plausible that elevations in LH seen in AD patients are responsible for the inappropriate cell‐cycle reentry in AD‐vulnerable neurons. In support of such a notion, we found significant elevations of LH in AD‐vulnerable neuronal populations when compared to aged‐matched control cases (Bowen et al., 2002). More important, such increases appear to be a very early change in disease history and parallel the ectopic expression of cell‐cycle and oxidative markers that represent one of the initiating pathological changes preceding neuronal degeneration by decades (Nunomura et al., 2001; Ogawa et al., 2003). Therefore, therapeutic strategies that are targeted toward the release of LH may prove successful in treatment of AD. In this regard, it is likely that the ineffectiveness of estrogen replacement therapy in the treatment of AD (Mulnard, 2000; Mulnard et al., 2000) may be due to the incapacity of this hormone to restore the HPG axis to its premenopausal state. Therefore, treatments such as leuprolide acetate—a gonadotropin‐releasing hormone agonist, which has been shown to suppress LH to undetectable levels by downregulating pituitary gonadotropin‐releasing hormone receptors—for mature patients (i.e., postmenopausal women) might be a more effective method for AD. The results of phase III clinical trials of leuprolide acetate for the treatment of AD are anticipated in late 2007.

5

Conclusions

As highlighted earlier, factors such as luteinizing hormone, which drive neuronal proliferation during development, may also play a key role in neuronal degeneration during AD. What irony, that factors used to build the brain are the same that deconstruct the brain after a lifetime of use. We hope that the recognition of this fact will enable efforts to forestall deconstruction and preserve cognitive integrity to the grave.

Parallels between neurodevelopment and neurodegeneration: A case study of Alzheimer’s disease

7

Acknowledgements Work in the authors’ laboratories is supported by the National Institutes of Health and the Alzheimer’s Association. CSA, GP, and MAS are consultants for and own equity in Voyager Pharmaceutical Corporation.

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Differentiation and De‐Differentiation—Neuronal Cell‐Cycle Regulation During Development and Age‐Related Neurodegenerative Disorders

T. Arendt

1 1.1 1.2 1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.3 1.4

The Eukaryotic Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Cyclin‐Dependent Kinases—Key Regulators of the Cell Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160 Regulation of Cyclin‐Dependent Kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Cyclins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Protein Phosphorylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 Cdk Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 Subcellular Sorting and Degradation of Cell‐Cycle Regulating Proteins . . . . . . . . . . . . . . . . . . . . . . . . 165 Regulation of ‘‘Noncell‐Cycle’’ Cdks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166 Effects of Cdk Activation—Cdk Substrates—The Retinoblastoma Protein . . . . . . . . . . . . . . . . . . . . . 166 Quality Control of Cell‐Cycle Progression by ‘‘Stop’’ and ‘‘Go’’ Signals: Restriction Point and Checkpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 1.5 Upstream Control of Cdk Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 1.5.1 Growth Factor Signaling—The Ras‐MAPK Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 1.5.2 Signaling Induced by Cell–Cell and Cell–Matrix Interactions—External ‘‘Positional’’ Cues . . . 170 1.5.3 Convergence of Ras and Integrin‐Dependent Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 2 2.1 2.2 2.3 2.3.1 2.3.2 2.3.3 2.3.4 2.3.5

Cell‐Cycle Regulators During Development and in Postmitotic Neurons . . . . . . . . . . . . . . . . . . . . . 172 Stem Cell Proliferation and Neurogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Do Cell‐Cycle Regulators Have Alternative Functions Unrelated to Cell‐Cycle Regulation? . . . 174 Coupling Cell Cycle to Cell Death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 The Bcl‐2 Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 P53 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180

3 3.1 3.1.1 3.1.2 3.1.3 3.2 3.2.1

Cell‐Cycle Regulators in Neurodegeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 The Link Between Neurodegeneration and Neuroplasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Coherent Hierarchical Pattern of Selective Neuronal Vulnerability and Neuroplasticity . . . . . . . 182 Potential Risks of Dynamic Stabilization of Neuronal Connectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 Aberrant Plasticity in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Cell‐Cycle Regulators Alternatively Execute Neuroplasticity or Cell Death . . . . . . . . . . . . . . . . . . . . . 185 Elevated Mitogenic Force and Activated Mitogenic Signaling in Alzheimer’s Disease . . . . . . . . . 185

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3.2.2 Oxidative Stress and the Autocrine Loop of Self‐Perpetuating Mitogenic Activation . . . . . . . . . . 186 3.2.3 The Replay of Developmental Programs in Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 3.2.4 Cell‐Cycle Activation in Alzheimer’s Disease and Other Neurodegenerative Disorders . . . . . . . . . . . . 190 4

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

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Abstract: The eukaryotic cell cycle is an evolutionary highly conserved process that results in cell division into two daughter cells. Nondividing cells rest outside the cell cycle. The cell cycle consists of an ordered set of events that are tightly regulated by defined temporal and spatial expression, subcellular localization, and degradation of cell cycle regulators. The transition through the cell cycle is controlled by ‘stop’ and ‘go signals’ at defined checkpoints. These checkpoints allow alternate decisions between further progression through the cell cycle and growth arrest, in order to provide potentials for DNA repair or induction of apoptosis. Postmitotic neurons in the CNS are generated from neuroepithelial stem cells, which are multipotent cells that differentiate into progenitor cells of neurons and glial cells. Differentiated neurons are postmitotic cells that have permanently withdrawn from the cell cycle. Currently accumulating evidence, however, shows that differentiated neurons express a number of cell cycle regulators which suggest a role of these proteins beyond cell cycle regulation. Under conditions of neurodegeneration in Alzheimer’s disease, neurons leave their differentiated stage and re-enter the cell cycle. This process which is driven by an abnormal activation of mitogenic pathways eventually results in cell death. Alzheimer’s disease should, thus, be added to the list of proliferative disorders such as cancer, cardiovascular disease, infections and autoimmune diseases, where cell cycle regulators are recognized targets for treatment. Here we attempt to give an overview on the current state of knowledge on the regulation of the cell cycle in neurons under physiological and pathophysiological conditions. List of Abbreviations: AD, Alzheimer’s disease; ATP, adenosine triphosphate; APC, anaphase-promoting complex; APP, amyloid precorsor protein; Akt/PKB, phosphoinositide 3-kinase/protein kinase B; ApoE, apoliporotein E; Abl, v-abl Abelson murine leukemia viral oncogene homolog 1 [Homo sapiens]; Aβ, amyloid-beta protein; ATM, ataxia telangiectasia mutated kinase; ATR, ATM-Rad-3 related kinase; Bad, Bcl-2-associated death protein; Bak, Bcl-2 agonist killer 1; Bax, Bcl-2-interactive cell death susceptibility regulator; Bcl-2, B-cell leukemia/lymphoma-2; Bcl-x, B-cell lymphoma 2-associated protein X; bFGF, basic fibroblast growth factor; Bid, BH3 interacting domain death agonist; Bim, Bcl-2-interacting mediator of cell death; BH, Bcl-2 homology; Bmf, Bcl-2 modifying factor; Bok, Bcl-2 related ovarian killer protein; CAK, cdk-activating kinase; Cdc, cell-division-control; Cdk, Cyclin dependent kinase; C/EBP, CCAAT/enhancerbinding protein transcription factor; Cip/Kip family, Cdk interacting protein/kinase inhibitory protein (Cip/Kip) family; c-myc, avian virus inducing Myelocytomatosis, c = cellular; CNS, central nervous system; DA, Dalton; Dab, Drosophila disabled homolog; DNA, deoxyribonucleic acid; DP5/Hrk, death protein5/ harakiri; ECM, extracellular matrix; E2F, transcription factor; EGF, epidermal growth factor; EGL, external germinal layer; ELISA, enzyme linked immunosorbent assay; Elk-1, member of ETS oncogene family (ETS erythroblast transformation specific domain); ERK, extracellular signal-regulated kinase; ERK1, Mitogenactivated protein kinase p44 extracellular signal-regulated kinase; ERK2, Mitogen-activated protein kinase p42 extracellular signal-regulated kinase; Ets-2, v-ets erythroblastosis virus E26 oncogene homolog 2 (avian); FAK, focal adhesion kinase; FKBP12, FK506-binding protein; FTD, Fronto-temporal dementia17 (FTD linked to 17q21); FAS, tumor necrosis factor ligand family, apoptose related molecule (CD95 or APO-1), encodes region of fatty acid synthase activity; FAS; multifunctional protein; GAP-43, growthassociated protein 43; GAPs, GTPase-activating proteins; G0, G1, G2, gap phase 0, 1, 2; GDP, guanine diphosphate; GEFs, guanine nucleotide exchange factors; GSK3β, glycogen synthase kinase 3-beta; GTP, guanine triphosphate; HCMV, human cytomegalovirus; HGF/SF, hepatocyte growth factor/scatter factor; HIV, human immunodeficiency virus; H3, histone 3; HP1, heterochromatin protein 1; HPV, human papilloma virus; HSV, herpes simplex virus; IL, interleukin; IGF-1, IGF-2, insulin-like growth factor-1, -2; INK4, inhibitors of cyclin-dependent kinase 4; JNK, c-Jun NH2-terminal kinase; Ki-67, cell-cycle related nuclear protein; LEF/TCF, lymphoid enhancer factor/T-cell factor; LTP, long term potentiation; M, mitosis; MAPK, mitogen activated protein kinase; MARCKS, myristoylated alanine-rich C-kinase substrate; MCI, Mild Cognitive Impairment; Myt1, myelin transcription factor 1; MEK, Map kinase extracellular signalregulated kinase; mTOR/FRAP, mammalian target of rapamycin/FKBP12 rapamycin associated protein; Mcl-1, myeloid cell leukemia sequence 1, antiapoptotic protein (Bcl-2 homologue); NCAM, neural cell adhesion molecule; NMDA receptor, N-methyl-D-aspartate receptor; NR2A, subunit of NMDA receptor; NO, nitric oxide; NOS, NO synthase; nNOS, Type I or neuronal isoform of NOS; iNOS, Type II or inducible isoform of NOS; eNOS, Type III or endothelial isoform of NOS; NGF, nerve growth factor; NF-kB, nuclear

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factor kappa-B, subunit 1; OA, okadaic acid; PCNA, proliferating cell nuclear antigen; p53, tumor protein p53; PHFs, paired helical filaments; pRb, phospho-retinoblastoma protein; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; PP1, protein phosphatase 1; PHAS-1, Phosphorylated heat- and acidstable protein 1; PLA-2, phospholipase A2; p70S6k, p70 ribosomal protein S6 kinase; PAK, p21-activated kinase; p75, p75 neurotrophin receptor; PC12, pheochromocytoma cell line; Puma, p53-upregulated modulator of apoptosis; PMAIP1/Noxa, phorbol-12-myristate-13-acetate-induced protein1/BH3-containing mitochondrial protein; PDGF, platelet derived growth factor; R, restriction point of the cell cycle; rhoA, rasrelated protein A; Ras, rat sarcoma virus; Raf, proto-oncogene serine/threonine-protein kinase; Rsk, ribosomal protein S6 kinase; S, DNA synthesis phase of cell cycle; SAA, serum amyloid; SSeCKS, Srcsuppressed protein kinase C substrate with metastasis suppressor activity; Skp2, S-phase kinase-associated protein 2 (p45); SUV39H1, Histone methyltransferase Suv39h1; SOS, son of sevenless protein; SMADs, Mothers against decapentaplegic homolog (vertebrates) (derived from MAD [Mothers against dpp CG12399-PA in Drosophila melanogaster] and SMA [in C. elegans]; STAT3, signal transducers and activators of transcription; SOD1G37R, Superoxide dismutase 1 (transgenic mice overexpressing mutant); Spike, small protein with inherent killing effect; Src, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) [Homo sapiens]; TGF, transforming growth factor; t-TGase, tissue-transglutaminase; TNF, tumor necrosis factor; TUNEL, TdT-mediated dUTP-biotin nick end labeling (TdT = terminal deoxynucleotidyl transferase); TrkA, tropomyosin receptor kinase A; Wnt, Wnt oncogene analog, “wingless” type

1

The Eukaryotic Cell Cycle

The cell cycle is an evolutionary, conserved, and ordered set of events resulting in the cell dividing into two daughter cells. Nondividing cells rest outside the cell cycle in G0. They account for the major part of nonproliferating cells. The cell cycle consists of two major consecutive events, basically characterized by DNA replication and segregation of replicated chromosomes into two separate cells. Corresponding phases of the cell cycle are the synthesis (S) phase in which DNA replication occurs and the mitosis (M) phase in which the cell undergoes division into two daughter cells. G0 and S are separated by gap1 (G1) phase in which cells increase in size and produce RNA and protein to get ready for replication, whereas S and M are separated by gap2 (G2) phase in which cells continue to grow and produce new protein for subsequent cell division (> Figure 8-1). The transition from one phase to another and, thus, the progression of the cell cycle is tightly regulated by defined temporal and spatial expression, subcellular localization, and degradation of cell cycle regulators.

1.1 Cyclin‐Dependent Kinases—Key Regulators of the Cell Cycle Key regulators of the cell cycle are the cyclin‐dependent kinases (Cdks), a family of small, serine/threonine protein kinases (30–35 kDa), whose members share greater than 40% identity. With the exception of Cdk3 and Cdk5, they are activated by a cyclin regulatory subunit (Morgan, 1997; Pavletich, 1999; Dhavan and Tsai, 2001; Knockaert et al., 2002). Cdks are numbered in order of their discovery. In dividing cells, Cdks regulate proliferation, differentiation, senescence, and apoptosis. A critical role in cell‐cycle regulation has been demonstrated for Cdk1, Cdk2, Cdk4, Cdk6, and Cdk7, whereas other members of the Cdk family subserve functions such as transcription (Cdk2, Cdk7, Cdk8, Cdk9, Cdk11), neurite outgrowth, neuron migration, neurotransmitter signaling (Cdk4, Cdk5, Cdk11), differentiation (Cdk2, Cdk5, Cdk6, Cdk9), and cell death (Cdk1, Cdk2, Cdk4, Cdk5, Cdk6, Cdk11) (Knockaert et al., 2002) (> Table 8-1). The orderly progression through the G1, S, G2, and M phases of the cell division cycle is driven by the sequential activation of Cdks (> Figure 8-2), which is controlled at multiple levels (1) by the accumulation of cyclins as positive regulators, (2) at the assembly level into a cyclin–Cdk complex, (3) by phosphorylation, and (4) by their association with inhibitory proteins, the cyclin‐dependent kinase inhibitors that can either block activation or block substrate/ATP access (Evans et al., 1983; Sherr, 1993, 1994; Heichman and

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. Figure 8-1 The phases of the cell cycle. The site of action of regulatory CDK/cyclin complexes is indicated

Roberts, 1994; Hunter and Pines, 1994; King et al., 1994; Sherr and Roberts, 1995; Poon et al., 1995; Rivard et al., 1996; Coats et al., 1996; Morgan, 1997; Pavletich, 1999).

1.2 Regulation of Cyclin‐Dependent Kinases 1.2.1 Cyclins The initial segment of the cell cycle, the first gap (G1) phase, is the site of integration of mitotic signals that result from ligand‐dependent activation of both growth factor receptors and integrins (Miyamoto et al., 1996; Assoian, 1997; Giancotti, 1997; Lin et al., 1997; Renshaw et al., 1997; Moro et al., 1998; Roovers et al., 1999; Assoian and Schwartz, 2001; Danen and Yamada, 2001), which converge on the activation of G1‐cyclin‐dependent kinases (Cdks) Cdk4/6 and Cdk2 (Sherr 1993, 1994). The resultant Cdk activities determine whether mitogenic signals are propagated downstream inducing phosphorylation of key substrates required for progression through G1 and entry into S‐phase. The major function of G1‐phase kinases is to partially phosphorylate the retinoblastoma protein (pRb), thereby contributing to the inactivation of its repressive function. Consequently, the E2F transcription factor can initiate transcription of cyclin E. Second, the cyclin D family sequesters the Cdk inhibitor p27Kip1 from the previously inactive cyclinE/Cdk2 complex (> Figure 8-3).

161

cyclinA cyclinE

cyclinE cyclin D

p35, p25 P39, p29

cyclin D

cyclin H

cyclin C

cyclinK cyclinT

cyclin L

CDK2

CDK3 CDK4

CDK5

CDK6

CDK7

CDK8

CDK9

CDK11

regulation of CDK7‐cyclin H

Cdk1, Cdk2 activation

G1phase

G1/S transition G1phase

Cell cycle prophase to metaphase transition regulation of topoisomerase 2 G1/S transition S phase G2 phase centrosome duplication

Modified after Knockaert et al. (2002)

cyclinB

MyoD‐mediated myocyte differentiation

lens cell differentiation myogenesis

Cell differentiation

RNA processing or transcription

basal transcription basal transcription RNA transcription

regulation of Sp1‐ mediated transcription

Transcription

dopamine and glutamate signaling

neurite outgrowth neurone migration induction of acetylcholine receptors dopamine and glutamate signaling neurotransmitter release

Specific neuronal functions

apoptosis

neuronal cell death

excitotoxin‐ induced neuronal death apoptosis

Cell death ß‐amyloid‐ induced cytotoxicity apoptosis in thymocytes, mesangial cells DNA damage‐ induced apoptosis

signal transduction

Golgi membrane traffic Insulin exocytosis by ß‐cells phototransduction

Other functions

8

CDK1

. Table 8-1 Functional implications of cyclin‐dependent kinases and associated cyclins

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. Figure 8-2 In proliferating cells, the temporal association of Cdks to cyclins is tightly regulated to control the progression of the cell cycle (modified after Nguyen et al., 2002)

When quiescent cells enter the G1 phase, genes encoding D‐type cyclins (D1, D2, and D3) are induced in response to mitogenic signals in conjunction with the appropriate external stimuli such as cell adhesion. During progression through G1, the level of D‐type cyclins increases, and, in a mitogen‐regulated manner, these proteins associate with, and activate their catalytic partners, Cdk4 or Cdk6 (Sherr, 1993; Matsushime et al., 1994; Vidal and Koff, 2000). The major function of D‐type cyclins is to provide a link between the mitogenic stimulation and the autonomously progressing cell‐cycle machinery. Unlike the other cyclins, cyclin D is not expressed periodically, but is synthesized as long as mitogenic stimulation persists (Assoian and Zhu, 1997) and, thus, is usually absent from the cell cycle that progresses independent of the presence of mitogenic signals. Conversely, constitutive activation of cyclin D can contribute to oncogenic cell transformation. Cyclin D1 mRNA translation can be inhibited by rapamycin, a mTOR/FRAP (target of rapamycin/FkBP12 rapamycin‐associated‐protein) inhibitor. In contact inhibited cells, cyclin D is sequestered to the cytoplasm by binding to SSeCKS/gravin protein, a major PKC substrate with tumor suppressor activity (Lin et al., 2000). The first wave of cyclin D‐dependent kinase activity is followed in late G1 by an increase in cyclin E, which associates with Cdk2 (Dulic et al., 1992; Koff et al., 1992). The Cdk2/cyclin E complex is not only responsible for the G1/S transition but also regulates centrosome duplication. Unlike the D‐type cyclin‐ dependent kinases, assembly of cyclin E and Cdk2 into active kinase is not mitogen‐dependent. Cdk2 reinforces Cdk4 to complete Rb phosphorylation and induces the degradation of p27Kip1. The cell cycle is now irreversibly committed to enter the S phase. CyclinE/Cdk2 phosphorylates a variety of substrates, thereby contributing to relieve the Rb‐mediated repression. Cyclin E targets components necessary for DNA synthesis, contributing to the assembly of the DNA replication complexes. Cyclin E, like cyclin D, is targeted for degradation by phosphorylation. The active cyclin E–Cdk2 complex itself phosphorylates cyclin E, which is subsequently recognized by the ubiquitin ligase SCF (Skp2) and targeted for degradation. Proteolysis of cyclin E marks the transition into the S phase (Won and Reed, 1996). Assembly of cyclin A with Cdk2 is required during the S phase. The Cdk2/cyclin A complex phosphorylates various substrates allowing DNA replication. At the S/G2 transition, cyclin A associates with Cdk1. In late G2 phase, cyclin B appears, complexes with Cdk1, and triggers the G2/M transition, where cyclin A is degraded (Peters, 1998). This resets the system and reestablishes the requirement for mitogenic cues to induce D‐type cyclins for the next cycle. Taken together, cyclins can influence when and where the Cdks are active. They are more than a conformational on/off switch for Cdk activity and influence Cdk activity by mediating critical interactions between cyclin/Cdk complexes and cellular proteins such as substrates, inhibitors, and activators.

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. Figure 8-3 The cell division cycle and its major regulatory elements

1.2.2 Protein Phosphorylation In addition to cyclin binding, activity of Cdks is regulated by phosphorylation. Phosphorylation/dephosphorylation events prime Cdks for activation of regulatory subunits. Inhibitory phosphorylation on N‐terminal threonine and tyrosine residues of Cdk maintains kinase complexes in an inactive state. Late in S phase, the dual specific kinases Wee 1 and Myt 1 phosphorylate Cdk1 on Thr14 and Tyr15, thereby inactivating the kinase. Positive regulation of Cdk activity occurs in the following two steps: dephosphorylation of the threonine and tyrosine residues by the Cdc25 phosphatase and phosphorylation

Neuronal cell‐cycle regulation during development and age‐related neurodegenerative disorders

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by the Cdk7–cyclin H complex, also called Cdk‐activating kinase (CAK) (Lew and Kornbluth, 1996). These phosphorylations induce conformational changes and further enhance binding of cyclins (Jeffrey et al., 1995).

1.2.3 Cdk Inhibitors Activity of Cdks is negatively regulated by Cdk‐inhibitors, which bind directly to Cdks or to Cdk/cyclin complexes (Biggs and Kraft, 1995; Cordon‐Cardo, 1995; Poon et al., 1995; Sherr and Roberts, 1995; Boice and Fairman, 1996; Coats et al., 1996; Guan et al., 1996; Rivard et al., 1996). Based on structural features, Cdk inhibitors are grouped into two families, the inhibitors of Cdk4 (INK4) family and the Cip/Kip family. The INK4 family currently consists of four members, p16INK4a, p15INK4b, p18INK4c, and p19INK4d, which commonly share the presence of ankyrin repeats (Serrano et al., 1993; Hannon and Beach, 1994; Guan et al., 1994; Chan et al., 1995; Hirai et al., 1995). The INK4A locus encodes two independent but overlapping genes, p16INK4a and p19ARF, the mouse homolog of p14ARF, which act in partly overlapping pathways (Carnero et al., 2000). All these proteins can inhibit cyclin D associated kinases, Cdk4 and Cdk6. INK4 proteins compete with D‐type cyclins for binding to the Cdk subunit (McConnell et al., 1999; Parry et al., 1999). The inhibition of cyclin binding is mediated by a conformational change in the kinase that prevents association of the cyclins and distorts the ATP binding site (Russo et al., 1998). The INK4‐family of cyclin‐ dependent kinase inhibitors might be involved in the regulation of pathways that control cell growth and proliferation as well as cell death. Deregulation of these Cdki proteins results in either uncontrolled proliferation and neoplastic transformation or activation of apoptosis (Freeman et al., 1994; Kranenburg et al., 1996; Liu et al., 1996a). The inhibitory action of the INK4 proteins is largely dependent on the presence of pRb in the cell. Three proteins currently comprise the following Cdk interacting protein/kinase inhibitory protein (Cip/ Kip) family: p21Cip1, p27Kip1, and p57Kip2 (Gu et al., 1993; Harper et al., 1993; Polyak et al., 1994a, b; Lee et al., 1995a). These proteins share a homologous inhibitory domain, which is necessary for binding to preformed complexes of cyclin‐Cdks, thereby directly inhibiting the kinases. Compared to the INK4‐family, they have a wider inhibiting specificity, affecting the activities of cyclin D‐, E‐, and A‐dependent kinases. In vivo they preferentially act on Cdk2 complexes. Through binding to cyclin D/Cdk4 complexes, p27Kip1 and p21Cip1 can be sequestered without inhibiting the activity of the complex (Sherr and Roberts, 1995). Kip/Cip proteins might even be able to promote activation of cyclin D/Cdk complexes by stabilizing the complexes and directing their nuclear translocation (LaBaer et al., 1997; Cheng et al., 1999). When mitogens are withdrawn and cyclin D is downregulated, this pool of Kip/Cip proteins is released, thereby inducing G1 phase arrest through inhibition of cyclin E/Cdk2. P21cip1 also associates with proliferating cell nuclear antigen (PCNA), an essential binding partner of DNA‐polymerase delta, which might be an additional mechanism through which p21Cip1 can inhibit DNA synthesis (Zhang et al., 1993; Luo et al., 1995). In addition to binding to Cdks, p21Cip1 and p57Kip2 also bind to the PCNA (Gulbis et al., 1996). PCNA is involved in DNA replication and binding of p21Cip1 or p57Kip2, prevents PCNA‐mediated DNA synthesis, and arrests the cell cycle (Watanabe et al., 1998). Cdkis are regulated by both external and internal signals. The expression of p21Cip1 is under the transcriptional control of the p53 tumor suppressor gene (el Deiry et al., 1993). Expression and activation of p15INK4b and p27Kip1 is increased by TGF‐ß, contributing to growth arrest (Reynisdottir et al., 1995). Both p21Cip1 and p27Kip1 undergo phosphorylation, which determines their subcellular location. Recognition of the phosphorylated form of p27Kip1 by the ubiquitin ligase SCF (Skp2) targets p27Kip1 for degradation.

1.2.4 Subcellular Sorting and Degradation of Cell‐Cycle Regulating Proteins Correct cell‐cycle progression is also regulated by the subcellular sorting of cell‐cycle regulating proteins. Cyclin B contains a nuclear exclusion signal and is actively exported from the nucleus until the beginning of

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the prophase. The Cdk inactivating kinases Wee1 and Myt1 are located in the nucleus and Golgi complex and protect the cell from premature mitosis (Liu et al., 1997). Subcellular sorting of different proteins is also regulated through sequestration by binding to the 14‐3‐3 protein (Peng et al., 1997; Yang et al., 1999). Many regulating proteins are subject to rapid degradation during the cell cycle. Proteins are irreversibly destroyed, which permits the orderly transition through the cycle, based on the completion of one phase before entry into the next phase. The specific degradation of many proteins, including Cdks, cyclins, and Cdk inhibitors, is achieved by targeted ubiquitination (Murray, 1995). Critical for the initiation of mitosis and cell‐cycle exit is a specific E3‐type ubiquitin ligase, referred to as the anaphase‐promoting complex (APC) or the cyclosome (Peters et al., 1996). APC selectively targets proteins for ubiquitination that contain a specific consensus sequence, which is found in cyclins and other mitotic proteins (Glotzer et al., 1991).

1.2.5 Regulation of ‘‘Noncell‐Cycle’’ Cdks Regulation of a Cdk that is not related to cell‐cycle progression, such as Cdk5, differs markedly from that of cell‐cycle Cdks (Nguyen et al., 2002). The association of Cdk5 with one of its neuron‐specific coactivators, p35 or p39, is required for neurite outgrowth, axonal migration, cortical lamination, control of cell adhesion, axonal transport, synaptic activity, neuronal adaptive changes, and motor functions (Dhavan and Tsai, 2001). Inhibition of Cdk5 or expression of a dominant negative form of the kinase in cultured neurons prevents neurite outgrowth, whereas overexpression of p35/Cdk5 induces the formation of longer neurites (Xiong et al., 1997; Nikolic et al., 1998). P35‐null mice display a loss of stratified cellular organization (Chae et al., 1997; Kwon and Tsai, 1998), and an even more severe disruption of embryonic cortical layering is observed in mice lacking Cdk5 (Ohshima et al., 1996). The cortical phenotypes of Cdk5‐, p35‐, or p39‐null mice resemble a severe developmental disorder in humans, referred to as type 1 lissencephaly (Reiner, 2000). P35/Cdk5 is also involved in associative learning (Fischer et al., 2002). The role of Cdk5 in plastic neuronal changes involves control of presynaptic function by phosphorylation of Munc18, amphysin, and synapsin I (Dhavan and Tsai, 2001). Cdk5 also regulates neurotransmission and long‐term potentiation by phosphorylating the NR2A subunit of NMDA receptors (Li et al., 2001). The involvement of Cdk5 in neurodegeneration has been suggested by reports of Cdk5 expression during apoptosis and by the presence of the kinase in neurofibrillary tangles in AD and fronto‐temporal dementia‐ 17 (FTD) (Yamaguchi et al., 1996; Pei et al., 1998). An abnormal localization of Cdk5 is also found in canine motor neuron disease, Parkinson’s disease, and amyotrophic lateral sclerosis (Nakamura et al., 1997; Bajaj et al., 1998; Green et al., 1998).

1.3 Effects of Cdk Activation—Cdk Substrates—The Retinoblastoma Protein The activities of G1 Cdk determine whether mitogenic signals are propagated downstream resulting in phosphorylation of key substrates required for progression through G1. One of the key substrates is the retinoblastoma protein (Rb), an inhibitor of progression through G1. Activation of cyclin D‐dependent‐ Cdks initiate Rb phosphorylation in mid‐G1 phase when cyclin E/Cdk2 becomes active and completes this process by phosphorylating Rb on additional sites (Sherr, 1993, 1994, 1996; Weinberg, 1995; Taya, 1997; Lundberg and Weinberg, 1998; Cheng et al., 1999). Cyclin A and B‐dependent Cdks, which are activated later during the cell cycle, maintain Rb in a hyperphosphorylated form (Ludlow et al., 1993). Resetting the pRb–E2F complex occurs in M‐phase by dephosphorylation, probably by PP1 (Durfee et al., 1993; Ludlow et al., 1993). In quiescent cells, Rb is hypophosphorylated, and transcription factors such as E2F and DP bind specifically to this hypophosphorylated form of pRb. Rb–E2F complexes bind to E2F‐binding sites in E2F‐ responsive genes and repress transcription (Kovesdi et al., 1986; Weintraub et al., 1992, 1995; Sherr, 1993, 1994; Weinberg, 1995; Luo et al., 1998; Zhang et al., 1999). Two other proteins, p130 and p107, are

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Rb‐related members of the pRb ‘‘pocket protein’’ gene family. They are also substrates of the G1‐phase Cdks, share structural and biochemical properties, and, like pRb, bind and modulate the activity of E2F transcription factors (Ewen, 1998; Ferreira et al., 1998; Mulligan and Jacks, 1998). P107 and p130 are structurally more closely related to each other than to Rb, and are thought to function redundantly in G0 and G1 to repress E2F‐responsive genes that are distinct from those regulated by Rb. The E2F transcription factor consists of a heterodimer between E2F and DP family proteins. Six members of the E2F family (E2F‐1 to E2F‐6) and three DPs (1, 2, 3) have been identified so far (Nervins, 1998). E2F‐1, E2F‐2, and E2F‐3 are structurally similar and form complexes exclusively with the retinoblastoma protein, whereas E2F‐4 and E2F‐5 interact primarily with p107 and p130. E2F‐1, E2F‐2, and E2F‐3 but not E2F‐4 and E2F‐5 contain cyclin‐binding motifs, which promote stable binding of cyclin A/Cdk2. E2F‐6 lacks both pocket protein‐binding and cyclin‐binding sequences and acts as a transcriptional repressor for E2F‐responsive genes (Morkel et al., 1997). Rb‐mediated repression occurs through at least three mechanisms (Brehm et al., 1998; Magnaghi‐Jaulin et al., 1998; Luo et al., 1998; Swanton, 2004). First, Rb induces modification of histones in the nucleosome by recruiting the histone deacetylase to E2F sites. Histone deacetylation causes tighter association between DNA and nucleosomes and restricts access of the transcriptional apparatus to DNA, thereby repressing transcription. Second, Rb regulates nucleosome structure by recruiting ATP‐dependent remodeling complexes to promoters. Third, Rb influences chromatine structure by recruiting the SUV39H1 methylase and HP1 to the cyclin E promoter leading to histone H3 methylation and silencing of cyclin E. Cdk‐dependent phosphorylation of Rb (pRb) disrupts its association with E2F family members, relieves pRb‐mediated repression, and allows E2F‐dependent transcription of several genes involved in cell cycle and growth regulation. Promoters of many genes have been found to contain consensus E2F binding sites, including enzymes of DNA metabolism, proto‐oncogenes, and cell‐cycle regulatory proteins such as cyclins E and A both of which are required to catalyze the G1/S transition (Sherr, 1994; DeGregori et al., 1995; Schulze et al., 1995; Weinberg, 1995; Dyson, 1998; Nevins, 1998; Black et al., 1999; Cheng et al., 1999). Hypophosphorylated pRb also binds to other proteins, such as members of the Abl family, which seem to be closely implicated in adhesion‐dependent growth control. The E2F‐dependent induction of cyclin E, which in turn stimulates Rb phosphorylation through Cdk2, provides a feedback loop that contributes to the G1/S transition. This event of pRb phosphorylation and release of E2F corresponds to progression through the restriction point R (Pardee, 1974). In addition to regulation of E2F activity through phosphorylation of pocket proteins, direct phosphorylation of E2F also influences its activity. Both cyclin A/Cdk2 and cyclin A/Cdc2 can phosphorylate E2F‐1 on multiple sites (Mudryj et al., 1991; Kitagawa et al., 1995; Adams et al., 1996; Dynlacht et al., 1997), which might contribute to its downregulation in late S/G2 phase. Besides its involvement in cell growth and cell‐ cycle regulation, E2F also plays a role in apoptosis. Similar to growth regulation, E2F‐mediated apoptosis is regulated by pocket proteins (Shan et al., 1996). Other Cdk substrates include histone H1 (Bradbury et al., 1974), DNA polymerase alpha primase (Voitenleitner et al., 1997), their own regulators Wee1 and Cdc25, and cytoskeletal proteins such as lamins, microtubules, and vimentin, which are required for correct mitosis (Hoffmann et al., 1993; Blangy et al., 1995).

1.4 Quality Control of Cell‐Cycle Progression by ‘‘Stop’’ and ‘‘Go’’ Signals: Restriction Point and Checkpoints Although initiation of the cell cycle depends on the presence of external cues, progression beyond G1 is largely regulated by internal control. The restriction point, R (Pardee, 1974, 1989), marks the ‘‘point of no return,’’ where cell‐cycle progression becomes independent of extracellular signals and the cells become irreversibly committed to continue cell‐cycle progression. The orderly sequence of cell‐cycle events resulting in successive waves of Cdk activation and inactivation is controlled further, depending on the completion of previous steps, through the so‐called checkpoint controls (Lew, 2000). DNA damage checkpoints and spindle checkpoints have been identified. This surveillance system is highly conserved from yeast to mammals, and its failure, especially in mammals,

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leads to genomic instability. These checkpoints allow to alternate decisions between further progression through the cycle and growth arrest, in order to provide potentials for DNA repair or induction of apoptosis. Apoptosis as an alternative to cell‐cycle progression is thus, aimed at preventing cell proliferation. In proliferating cells, this might be a protective mechanism against transmission of nonreparable DNA. Depending on the chemical nature of the damage on DNA, different types of repair programs and cellular responses are evoked (Laiho and Latonen, 2003). In highly differentiated cells, such as neurons, this mechanism might be preserved to prevent inappropriate generation of new cells that cannot easily be integrated into functional circuitry (Copani et al., 2001; Liu and Greene, 2001). Thus, an apoptotic response might be inherent to an inappropriate activation of the cell cycle (Evan et al., 1995; Evan and Littlewood, 1998). DNA quality controls, i.e., damage checkpoints, are positioned before DNA replication, at the G1‐S transition, and prior to mitosis, at the G2‐M transition. Further checkpoints are positioned during the S and M phase. Cell‐cycle arrest induced by DNA damage at the G1‐S checkpoint is p53‐dependent. DNA damage activates the product of the ataxia‐teleangectasia gene (ATM/ATR) and DNA‐dependent protein kinase family, which leads to posttranslational stabilization of a normally labile p53 protein (Giaccia and Kastan, 1998) (> Section 2.3.3). S‐phase checkpoints control initiation and elongation phase of DNA replication (Paulovich and Hartwell, 1995). Progression of the mammalian cell cycle requires the activity of Cdc25 family of phosphatases, which dephosphorylate the inhibitory phosphorylations of Cdks and, thus, enable Cdk activities required for S phase and mitosis. A G2 checkpoint prevents entry into mitosis by Cdk1 inhibition through phosphorylation or sequestration outside the nucleus. The G2‐M checkpoint also involves p53, which induces the Cdk inhibitor p21Cip1 as well as a member of the 14‐3‐3 family, which sequesters cyclin B outside the nucleus (Hermeking et al., 1997). In mitosis, the error‐free segregation of sister chromatides is essential for division. In response to unreplicated or damaged DNA, the cell arrests at a DNA structure checkpoint in mitosis. The ‘‘spindle assembly checkpoint’’ induces mitotic arrest in case of improper alignments of chromosomes or defects in microtubule attachment (Amon, 1999). A further checkpoint, dependent on both p53 and pRb, acts subsequent to mitotic errors to block proliferation of cells that have entered G1 with tetraploid status (Borel et al., 2002).

1.5 Upstream Control of Cdk Activation 1.5.1 Growth Factor Signaling—The Ras‐MAPK Pathway Extracellular physiological signals affect the decision to transit the restriction point and this decision is mediated through phosphorylation of pRb. This means, extracellular signals directly control phosphorylation of pRb. Mitogen‐dependent induction of these mechanisms is often mediated through the Ras‐MAP‐ kinase pathway (Filmus et al., 1994; Liu et al., 1995). Small GTPases of the Ras family, which act as molecular switches in intracellular signaling (Bourne et al., 1990), regulate cellular proliferation and mediate the mitogenic response of a variety of growth factors (Mulcahy et al., 1985). In mammals, the Ras superfamily of GTPases contains more than 65 members. According to their function and structure, this superfamily has been subdivided into six families represented by Ras, Rho, Rab, Ran, Rad, and Arf (Barbacid, 1987; Valencia, 1991). In mammals, three genes encode the ras family: H‐ras, K‐ras, and N‐ras. The characteristic of Ras function is its regulated transit between an inactive state, where it is bound to guanine dinucleotides (GDPs), and an active state, bound to guanine trinucleotides (GTPs). The GDP/GTP exchange is regulated by guanine nucleotide exchange factors (GEFs), which catalyze the GDP/GTP exchange, and GTPase‐activating proteins (GAPs), which enhance the intrinsic capacity of Ras to hydrolyze GTP into GDP, thereby returning Ras to the inactive state (Lowy and Willumsem, 1993; Schlessinger, 2000). To be functional, Ras proteins must be recruited to the inner side of the plasma membrane. Upon ligand

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binding, growth factor receptors dimerize, autophosphorylate tyrosine residues in their own cytoplasmic domain, and recruit intracellular signaling molecules to the membrane. Among these recruited molecules are GEFs for Ras protein, which promote binding to GTP (Schlessinger, 2000). Through conformational changes that occur upon the exchange of GDP for GTP, the GTP‐bound form of Ras interacts with Raf, phosphatidylinositol 3‐kinase (PI3K), and RalGdS, driving intracellular signaling cascades (> Figure 8-4). Activated Raf stimulates MAP‐kinase extracellular signal‐regulated kinase (MEK) (Kyriakis et al., 1992), which in turn activates mitogen‐activated protein kinases p44 extracellular signal‐regulated kinase (ERK1) and p42 (ERK2) (Gomez and Cohen, 1991). ERKs have a wide spectrum of substrates including cytoplasmic proteins such as SOS, MEK, Rsk, PHAS‐1, or PLA2, and nuclear proteins that are phosphorylated upon ERK translocation to the nucleus, mainly transcription factors such as Elk‐1, Ets‐2, C/EBP, and SMADs (Whitmarsh and Davis, 1996). The main substrates of PI3K are Akt/PKB and p70 ribosomal protein S6 kinase (p70S6K) (Downward, 1998). Activation of Akt generates an antiapoptotic signal and regulates glycogen synthase kinase 3b (GSK3ß) among other substrates. MAP‐kinase and PI3K pathways activate transcription factors to induce numerous genes, including those of cyclin D1 (Hill and Treisman, 1999). The promoter of the cyclin D1 gene contains mitogen‐ responsive Ets and AP‐1 binding sites (Albanese et al., 1995), known to respond to Ras signaling (Galang and Hauser, 1994). Ras signaling promotes nuclear location of cyclin D1 by negatively regulating GSK3ß, which phosphorylates cyclin D1. This modification redistributes cyclin D1 to the cytoplasmic compartment and labels cyclin D1 for degradation by the ubiquitin‐proteosome (Diehl et al., 1998). Ras also regulates the cyclin D assembly. In quiescent cells, stimulated with growth factors, Ras signaling promotes transition from G0 to G1 and from G1 to S. Proliferating cells make an Ras‐dependent commitment for completion of the next cycle while they are in G2 phase of the preceding cycle (Hitomi and Stacey, 2001). Thus, Ras activity during G2 phase induces cyclin D1 expression, which apparently persists independently of Ras until the beginning of the next S phase (Sa et al., 2002). Ras also controls cell‐cycle progression through control of the expression of other cyclins such as D3, A, and E, and members of the E2F family of transcription factors (Fan and Bertino, 1997) as well as through downregulation of p27Kip1 (Aktas et al., 1997). Mutations conferring constitutive Ras activation are found in nearly 30% of human tumors (Bos, 1989). Oncogenic Ras promotes uncontrolled mitogenesis independently of the presence of growth factors (Feramisco et al., 1984) but is unable to transform cells. Constitutively activated Ras, moreover, not only regulates cellular proliferation, but also renders cells susceptible to apoptosis (Chen and Faller, 1995; Gulbins et al., 1995; Mayo et al., 1997; Serrano et al., 1997; Downward, 1998; Joneson and Bar‐Sagi, 1999; Frame and Balmain, 2000; Liou et al., 2000; Khokhlatchev et al., 2002). Depending on the activation of other signaling pathways, activation of Ras signaling pathways can also arrest the cell cycle rather than activate proliferation (Kohl and Ruley, 1987; Ridley et al., 1988; Marshall, 1995; Serrano et al., 1997; Sewing et al., 1997; Woods et al., 1997). As a result, Ras suppresses oncogenic transformation and induces a condition phenotypically identical to premature cellular senescence (Serrano et al., 1997; Lin et al., 1998; Hahn et al., 1999). It selectively inhibits genes involved in mitosis, DNA replication, segregation, and repair, and at the same time upregulates genes associated with cell death and potentially involved in neurodegenerative disorders and tumorigenesis such as APP, serum amyloid (SAA), and tissue transglutaminase (t‐TGase) (Chang et al., 2000). This process involves the upregulation of the cyclin‐dependent‐kinase inhibitors p15INK4b, p16INK4a, p21Waf1/Cip1, p19ARF, and p53 (Serrano et al., 1997; Olson et al., 1998; Palmero et al., 1998; Ferbeyre et al., 2000; Fogal et al., 2000; Malumbres et al., 2000; Guo et al., 2000a; Pearson et al., 2000; Zhong et al., 2000a, b; Gottifredi and Prives, 2001) and, thus, resembles changes seen in early AD (Ga¨rtner et al., 1995, 1999; Arendt et al., 1996, 1998d, e). Activation of the p21Ras cascade, thus, plays an essential role in oncogenesis and cellular senescence as well as AD (> Section 3.2.1). Cellular effects of Ras activation are strictly cell type dependent and show pleiotropic features, depending on the activity level of Ras signaling (Crespo and Leo´n, 2000). Whereas high levels of Raf activity are able to induce cellular senescence, less intense Raf activation induces cyclin D1 expression and drives cells into proliferation (Woods et al., 1997).

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1.5.2 Signaling Induced by Cell–Cell and Cell–Matrix Interactions—External ‘‘Positional’’ Cues Activation of the cell cycle through growth factors also requires signals by the extracellular matrix. Entry into G1 phase and further progression toward the S phase is a major downstream event of synergistic signaling by mitogenic compounds and integrin‐dependent cell adhesion. All of the important mitogenic signaling cascades downstream of the Ras and Rho family small GTPases and the PI3‐kinase‐PKB/Akt pathway are regulated by integrin mediated cell adhesion (Danen and Yamada, 2001) (> Figure 8-4). They control critical molecular switches such as induction of cyclin D1 resulting in activation of Cdk4/6, the suppression of p21Cip1 and p27Kip1 inducing Cdk2 activity (Fang et al., 1996; Schulze et al., 1996; Stromblad et al., 1996; Zhu et al., 1996; Assoian, 1997; Resnitzky, 1997; Wu and Schonthal, 1997) and the subsequent phosphorylation of Rb (Weinberg, 1995; Sherr and Roberts, 1999). Some of these matrix‐related effects involve matrix‐dependent organization of the cytoskeleton. Adhesion‐dependent cell‐cycle progression involves both integrin binding to the ECM and integrin‐mediated cytoskeletal organization (Hansen et al., 1994; Assoian and Zhu, 1997; Chen et al., 1997). An organized cytoskeleton is, thus, a requirement throughout the mitogen‐dependent portion of the G1‐phase; and the adhesion‐dependent expression of cyclin D and the phosphorylation of pRb are mediated by the cytoskeleton (Bo¨hmer et al., 1996; Assoian and Zhu, 1997). Cell adhesion to extracellular matrix (ECM) is mediated by binding to the cell surface integrin receptors, which activate intracellular signaling cascades and mediate tension‐dependent changes in cell shape and cytoskeletal structure. Although growth control has focused on integrin and growth factor signaling, the cell shape might play an equally critical role in cell‐cycle progression, which acts by subjugating the molecular machinery that regulates the G1/S transition (Huang et al., 1998). Adhesion to substratum has two separate effects on cells both of which could underlie the anchorage requirement for cell‐cycle progression, i.e., the initial adhesion event and the subsequent clustering of occupied integrins. Integrins might act as mechanoreceptors, transmitting mechanical information from the extracellular matrix to the cytoskeleton (Wang et al., 1993; Hansen et al., 1994). Alternatively, an organized cytoskeleton might be required to force integrins to remain clustered at focal contacts, and integrin clustering seems to be a prerequisite for integrin signaling.

1.5.3 Convergence of Ras and Integrin‐Dependent Signaling Signaling through Ras and integrin regulates cyclin D/Cdk4 and cylin E/Cdk2 at different levels. (1) Expression of cyclin D1, often the rate/limiting step in the activation of cylin D/Cdk4/6, critically depends on synergistic . Figure 8-4 Synopsis of current models of information flow (light gray) from cell surface events (dark gray) involved in intercellular communication to gene expression. These pathways mediate structural plasticity and dynamic regulation of intercellular connectivity as well as cell‐cycle activation and cellular proliferation (speckled). One of the major downstream targets is cyclin D1, which is under positive control of b‐catenin/LEF/TCF, RhoA, PAK, JNK, and ERK1/2. RhoA also represses p21Cip1 and p27Kip1, inhibitors of Cdk2. Thus cyclin D/Cdk4/6 and cyclin E/ Cdk2 become active and phosphorylate E2F–pocket protein complexes (E2F–pRb and E2F–p107), allowing for E2F‐dependent expression of cyclin A. This phosphorylation of pRb correlates with cell‐cycle progression out of the growth factor dependent portion of the G1 phase. At multiple levels, these pathways are also potentially involved in t phosphorylation (Cdk5, GSK‐3ß, JNK, ERK1/2) (hatched). (The individual regulatory relationships depicted in this cartoon have been demonstrated in various published reports using different types of cells. These signaling pathways, however, have neither been demonstrated in their entirety within a single experimental system, nor is their action firmly established for neurons; for reference see: Assoian and Zhu, 1977; Ben‐Ze’ev, 1977; Giancotti, 1997; Yamada and Geiger, 1997; Howe et al., 1998; Schlaepfer and Hunter, 1998; Schlaepfer et al., 1999; Schoenwaelder and Burridge, 1999; Schwartz and Baron, 1999; Novak and Dedhar, 1999; Orford et al., 1999; Aplin et al., 1999; Dedhar et al., 1999; Patapoutian and Reichert, 2000; Petit and Thiery, 2000; Dedhar, 2000; Welsh and Assioan, 2000; Assoian and Schwartz, 2001; Baki et al., 2001) Modified after Arendt (2003)

. Figure 8-4 (continued)

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signaling through the Ras/MAPK cascade, integrin, cadherin, and Wnt dependent signaling (Albanese et al., 1995; Lavoie et al., 1996; Miyamoto et al., 1996; Sherr, 1996; Winston et al., 1996; Zhu et al., 1996; Aktas et al., 1997; Kerkhoff and Rapp, 1997; Lin et al., 1997; Renshaw et al., 1997; Weber et al., 1997; Cheng et al., 1998; Huang et al., 1998; Short et al., 1998; Aplin et al., 1999; Gille and Downward, 1999; Roovers et al., 1999; Roovers and Assoian, 2000) (> Figure 8-4). (2) Assembly of newly synthesized cyclin D1 with Cdk4 similarly depends on the Ras/MAPK cascade (Peeper et al., 1997; Cheng et al., 1998). (3) Turnover of D‐type cyclins, moreover, is Ras‐dependent and mediated by the PI3‐kinase‐Akt‐pathway, which regulates the phosphorylation of cyclin D1 by GSK‐3b (Diehl et al., 1998). Inhibition of this pathway leads to cyclin D phosphorylation, enhancing its nuclear export and the ubiquitin‐dependent proteasomal degradation. (4) Cell adhesion is a prerequisite for efficiently downregulating the steady‐state levels of p21Cip1 and p27Kip1 (Fang et al., 1996; Schulze et al., 1996; Zhu et al., 1996; Welsh and Assoian, 2000; Welsh et al., 2001). P27Kip1 was first identified as a Cdk2‐inhibitory factor detected in contact‐inhibited cells (Koff et al., 1993; Polyak et al., 1994a, b) and many factors, including cell–cell contact, induce accumulation of p27Kip1 (Hengst and Reed, 1998). Under these conditions, p19INK4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state (Zindy et al., 1999). Adhesion lowers the levels of p21Cip1 and p27Kip1, thus permitting activation of cyclin E/Cdk2 (Fang et al., 1996; Zhu et al., 1996) whereas high levels of p27Kip1 persist in cells that were prevented from spreading by restriction of the size of the substratum, despite normal MAPK activation (Huang et al., 1998). Key intermediates of signaling of integrin‐dependent cell adhesion upon cell‐cycle regulation are the integrin‐linked kinase (Radeva et al., 1997), which, through inhibition of GSK‐3ß (Delcommenne et al., 1998; Troussard et al., 1999), can also regulate both the expression and degradation of cyclin D1 (Diehl et al., 1998) and Focal adhesion kinase (FAK), which controls cell‐cycle progression through G1 via JNK (Oktay et al., 1999) and through Ras‐ERK1/2 (Chan et al., 1994; Cobb et al., 1994; Schaller et al., 1994; Schlaepfer et al., 1994; Xing et al., 1994; Schlaepfer and Hunter, 1996; Zhao et al., 1998; Sastry et al., 1999; Barberis et al., 2000; Assoian and Schwartz, 2001) (> Figure 8-4). Downstream G1 events, including phosphorylation of pRb leading to release of E2F‐transcription factors from their complex with Rb and cyclin A expression also require both soluble mitogens and cell interaction with the extracellular matrix (Kang and Krauss, 1996; Bo¨hmer et al., 1996; Koyama et al., 1996; Schulze et al., 1996; Zhu et al., 1996; Assoian, 1997; Aplin et al., 1999).

2

Cell‐Cycle Regulators During Development and in Postmitotic Neurons

2.1 Stem Cell Proliferation and Neurogenesis Postmitotic neurons in the CNS are generated from neuroepithelial stem cells, which are multipotent cells that differentiate into progenitor cells of neurons and glial cells. Stem cells are undifferentiated cells capable of self‐renewal and asymmetric divisions to generate both differentiated and undifferentiated cells. The vertebrate CNS develops from the neuronal tube where the positions of the nuclei of stem cells are correlated with the cell‐cycle phases (Sauer, 1935; Fujita, 1960) (> Figure 8-5). Cellular nuclei are located in the outer part of the ventricular zone during S phase, where DNA replication occurs and move toward the ventricular surface in G2 phase, where they complete mitosis. Daughter cells generated by this symmetric division retain the characteristics of stem cells and enter into the G1 phase with their nuclei in the inner part of the ventricular zone, which subsequently move further outward for a new round of DNA replication. During neurogenesis, asymmetric division generates two daughter cells with one of them differentiating into a neuroblast, which is incapable of replication and begins to migrate to the upper marginal zone whereas the other daughter cell reenters the cell cycle as a stem cell. Symmetric divisions of neuronal stem cells, which produce two identical daughter cells that reenter the cell cycle, are characterized by vertically oriented cleavage, whereas asymmetric division generating two daughter cells with different fates is characterized by horizontally oriented cleavage. During neurogenesis, up to 50% of cells in both the ventricular zone and the postmitotic cortical plate are estimated to undergo apoptosis (Raff et al., 1993). Apoptosis in the early stages of neurogenesis may

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. Figure 8-5 Proliferation of stem cells during early stages of neuronal tube development (symmetric) and during neurogenesis (asymmetric). Modified after Yoshikawa (2000)

occur as a result of erroneous DNA replication. In later stages, neuronal apoptosis might be due to limited availability of trophic support or lack of synaptic input from target cells (Oppenheim, 1991). Neurons of particular types arise from cells that divide at particular times. Different neuronal fates, thus, appear to be determined at specific developmental stages or after a certain number of cell divisions. Some fates are determined independently of active cycling, and the mature phenotype of a neuron is often determined even postmitotically. Other fates, however, are determined when a cell is actively proliferating, and the cell‐cycle status might have a critical influence over the determination process. The balance between proliferation and differentiation in the nervous system is, thus, critical for histogenesis, and there might indeed exist bidirectional mechanisms for coordinating the cell cycle with the process of neuronal determination and differentiation (Edlund and Jessell, 1999; Ohnuma et al., 2001). A variety of molecules such as Cdc2, Cdk1, Cdk5, D‐type cyclins, p27Kip1, p57Kip2, or Rb may have complementary but separable roles in regulating the cell cycle and promoting neuronal determination/

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differentiation (Hamel et al., 1992; Okano et al., 1993; Ohnuma et al., 1999; Dyer and Cepko, 2000; Levine et al., 2000) (see also > Table 8-1). In addition, various determination/differentiation factors such as TGF‐a, TGF‐b, EGF, or Notch might regulate cell‐cycle factors through transcriptional or posttranscriptional mechanisms (Dorsky et al., 1997; Hanahan and Weinberg, 2000).

2.2 Do Cell‐Cycle Regulators Have Alternative Functions Unrelated to Cell‐Cycle Regulation? Differentiated neurons are postmitotic cells that have permanently withdrawn from the cell cycle. Most mammalian CNS neurons reach their postmitotic state during the embryonic period when they enter into an ‘‘extended G0‐phase.’’ At this stage, neurons are incapable of dividing and they are also not able to regenerate (Arendt, 2003). However, they express a number of cell‐cycle regulators (Yoshikawa, 2000; Schmetsdorf et al., 2005). Why these genes that potentially promote cell‐cycle progression are expressed in permanent mitotic quiescence remains elusive. From a phylogenetic point of view, the evolution of the cyclin-Cdk‐based engine to control the cell cycle from other, older protein kinases had been suggested (Murray, 2004). It might, thus, be speculated that control of cell cycle is a phylogenetically newly acquired role of these regulators that still might subserve a wider range of cellular functions, such as coordinating tissue remodeling and neuronal plasticity (Nabel, 2002; Arendt, 2003; Schmetsdorf et al., 2005). Functions unrelated to Cdk binding have recently been suggested for some cyclins and Cdk inhibitors (Tannoch et al., 2000; Coqueret, 2003; Schmetsdorf et al., 2005). These additional functions of cell‐cycle regulators rely on different cellular localizations and different targets, present either in the cytoplasm, in the nucleus, and probably also on DNA (Coqueret, 2003). Cip/Kip proteins, for example, might act as assembly‐promoting factors of cyclin D1/Cdk4 complexes, and besides their nuclear inhibitory function, also activate D‐type cyclin complexes through enhanced association and nuclear accumulation. Both p21Cip1 and p27Kip1 might also have anti‐apoptotic pro‐survival effects, which are associated with its relocation to specific cytoplasmic compartments and, in addition, might be required to maintain the differentiated state of certain cells (Bunz et al., 1998 ; Chang et al., 2000; Levine et al., 2000; Blagosklonny, 2002). Further, both cyclin D1 and Cdk inhibitors of the Cip/Kip family such as p21Cip1 and p27Kip1 might act as transcriptional cofactors, thereby controlling the transcriptional activation of various genes in a Cdk‐ independent manner (Zwijsen et al., 1997; McMahon et al., 1999; Bienvenu et al., 2005). p21Cip1, for example, regulates activity of NF‐kB, STAT3, Myc, C/EBP, and E2F (Coqueret and Gascan, 2000; Kitaura et al., 2000; Harris et al., 2001), and it inhibits expression of several genes involved in cell‐cycle progression such as those encoding for DNA polymerase a, topoisomerase II, cyclin B1, and Cdk1 (Chang et al., 2000).

2.3 Coupling Cell Cycle to Cell Death 2.3.1 Apoptosis Apoptosis is a highly conserved mechanism by which eukaryotic cells actively commit suicide. Apoptosis in the nervous system has been recognized as an essential process during development where it plays an essential role in the control of the final number of neurons (Oppenheim, 1991). Apoptosis might also be responsible for the loss of neurons during normal aging (Tagliatella et al., 1996; Kaufmann et al., 2001). Apoptosis involves a series of stereotyped, morphologically well‐defined phases. Its morphological manifestations include nuclear and cytoplasmic condensation, intranucleosomal DNA cleavage, nuclear membrane breakdown and plasma membrane blebbing, and the formation of apoptotic bodies. In proliferating cells, apoptosis acts to prevent transmission of nonrepairable DNA modifications to the progeny. Apoptosis, thus, prevents cell proliferation if it is inappropriate. This role is potentially maintained in postmitotic cells such as differentiated neurons in which unscheduled proliferation would otherwise generate neurons that cannot be easily integrated into functional circuitry. In proliferating cells, it is mostly DNA damage that triggers cell‐cycle arrest, and if DNA damage is too extensive to be repaired, apoptosis.

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During development, ‘‘death is the default’’ (Benn and Woolf, 2004). Immature neurons are ‘‘competent to die’’ (Deshmukh and Johnson, 1998) by an active apoptotic pathway unless it is shut off by sufficient trophic support (Oppenheim, 1991). Thus, apoptosis during development results from the absence of enough trophic factor support or can actively be triggered by signaling through the low‐affinity neurotrophin receptor p75NTR (Carter and Lewin, 1997; Miller and Kaplan, 2001) or the FAS receptor (Raoul et al., 1999, 2000). In mature neurons, two main pathways lead to apoptosis, an ‘‘extrinsic’’ or death receptor‐initiated pathway, and an ‘‘intrinsic’’ or mitochondrial pathway (Green, 1998; Strasser et al., 2000; Benn and Woolf, 2004). Both pathways converge at the mitochondrion on the release of apoptogenic proteins (Shimizu et al., 2001; Rostovtseva et al., 2004; Sharpe et al., 2004). Extrinsic apoptosis is initiated by the activation of plasma membrane ‘‘death receptors’’ of the tumor necrosis factor (TNF) receptor superfamily. The extrinsic death receptor pathway either directly activates effector caspases through JNK or converges with the intrinsic pathway at the mitochondrion. This intrinsic pathway results from mitochondrial damage that leads to the release of cytochrome c from the mitochondria, which triggers the formation of the apoptotic complex and the subsequent activation of effector caspases. Death pathways differ between neurons. The specific death‐signaling pathway that leads to degeneration depends on the neuronal population that is affected, as well on the nature, stage, cause, and extent of the death‐inducing insult. Apoptosis, proliferation, and differentiation are coupled through potentially ‘‘multifunctional’’ upstream signaling pathways. According to the cellular context, activation of identical pathways is translated into such diverse cellular responses as growth arrest, cycling, cell death, or survival (Blagosklonny, 2003). The cell cycle and apoptosis share both identical molecular regulators such as Rb, E2F, and p53 and cellular phenomena such as detachment from the matrix, condensation of chromatin, disassembly of nuclear lamina, and membrane blebbing (Ucker et al., 1991; Raff et al., 1993; Evan et al., 1995; Liu and Green, 2001). Deregulation of the cell cycle can either directly induce apoptosis or can increase the sensitivity to apoptotic inducers (Evan et al., 1995; Evan and Littlewood, 1998). Successful proliferation, as an alternative to apoptosis, requires active suppression of the apoptotic program. The activation of various components of the cell cycle in postmitotic neurons might contribute to apoptosis (King and Cidlowski, 1995). The induction of proteins that control the G1/S phase transition in proliferating cells such as cyclin D1 occurs in dying neurons (Freeman et al., 1994; Kranenburg et al., 1996), and G1 cyclin‐dependent kinases can trigger neuronal death (Park et al., 1997a, b, 1998a). Similarly, proteins that control the G2/M phase transition such as Cdc2 might be involved in apoptosis (Shi et al., 1994; Vincent et al., 1997; Yu et al., 1998). Neuronal activity deprivation in cerebellar granule neurons can stimulate the activity of Cdc2, which triggers apoptosis (Konishi et al., 2002). This process involves phosphorylation of the BH3‐only protein Bad, thereby activating Bad‐mediated apoptosis by inhibiting its sequestration through 14‐3‐3 protein. In neuronal apoptosis, induced in a variety of experimental in vitro paradigms, changes in Cdk activities, levels of cyclins and Cdkis, and deregulation of Rb/E2F activity have been observed (Park et al., 1997a, b, 1998a, b; Padmanabhan et al., 1999; O’Hare et al., 2000) (> Table 8-2). Induction of Cdks, for example, occurs in vivo in mature adult neurons in mouse, rat, or rabbit during ischemia, excitotoxic cell death, spinal cord injury, or various mutant mouse models associated with neuronal death such as mutant SOD1G37R, weaver mouse, harlequin mouse, or staggerer mouse. It is also seen in vitro in neuroblastoma cells or primary neuronal cultures deprived of trophic factor support or treated with DNA damaging agents or ß‐amyloid (Giovanni et al., 1999, 2000; Park et al., 2000). The induction of Cdks in neurons is associated with their de‐differentiation and triggers neuronal death that can be blocked by Cdk inhibitors or dominant negative Cdks (Park et al., 2000; Liu and Greene, 2001). NGF deprivation, for example, leads to increased Cdc2 activity and cyclin B expression in PC12 cells (Gao and Zelenka, 1995), as well as elevated cyclin D1 levels in sympathetic neurons (Freeman et al., 1994) whereas pharmacological inhibitors of Cdks, upregulation of Cdk inhibitors, or dominant negative Cdk4 or Cdk6 promote survival of NGF‐deprived sympathetic neurons (Park et al., 1997a, b, 1998a, b). Apoptosis, induced by DNA damage, requires Cdk4 and Cdk6 activity and subsequent phosphorylation of pRb and activation of the pRb/E2F/DP pathway (Park et al., 2000; Liu and Greene, 2001) and is suppressed by the G1/S transition blockers deferoxamine and mimosine as well as by the Cdk inhibitors flavopiridol and olomoucine

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. Table 8-2 Evidence of cell‐cycle activation under experimental conditions of in vivo and in vitro cell death In Vivo Paradigms of Cell Death Ischemia (transient focal or global ischemia in mouse, rat, or rabbit) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk4 Cdk2 cyclin D1 cyclin B1 cyclin G1 p21Cip1 MPM‐2 PCNA increased phosphorylation of Rb neuroprotection through post/periischemic administration of Cdk inhibitors Combined ischemia/hypoxia increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk2 Ki67 increased phosphorylation of Rb DNA‐synthesis (BrdU incorporation) Kainate‐induced seizures increased neuronal expression of cell‐cycle regulators Cdk4 cyclin D1 increased phosphorylation of Rb Spinal cord injury (rat) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk4 cyclin D1 cyclin G Gadd45a c‐myc PCNA increased phosphorylation Rb, E2F5 Amyotrophic lateral sclerosis caused by mutant SOD1G37R mice increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk4 cyclin D1 increased phosphorylation of Rb Weaver mouse (apoptosis in the external germinal layer (EGL) of the cerebellum) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk4

Guegan et al. (1997) Hayashi et al. (2000) Katchanov et al. (2001) Li et al. (1997a, b) Osuga et al. (2000) Sakurai et al. (2000) Timsit et al. (1999) Tomasevic et al. (1998) van Lookeren Campagne and Gill (1998) Gill (1998) Wang et al. (2002) Wen et al. (2004) Kuan et al. (2004)

Ino and Chiba (2001) Park et al. (2000) Timsit et al. (1999)

Di Giovanni et al. (2003)

Nguyen et al. (2003)

Migheli et al. (1999)

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. Table 8-2 (continued) cclin D cyclin A PCNA harlequin mouse mutation (progressive degeneration of terminally differentiated cerebellar and retinal neurons) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins PCNA Target‐related neuronal death during development (apoptotic staggerer granule cells) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins cyclin D PCNA In Vitro Paradigms of Cell Death Apoptotic cell death induced by: Trophic withdrawal/serum starvation KCl withdrawal DNA damage induced by camptothecin or UV radiation Cisplatin ß‐amyloid (murine Balb/c‐3T3 cells, neuroblastoma cells, PC12 cells, sympathetic neurons, and rat cortical neurons) increased neuronal expression of cell‐cycle regulators/proliferation associated proteins Cdk1 Cdc2 Cdk4 cyclin B cyclin D1 PCNA increased phosphorylation of Rb BrdU incorporation Paradigms of experimental neuroprotection in vitro CDK inhibitor flavopiridol and olomoucine suppress pRb and p107 phosphorylation in paradigms, DNA damage induced by camptothecin suppress death of PC12 cells and sympathetic neurons after trophic factor withdrawal Ectopic expression of p16Ink4a, p21Waf/Cip1, and p27Kip1, as well as DN‐Cdk4 and 6 protect rat sympathetic and cortical neurons against DNA damage and apoptosis induced by camptothecin or UV radiation in vivo intraventricular infusion of Cdk4 or cyclin D1 antisense oligonucleotides suppresses excitotoxin‐ induced neuronal cell death

Klein et al. (2002)

Herrup and Busser (1995)

Freeman et al. (1994) Gao and Zelenka (1995) Giovanni et al. (1999) Gill and Windebank (1998) Kranenburg et al. (1996) Padmanabhan et al. (1999) Pandey and Wang (1995) Park et al. (1998, 2000)

Ino and Chiba (2001) Park et al. (1996, 1998a, b, 2000)

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(Park et al., 1997). Activation of the cyclin D/Cdk4/6‐Rb‐E2F pathway might, thus, be a critical component of the apoptotic mechanism induced by DNA‐damage or trophic factor deprivation in postmitotic neurons (Liu and Greene, 2001; Kruman et al., 2004). 2.3.1.1 Caspases Caspases are the ‘‘executioners’’ of the apoptotic program (Thornberry and Lazebnik, 1998). Caspases stands for cysteine aspartate proteases. They cause cell death by degrading critical structural elements such as lamins and gelsolin, and by activating latent enzymes, such as DNAses, through proteolysis of their inhibitors. Caspases are present in healthy cells as inactive precursors (zymogens) that can be activated by caspase‐mediated cleavage. Proteolysis releases two fragments of about 10 kDa and 20 kDa that are assembled into the active tetrameric enzyme (O’Connor et al., 2000). There are 14 members in the mammalian family of caspases, divided into three subfamilies on the basis of the peptide‐sequence preferences of their substrates. Certain ‘‘initiator’’ caspases, such as caspase‐8 and caspase‐9 have low enzymatic activity in their zymogen form and contain regulatory amino‐terminal prodomains that can bind to specific adapter proteins (Salvesen and Dixit, 1997; Thornberry and Lazebnik, 1998). Caspase‐8 is associated with apoptosis involving death receptors, and loss of adhesion, whereas caspase‐9 is involved in stress‐induced apoptosis (Earnshaw et al., 1999). Stressed cells might receive conflicting signals for cell division and growth arrest, which might trigger apoptosis (Arsura and Sonenshein, 1996; O’Connor et al., 2000). In response to apoptotic stimuli, adapter proteins bind these initiator caspases and form oligomeric aggregates, which facilitate autocatalytic processing of the cymogens (Martin et al., 1998; Srinivasula et al., 1998). Initiator caspases subsequently process ‘‘effector’’ caspases, creating a proteolytic cascade that executes cell death. In addition, there are noncaspase proteases such as cathepsins, calpains, and granzyme B, involved in apoptosis. 2.3.1.2 Anoikis The adhesion‐related form of apoptosis is called anoikis (Frisch and Francis, 1994). It is triggered by inappropriate or inadequate contacts between the cell and the extracellular matrix and essentially displays all the features of apoptosis such as membrane blebbing, nuclear fragmentation, and DNA degradation. Integrin‐mediated cell anchorage, furthermore, has a vital role in the control of apoptosis (Frisch and Ruoslahti, 1997; Aplin et al., 1998). They regulate cell viability through their interaction with the extracellular matrix and can convert cell–matrix interactions into intracellular signals. These mechanisms involve FAK (Frisch et al., 1996b) and Ras‐dependent signaling (Chen and Guan, 1994; Datta et al., 1997; King et al., 1997; Khwaja et al., 1997) as well as the Wnt‐1/ß‐catenin pathway (Frisch and Ruoslahti, 1997; Ku¨hl and Wedlich, 1997). FAK when activated can suppress anoikis. Phosphatidylinositol 3‐kinase/Akt and mitogen‐ activated protein kinase may mediate the anoikis‐suppressing effects. Activation of TrkB, the major receptor for brain‐derived neurotrophic factor, can also suppress anoikis through stimulation of PI3 kinase signaling (Douma et al., 2004). Conversely, the stress‐activated protein kinase/Jun amino‐terminal kinase pathway promotes anoikis (Zhan et al., 2004). Control of anoikis may vary substantially from one cell lineage to another and Ras may fulfill both positive and negative regulatory functions depending on the cell type (Meredith et al., 1993; Frisch and Francis, 1994; Hall et al., 1994; Polakowska et al., 1994; Ruoslahti and Reed, 1994; Coucouvanis and Martin, 1995; Boudreau et al., 1995; Hungerford et al., 1996; Vachon et al., 1996, Assoian, 1997; Frisch and Ruoslahti, 1997; Khwaja et al., 1997; Meredith and Schwartz, 1997; McGill et al., 1997; Eckert et al., 2004). Anoikis may also involve phosphorylation of FAK and p53‐dependent mechanisms (Zhang et al., 2004). The proapoptotic protein Bim, a member of the Bcl‐2‐family, is a critical mediator of anoikis in epithelial cells (Reginato et al., 2003). Detachment‐induced expression of Bim requires a lack of ß1‐integrin engagement, downregulation of EGF‐receptor expression, and inhibition of ERK signaling (Reginato et al., 2003).

2.3.2 The Bcl‐2 Family The B‐cell leukemia/lymphoma‐2 (Bcl‐2) families of proteins are major gatekeepers of apoptosis (Cory and Adams, 2002). They control cell survival through both caspase‐dependent and caspase‐independent

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pathways and can act via regulation of permeabilization of the outer mitochondrial membrane (Cory and Adams, 2002; Akhtar et al., 2004). The Bcl‐2 family consists of three main subgroups, which are categorized according to their anti‐ or proapoptotic function, and the presence or absence of the Bcl‐2 homology (BH) domains (Korsmeyer, 1999). Antiapoptotic members, such as Bcl‐2, Bcl‐xL, Bcl‐w, and myeloid cell leukemia‐1 (Mcl‐1) contain four Bcl‐2‐homology domains. They can also delay entry into the cell cycle (O’Reilly et al., 1996). The proapoptotic members of this family fall into two categories: Bax‐like proteins, such as B‐cell lymphoma 2‐associated protein X (Bax), Bcl‐2 agonist killer 1 (Bak), Bcl‐2‐related ovarian killer protein (Bok), and Bcl‐xS that contain multiple BH domains, or BH3‐only members, such as Bcl2‐associated death protein (Bad), BH3 interacting domain death agonist (Bid), Bcl‐2‐interacting mediator of cell death (Bim), Bcl‐2 modifying factor (Bmf) death protein 5/harakiri (DP5/Hrk), Puma (p53‐upregulated modulator of apoptosis), small protein with inherent killing effect (Spike), and phorbol‐12‐myristate‐13‐acetate‐induced protein (PMAIP1/Noxa) (Puthalakath and Strasser, 2002; Akhtar et al., 2004). Although dimerization among the pro‐ and anti‐apoptotic members of the Bcl‐2 family plays a major role in the regulation of apoptosis, the expression levels of Bcl‐2 determine the cell’s fate. Bcl‐2 family members are involved in neuronal programmed cell death and play a significant role in diseases of the nervous system such as hypoxic‐ischemic cell death, seizure‐induced neurodegeneration, motor neuron disease, and neurotoxic and mechanical neuronal injury (for review see Akhtar et al., 2004).

2.3.3 P53 Another major element in the regulation of apoptosis is p53. Its activation prevents cell proliferation by inducing cell‐cycle arrest at the G1‐S checkpoint or apoptosis through activation of proapoptotic genes such as bax. Although the constitutive level of p53 is low, it can rapidly be induced through DNA damage (Levine, 1997), which stimulates the transcription of regulators such as p21Cip1, mdm2, and bax (Agarwal et al., 1998). P21Cip1 inhibits Cdk activation and results in cell‐cycle arrest, thus, preventing replication of damaged DNA (Ko and Prives, 1996). This function of p53 in shutting down the proliferation in response to DNA damage reflects its role as ‘‘guardian of the genome’’ (Lane, 1992). Mdm2 provides a feedback loop regulating degradation of p53 by ubiquitination (Oren, 1999). In severely damaged cells unable to repair DNA, p53 induces bax, fas, and other genes involved in apoptotic signaling (Miyashita and Reed, 1995; Yin et al., 1997; Gottlieb and Oren, 1998).

2.3.4 Nitric Oxide Nitric oxide (NO) is a signaling free radical, which displays antiadhesive properties (Radomski et al., 1990) and can be considered as a cause for anoikis (Monteiro et al., 2004). It has the capacity to signal and trigger proapoptotic events in a variety of cell types. NO can inhibit cell adhesion, interfere with the assembly of focal adhesion complexes, and disrupt the cell–extracellular matrix interactions. NO is enzymatically derived from L‐arginine and O2. For the enzymes NO synthases (NOS), three isoforms can be distinguished, Type I or neuronal isoform (nNOS), Type II or inducible isoform (iNOS), and Type III or endothelial isoform (eNOS). Lipopolysacharides, cytokines, and toxins can induce the expression of the iNOS with the production of large amounts of NO whereas the neuronal and endothelial isoforms are Ca2þ/calmodulin‐dependent, constitutively expressed enzymes that produce lower amounts of NO (Nathan, 1992). NO might have pleiotropic effects on the induction of apoptosis and cell survival, depending on factors such as levels of NO production, coactivation of other signaling pathways, and other cell type‐specific features. In a variety of cell types such as lymphocytes, hepatocytes, and endothelial cells, NO, at tissue concentrations, can effectively block lipid peroxidation, and protect against oxidative damage mediated cell death and apoptosis (Padmaja and Huie, 1993; Liu and Stamler, 1999). Among other mechanisms, these

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effects involve the activation of p21Ras signaling through both MAP‐kinase and PI3 pathway (Lander et al., 1995; Deora et al., 1998; McFall et al., 2001). As opposed to the protective role of NO when produced at low fluxes, NO generated at high fluxes through iNOS is cytotoxic (Brune et al., 1999). NO has the capacity to trigger proapoptotic events. It can inhibit cell adhesion, and it interferes with the assembly of focal adhesion complexes, thereby disrupting cell–extracellular matrix interactions leading to anoikis. NO can potentially uncouple cooperative signaling by growth factors and matrix receptors, which ultimately leads to apoptosis (Frisch and Screaton, 2001; Monteiro et al., 2004). This underlies the importance of coupling integrin‐ and growth factor‐signaling pathways for regulating cellular survival.

2.3.5 Transcription Factors 2.3.5.1 C‐Myc C‐Myc is a nuclear phosphoprotein that acts as a transcription factor stimulating both cell‐cycle progression and apoptosis (Penn et al., 1990; Facchini and Penn, 1998). Activity of c‐Myc is regulated through phosphorylation and interaction with other cellular proteins such as Max (Lutterbach and Hann, 1994). It can exert its effects on the cell cycle by controlling transcription of genes coding for Cdc25A, cyclin D1, cyclin D2, cyclin E, cyclin A, Cdk1, Cdk2, Cdk4, and E2F (Born et al., 1994; Kim et al., 1994; Beier et al., 2000). It can also suppress transcription of the genes for Cdk inhibitors p15INK4b, p21Cip1, and p27Kip1 (Grandori et al., 2000). As c‐Myc responds directly to mitogenic signals, it might have a critical role especially in the transition from G0 to S phase (Spencer and Groudine, 1991). C‐Myc expression, however, is maintained throughout the cell cycle and might have a role in G2 (Hann et al., 1985; Mateyak et al., 1997). C‐Myc also plays a key role in apoptosis, which might involve p53‐dependent and independent pathways. C‐Myc can transactivate the p53 gene promoter and increase the half‐life of p53 (Hermeking and Eick, 1994). C‐Myc‐induced apoptosis might also involve activation of Fas receptor (Wang et al., 1998) and inhibition of Bcl‐2 and Mcl‐1 (Reynolds et al., 1994). Phosphorylated c‐Myc can be detected immunocytochemically in dystrophic neurites and neurons with neurofibrillary degeneration in AD, and in neurons and glial cells with abnormal t deposition in Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration (Ferrer et al., 2001). 2.3.5.2 E2F A number of cell‐cycle molecules that have been associated with neuronal death converge on regulation of the E2F family of transcription factors. E2F acts as a gene silencer in neurons and that repression is required for neuronal survival (Liu and Greene, 2001). Induction of apoptosis induced by DNA damaging agents or trophic factor withdrawal is characterized by depression of E2F responsive genes (Liu and Greene, 2001). Among those genes that are derepressed in the process of cell death are the transcription factors B‐ and C‐myb. E2F can also induce the expression of the proapoptotic factor Apaf‐1 (Moroni et al., 2001). E2F1‐ deficient mice are more resistant to focal ischemia (MacManus et al., 1999). PRb/E2F and p53 pathways may be directly linked in the cell cycle and apoptosis. Activated p53 induces p21Cip1, thereby preventing Cdk activation and subsequent phosphorylation of pRb. In contrast, free E2F induces p53 transactivation, connecting the pRb/E2F directly to p53‐dependent apoptosis (Hiebert et al., 1995). 2.3.5.3 STATs Many cytokine receptors transduce signals that promote cell division, block apoptosis, and induce differentiation. These pathways involve distinct members of the JAK family of kinases and the STAT family of transcriptional regulators (Ihle, 1996). Depending on the specific stimulus or cell type, STATs can mediate either proapoptotic or antiapoptotic signals (Battle and Frank, 2002). Regulation of apoptotic pathway by STATs is largely due to transcriptional activation of genes, which encode proteins that are involved in cell death processes, such as Bcl‐xL, caspases, Fas, and TRAIL as well as those that regulate cell‐ cycle progression such as p21Waf1. 2.3.5.4 NF‐kB The transcription factor NF‐kB is activated by a broad range of signals including cytokines, mitogens, free radicals, and other stress signals. NF‐kB upregulates several survival factors, and might also directly promote antiapoptotic mechanisms, such as upregulation of Bcl‐2 family members (Baichwal and Baeuerle, 1997;

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Hinz et al., 1999; Glasgow et al., 2001). In contrast, there is also evidence for apoptosis‐promoting activity of NF‐kB, depending on the nature of the apoptosis‐inducing stimulus (Kaltschmidt et al., 2000).

3

Cell‐Cycle Regulators in Neurodegeneration

Cell‐cycle regulators are recognized targets for the treatment of proliferative disorders such as cancer, cardiovascular disease, infections, or autoimmune diseases (Brooks and La Thangue, 1999; Nabel, 2002; Vermeulen et al., 2003a) (> Table 8-3). In cancer, cell‐cycle deregulation occurs through mutations affecting proteins critically involved in different levels of the cell cycle such as Cdk, cyclins, Cdk‐activating enzymes, Cdk inhibitors, Cdk substrates, and checkpoint proteins (Sherr, 1996; McDonald and el Deiry, 2000).

. Table 8-3 Disorders with involvement of cell‐cycle regulators that are potential targets for treatment with cell‐cycle inhibitors Cancer Cardiovascular disease atherosclerosis cardiac hypertrophy Nervous system Alzheimer’s disease amyotrophic lateral sclerosis stroke Viral infections human cytomegalovirus (HCMV) human papillomavirus (HPV) human immunodeficiency virus (HIV) herpes simplex virus (HSV) Fungal infections Protozoan disease malaria leishmaniosis trypanosomes Psoriasis Glomerulonephritis

Recent evidence indicates that the control of proliferation and differentiation, i.e., cell‐cycle control, is also involved in degeneration of nondividing cells, such as mature neurons that might be of critical relevance to the pathomechanism of AD and related neurodegenerative disorders (Arendt, 1993; Heintz, 1993; Nagy, 2000; Copani, 2001). The control of cell division and cellular differentiation has repeatedly been linked in the past to neurodegeneration in AD, and, based on this potential relationship, a variety of hypotheses have been formulated such as the concept of ‘‘hyperdifferentiation’’ (Bouman, 1934), ‘‘retro‐differentiation’’ (Scheibel and Scheibel, 1975), ‘‘dys‐differentiation’’ (Woolf and Butcher, 1990), or ‘‘de‐differentiation’’ (Arendt, 1993; Heintz, 1993). About 10 years ago, it became increasingly clear that in AD, the mitogenic force is elevated and neuronal mitogenic pathways are aberrantly activated in the early stages of the disease (Ga¨rtner et al., 1995). From that time on, further insight into the pathomechanism progressed further downstream, suggesting a potential involvement of cell‐cycle control as a result of abnormal mitogenic signaling (Arendt et al., 1996; Vincent et al., 1996; Nagy et al., 1997).

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3.1 The Link Between Neurodegeneration and Neuroplasticity 3.1.1 Coherent Hierarchical Pattern of Selective Neuronal Vulnerability and Neuroplasticity Cell death in the nervous system, irrespective of whether caused by inflammation, ischemia, or primary degeneration, selectively affects special types of neurons whereas the others are spared. In AD, this selective neuronal vulnerability has systemic character. Neurodegeneration, initially very constantly affects circumscribed brain areas such as cholinergic basal forebrain projection neurons and the entorhinal cortex and progresses from there throughout the brain, subsequently involving other areas in a defined sequence. The reasons for this selective neuronal vulnerability are unknown, and several explanations have been given. Paul Flechsig (1896) stressed the ontogenetic aspect of selective neuronal vulnerability when he advanced the idea that variations in vulnerability of different groups of neurons are to be traced back in large part to developmental conditions, a concept later defined by Cecile and Oscar Vogt (1951) as the ‘‘principle of pathoclisis.’’ More recently, the phylogenetic dimension of selective neuronal vulnerability was recognized (Ashford and Jarvik, 1985; Rapoport, 1989; DiPatre, 1991; Braak and Braak, 1996; Arendt et al., 1998; Arendt, 2003). Brain areas and neuronal types highly vulnerable against neurofibrillary degeneration in AD not only mature rather late during ontogenic development, but also have been acquired late during phylogenetic development (or have completely been reorganized lately). Accordingly, these brain structures typically subserve higher brain functions such as learning, memory, self‐awareness, and others and as such exhibit a particularly high degree of synaptic plasticity (> Figure 8-6).

3.1.2 Potential Risks of Dynamic Stabilization of Neuronal Connectivity During development, after proliferation, migration and differentiation are completed, neurons become integrated into a neuronal network. This integration is based on intercellular communication, which is largely regulated through external cues. Connectivity and attachment of a cell are mechanisms that, during evolution from single cellular systems to multicellular systems, have been acquired to assess signals from the environment and respond appropriately by proliferation, differentiation, and cell death. In a multicellular organism, external cues are thus, used for morphoregulation, i.e., the assembly of individual cells into highly ordered tissues. Terminally differentiated neurons that have permanently withdrawn from the cell cycle, however, make use of these mechanisms primarily developed to control connectivity for their genuine function, i.e., for information processing in a multicellular network. Thus, cell–cell interactions in the nervous system subserve a phylogenetically newly acquired purpose, the formation of a dynamic network. Once the network has been set up, the cell cycle is shut down and information from neighboring cells is translated into continuous changes of synaptic strength and morphology, a process referred to as synaptic plasticity. This means that, at least partially, identical signaling mechanisms are used to control and regulate such divergent effectors as cell‐cycle control and synaptic plasticity. From the phylogenetic perspective, it might be a great achievement for a neuron to control its synaptic plasticity; to do this on the potential expense of differentiation control, however, might put the neuron on a permanent risk. Neurons acquired particularly late during evolution of the human brain (e.g., cortical associative circuits), which subserve ‘‘typical human higher cortical functions’’ such as learning, memory, reasoning, consciousness, self‐awareness, etc., need to display a particularly high degree of synaptic plasticity, which might explain the particularly high sensitivity toward the loss of differentiation control and cell death (> Figure 8-6). What makes the human brain so special, a large association cortex characterized by a high degree of plasticity—a prerequisite for self‐consciousness, i.e., the development of a belief of itself—at the same time creates a vulnerability unique to humans (> Figure 8-2). Identical signaling pathways might be used in the first stage of synaptic change in both development and maturity, and higher cognitive functions such as learning and memory might be based on adaptive modifications of an ancient mechanism initially evolved to wire the brain. The activity‐dependent modification of synapses

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. Figure 8-6 Hierarchy of cortical vulnerability to neurofibrillary degeneration in the cerebral cortex. a: Neuropathological staging of AD according to Braak and Braak (1991). b: This progression of neurofibrillary degeneration throughout different cortical areas in AD follows a certain sequence (stage I to VI) that represents systematic differences in the vulnerability (hierarchy of vulnerability), which matches inversely the hierarchic pattern of complexity of cortical information processing, structural plasticity, and phylogenetic and ontogenetic age. Those neuronal systems that play a crucial role in ‘‘higher brain functions’’ and therefore become increasingly predominant as the evolutionary process of encephalization progresses, such as the hippocampus and the neocortical association areas, retain a high degree of structural plasticity throughout their life. These areas of the brain take the longest to mature during childhood and adolescence. Exactly the same brain structures display the highest degree of vulnerability during aging and in AD. Modified after, Arendt et al. (1998); Braak and Braak (1991, 1996)

is, thus, a powerful mechanism for shaping and modifying the response properties of neurons, but it is also dangerous (Abbott and Nelson, 2000) as it still hides its ‘‘beasty potency,’’ the risk of phylogenetic regression into cell‐cycle activation, which ultimately results in cell death (Arendt, 2003). Synaptic plasticity might thus, not only be beneficial providing the basis for information processing and memory formation in the

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brain, it might also be crucially involved in a variety of pathological conditions, including amnesia, dementia, epilepsy, and psychosis (BenAri and Represa, 1990; McEachern and Shaw, 1996; Jeffery and Reid, 1997).

3.1.3 Aberrant Plasticity in AD Alterations of neuroplasticity are also a constant feature of AD (Ashford and Jarvik, 1985; Cotman and Anderson, 1988; Butcher and Woolf, 1989; Phelps, 1990; Flood and Coleman, 1990; DiPatre, 1991; Geddes and Cotman, 1991; Masliah et al., 1991c; Swaab, 1991; Walsh and Opello, 1992; Roberts et al., 1993; Neill, 1995; DeWitt and Silver, 1996, Mirmiran et al., 1996; Foster, 1999; Kondo et al., 1999; Mesulam, 1999; Teter and Ashford, 2002). Neurodegeneration in AD is associated with aberrant neuritic growth (Arendt et al., 1986). Abnormal growth profiles preferentially affect neurons that are potentially vulnerable to neurodegeneration, such as cholinergic basal forebrain neurons and cortical pyramidal cells (Arendt et al., 1995a, b, e) (> Figure 8-7). . Figure 8-7 Aberrant morphological growth profiles in AD on neurons of the cholinergic basal nucleus of Meynert. Growth profiles are localized on dendritic endings (d) or cover the soma (b, c). The appearance of these growth profiles is associated with an upregulation of the NGF receptors TrkA (a) and p75 (b). (a: TrkA in situ hybridization; b: p75 immunocytochemistry; c, d: Golgi impregnation). Scale bar: 30mm. Modified after Arendt and Bru¨ckner (1992); Arendt et al. (1995a)

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Aberrant sprouts can be detected in early stages of the disease (Ihara, 1988) and precede the formation of paired helical filaments or occur even without massive neuronal loss (Arendt and Bru¨ckner, 1992b). They might thus, represent a very early event of primary significance, inherent to the pathomechanism rather than a secondary event triggered by ongoing degeneration. As opposed to the continuous growth of dendritic elements during aging (Buell and Coleman, 1979), growth in AD is aberrant with respect to its localization, morphological appearance, and composition of cytoskeletal elements (Arendt et al., 1986, 1995a, b, d; McKee et al., 1989). Dystrophic neurites, mainly dendritic but occasionally also axonal in origin, form a constant component of AD pathology. The original identification of these neurites, in the early years of research on ‘‘Alzheimer’s disease’’ by Fischer (1907), has been corroborated more recently by Golgi studies, ultrastructural evidence, and the identification of elevated growth‐associated proteins (for review see Arendt, 2003). Growth profiles on cholinergic basal forebrain neurons, for example, are typically associated with receptors for NGF, indicating the potential ability to respond to an increased trophic force (Arendt and Bru¨ckner, 1992a, b) (> Figure 8-7).

3.2 Cell‐Cycle Regulators Alternatively Execute Neuroplasticity or Cell Death 3.2.1 Elevated Mitogenic Force and Activated Mitogenic Signaling in Alzheimer’s Disease The presence in the AD brain of growth‐associated and growth‐promoting proteins, as well as their receptors, might be an indication of an increased mitogenic force particularly pronounced within the microenvironment of plaques. The growing list of compounds contains GAP‐43, MARCKS, spectrin, heparansulfate, laminin, NCAM, various cytokines, and neurotrophic factors such as NGF, bFGF, EGF, IL‐1, IL‐2, IL‐6, IGF‐1, IGF‐2, PDGF, vascular endothelial growth factor, and HGF/SF (for review see Arendt, 2003). 3.2.1.1 Ras A number of these neurotrophic and potentially mitogenic compounds that are elevated early

in the course of the disease mediate their cellular effects through activation of receptor tyrosine kinases, which recruit p21Ras, a GTPase that belongs to the Ras superfamily of small‐G‐proteins and lead to the sequential activation of Raf, MEK, and ERK (Seger and Krebs, 1995) (> Section 1.5.1). Ras proteins are involved in the central coordination of a variety of functions including cytoskeletal organization, gene expression, cell‐cycle progression, membrane trafficking, cell adhesion, migration and polarity, and synaptic plasticity (Heumann et al., 2000; Arendt et al., 2004). During brain development, p21Ras participates in the regulation of the G0/G1 transition of the cell cycle and might thus, be a critical regulator for cellular proliferation and differentiation (Borasio et al., 1989). In the adult nervous system, it plays a role in reactive dendritic proliferation and neosynaptogenesis (Phillips and Belardo, 1994; Holzer et al., 2001a, b). Thus, Ras is a central regulator of synaptic plasticity in the adult brain (Arendt et al., 2004). The Ras‐ERK/MAPK pathway is an evolutionary, conserved signaling mechanism that plays a fundamental role in the regulation of cellular proliferation and differentiation and control of neuroplasticity, and is also involved in modulating the expression and posttranslational processing of APP and t protein (Greenberg et al., 1994; Mills et al., 1997; Sadot et al., 1998a), two proteins critically involved in AD pathomechanism. In AD, p21Ras is highly expressed in vulnerable brain areas prior to its affection by neurofibrillary degeneration (Ga¨rtner et al., 1999), which indicates an activation of the Ras‐MAPK pathway in the early stages of the disease (Arendt et al., 1995c) (> Figure 8-8). 3.2.1.2 MAPK The activation of MAPK that is associated by its nuclear translocation plays an essential role in the expression of many immediate, early, and late response genes (> Section 1.5.1). MAPK activation and nuclear translocation are required for long‐term facilitation in Aplysia (Martin et al., 1997b) and LTP in vertebrates (Impey et al., 1998). MAPK signaling might thus be a critical regulator for both short‐term synaptic function and transcription of genes required for long‐term plasticity. Ras/MAPK signaling components are highly enriched in the adult CNS, and expression of many MAPK regulators is largely

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restricted to the CNS, where they are highly abundant in association areas implicating a role in memory consolidation and synaptic plasticity. ERK1/2 and other members of the MAPK family (Drewes et al., 1992; Goedert et al., 1992) are able to phosphorylate the microtubule‐associated protein t on threonine and serine residues found phosphorylated in PHFs. In AD, both MAPKK and MAPK are activated in the early disease stages (Arendt et al., 1995c) (> Figure 8-8). This activation of the MAPK pathway can be modeled in transgenic mice expressing permanently activated ras (> Figure 8-9).

3.2.2 Oxidative Stress and the Autocrine Loop of Self‐Perpetuating Mitogenic Activation Neuronal nitric oxide synthase (nNOS) was originally thought to be a constitutively expressed enzyme. It becomes increasingly clear now, however, that its levels are dynamically regulated in response to neuronal development, plasticity, and injury (Dawson et al., 1998). The transcriptional induction of nNOS, which is controlled by neurotrophins and other growth factors, is in turn involved in regulating the expression of immediate early genes in neurons, thereby controlling neuronal growth and differentiation (Peunova and Enikolopov, 1993, 1995). NO may play a major role in nervous system morphogenesis and plasticity and may be involved in activity‐dependent establishment of connections in both developing and regenerating neurons (Dawson et al., 1998; Lu¨th and Arendt, 1998a; Bogdan, 2001). Under developmental conditions, NO may trigger growth arrest, a process that at least in certain cell types might involve inhibition of Cdk2, a key regulator of the G1 and S phases of the cell cycle (see later). These antiproliferative effects of NO involve the repression of cyclin A reexpression as well as an induction of the cyclin‐dependent kinase inhibitor p21Cip1 (Gansauge et al., 1998). The high degree of coexpression of nNOS with p16INK4a (Lu¨th et al., 2000) indicates that further regulators of the G1‐S transition might be involved in the NO‐induced cell‐cycle arrest or that additional mechanisms of proliferation and differentiation‐regulating mechanisms are activated in parallel in the course of neurodegeneration in AD. Thus NO serves as an inducer of cell‐ cycle arrest, initiating the switch to cytostasis during differentiation (Peunova and Enikolopov, 1995; Dawson et al., 1998), a process that can alternatively lead to apoptosis (Gansauge et al., 1998). Although the molecular mechanism for the control of NO in proliferation, differentiation, cellular survival, and death is not understood in detail, recent evidence indicates that activation of p21Ras is critically involved in downstream signaling as a potential endogenous NO‐redox‐sensitive effector molecule (Lander et al., 1997b; Yun et al., 1998). Endogenous NO and intermediates generated through oxidative stress can drive the Ras/MAPK cascade directly by direct activation of Ras‐GTPase activity (Lander et al., 1997; Bogdan 2001). NO might thus be a key mediator linking cellular activity to gene expression and long‐ lasting neuronal responses through the activation of p21Ras by redox sensitive modulation (Dawson et al., 1998), a process with potential implications both in development and degeneration. In AD, nNOS is aberrantly expressed in potentially vulnerable neurons of the isocortex and entorhinal cortex. Since these neurons express nNOS before they are affected by neurofibrillary degeneration (Lu¨th and Arendt 1998b; Lu¨th et al., 2000), transcriptional induction of nNOS might be an early event in the process of neurodegeneration. As expression of nNOS in AD is highly colocalized with p21Ras (Lu¨th et al., 2000) (> Figure 8-10), an autocrine loop may exist within cells, whereby NO activates p21Ras, which in turn leads to cellular activation and stimulation of NOS expression (Lander et al., 1997). In parallel,

. Figure 8-8 Activation of p21Ras‐dependent MAP‐kinase pathway in AD. Expression of p21Ras, b‐Raf, MAP kinase kinase (MEK1), and the MAP kinases ERK1/2 quantified by ELISA and detected by immunohistochemistry in Brodmann area 10. All four markers are elevated early in the course of the disease. P21Ras is highly enriched in plaques, plaque‐associated astrocytes, as well as in tangle‐bearing neurons. B‐Raf and the associated protein 14‐3‐3 are also found in tangles. The subcellular translocation of MEK1 and ERK1/2 into neuronal nuclei indicate enzyme activation. Modified after Arendt et al. (1995d); Arendt (2003); Ga¨rtner et al. (1995)

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. Figure 8-9 Changes in the subcellular distribution of MAP kinase (ERK1/2) in AD, and in a transgenic mice model expressing permanently activated p21Ras under control of the synapsin promoter (synRas mouse). Both in AD and transgenic mice, MAPK is translocated from the cytoplasm (asterisk) to the nucleus (arrows) indicating an activation of the enzyme. Scale bar: 10 mm. Modified after Arendt et al. (1995c); Holzer et al. (2001)

. Figure 8-10 Double immunofluorescence for nNOS/p21Ras and nNOS/p16INK4a. The aberrant expression of nNOS in pyramidal neurons is highly colocalized with p21Ras (top row) and p16INK4a (bottom row), respectively. Interneurons expressing nNOS constitutively (arrow), on the contrary, neither express p21Ras nor p16INK4a, modified after Lu¨th et al. (2000)

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downstream mechanisms such as the cell cycle might be activated as indicated by the coexpression of cell‐cycle regulators (> Figure 8-10). The coexpression of NOS and p21Ras in neurons vulnerable to neurofibrillary degeneration early in the course of AD might thus provide the basis for a feedback mechanism that might exacerbate the progression of neurodegeneration in a self‐propagating manner (> Figure 8-11). This self‐perpetuation of a process likely to be associated with limited prospects of physiological control and termination might be the critical switch converting two potentially neuroprotective mechanisms such as NO (Farinelli et al., 1996) and p21Ras (Heumann et al., 2000) dependent signaling into a disease process, leading to slowly but continuously progressing neuronal death.

3.2.3 The Replay of Developmental Programs in Alzheimer’s Disease Several genes and proteins and their posttranslational modification associated with AD have recently been found to play critical roles in neuronal development, particularly neuronal migration, and axon extension (Arendt, 2000, 2001a, b; Bothwell and Giniger, 2000; Mehler and Gokhan, 2001). Neurodegeneration,

. Figure 8-11 Schematic illustration of the intracellular signaling events triggered by Morphodysregulation in AD that involve an aberrant activation of p21Ras/MAP‐kinase signaling, a loss of differentiation control, the subsequent reentry and partial completion of the cell cycle, and eventually result in cell death. Modified after Arendt et al. (1998), J Neural Transm (Suppl 54), 147–158; Arendt (2003)

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furthermore, is associated with a complex structural reorganization, which involves a replay of developmental programs. The aberrant neuritic growth in AD, as a likely indication of defect synapse turnover, is accompanied by microtubular reorganization (McKee et al., 1989) associated with the reexpression of a number of developmentally regulated proteins involved in morphoregulation indicating that molecules, overexpressed in AD, might play a role in structural remodeling of the adult brain (Arendt, 2001b). Based on this evidence, we have suggested previously that the process of continuous synaptic reorganization becomes defective in AD (Arendt, 1993). In this pathogenetic process, a subset of neurons retaining a high degree of plasticity and that are presumably in a ‘‘labile state of differentiation,’’ are forced into a condition of de‐differentiation that is characterized by an expression of developmental regulated genes, posttranslational modifications, and an accumulation of gene products to an extent that goes beyond those observed during regeneration. This replay of developmentally regulated mechanisms might be the end stage of disturbed structural brain self‐organization and a slowly progressing ‘‘morphodysregulation.’’ This process of de‐differentiation involves molecular events that, in dividing cell populations, would lead to cellular transformation and is thus, not compatible with the state of a neuron being irreversibly blocked from reentry into the cell cycle. It might, therefore, lead to neuronal death. From this hypothesis, it can be predicted that these molecular events that are involved in neoplastic transformation might also play a key role in the pathomechanism of AD (Heintz, 1993). These mechanisms are notably a dysfunction of mitogenic signal transduction and cell‐cycle control.

3.2.4 Cell‐Cycle Activation in Alzheimer’s Disease and Other Neurodegenerative Disorders Proteins that normally function to control the cell‐cycle progression in actively dividing cells may play roles in the death of terminally differentiated postmitotic neurons (Farinelli and Greene, 1996; Park et al., 1996). Thus, a dysregulation of Cdks and their regulating partners, such as cyclins and Cdk inhibitors, indicating an activation of the cell cycle in postmitotic neurons has been observed in AD (Smith et al., 1995; Arendt et al., 1996, 1998b, c, 2000; McShea et al., 1997; Vincent et al., 1997; Nagy et al., 1997a, 1988; Jordan‐Sciutto et al., 1999; Arendt, 2000; Dranovsky et al., 2001), cerebral ischemia, and during trophic factor deprivation (Gill and Windebank, 1998; Tomasevic et al., 1998; van Lookeren Campagne and Gill, 1998; Timsit et al., 1999; Osuga et al., 2000; Sakurai et al., 2000; Katchanov et al., 2001) (> Figures 8-12 and > 8-13, > Table 8-4). Correspondingly, pharmacological inhibitors of cell cycle or ectopic expression of Cdkis can protect neurons against death (Farinelli and Greene, 1996; Kranenburg et al., 1996; Park et al., 1996; Giovanni et al., 1999; Padmanabhan et al., 1999; Osuga et al., 2000; Katchanov et al., 2001; Knockaert et al., 2002) (> Figure 8-14). 3.2.4.1 Cyclin‐Dependent Kinases Several cyclin‐dependent kinases critical for progression through the cell cycle (Koh et al., 1995) such as Cdk1 (Cdc2), Cdk4, and Cdk5 are deregulated in AD (Ledesma et al., 1992; Vincent et al., 1997; Busser et al., 1998; Nagy et al., 1998; Patrick et al., 1999; Arendt et al., 2000). The Cdk1 (Cdc2) kinase, moreover, is able to phosphorylate t protein at sites known to be phosphorylated in AD, and thus potentially contributes to the generation of PHF‐t (Ledesma et al., 1992; Kobayashi et al., 1993). APP, furthermore, is phosphorylated both in vitro and in intact cells by a Cdk1 (Cdc2)‐like kinase in a cell‐cycle‐ dependent manner, which is associated with altered production of potentially amyloidogenic fragments containing the entire b/A4 domain (Suzuki et al., 1994). Ab in turn might potentially act as a proliferating signal, driving cultured rat primary neurons into the cell cycle (Copani et al., 1999), although in vivo, this has not been replicated (Ga¨rtner et al., 2003). 3.2.4.2 Cyclins The level of cyclin D1, a critical regulator of the transition from the G0 to G1 phase of the cell cycle that acts through activation of Cdk4, is increased in neurons prone to neurodegeneration in AD (Busser et al., 1998; Arendt et al., 2000). Cyclins other than D1 such as cyclins E and A involved in regulation of G1/S transition as well as cyclin B regulating G2/M transition (Nagy et al., 1997a; Vincent et al., 1997; Smith et al., 1999; Arendt et al., 2000) are also elevated.

Neuronal cell‐cycle regulation during development and age‐related neurodegenerative disorders

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. Figure 8-12 The cell division cycle and its major regulatory elements. Main switches for the activation and progression of the cell cycle are elevated in neurons prone to neurofibrillary degeneration early in the course of AD (shown in dark gray)

INK4a

3.2.4.3 Cdk Inhibitors p16

, a prominent representative of the INK4 family, involved in pathways for control of cell growth and proliferation (Kamb et al., 1994), is also increased in AD as are other members of the INK4 family of the cyclin‐dependent kinase inhibitors interacting with Cdk4/6 such as p15INK4b, p18INK4c, and p19INK4d. However, alterations of p21Cip1 and p27Kip1 are less constant (Arendt et al., 1996, 1998b, c).

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. Figure 8-13 Major regulators of the activation and orderly progression through the cell cycle are expressed in vulnerable neurons in AD (compare also > Figure 7-11). They are already highly abundant in pyramidal neurons prior to PHF formation. In more advanced stages of the disease, they are associated with PHFs (Brodmann area 22). Modified after Arendt et al. (2000), Neurobiol Aging 21, 783–796

3.2.4.4 Retinoblastoma Protein In AD, increased immunoreactivity for hyperphosphorylated pRb and for E2F has been described (Jordan‐Sciutto et al., 2002; Hoozemans et al., 2004). The upregulation of E2F‐1 as it has been detected in pyramidal neurons of Down’s patients exhibiting the neuropathological features of AD at the same time as Ab deposition occurs might thus, be of potential relevance to the process of neurodegeneration (Motonaga et al., 2001). 3.2.4.5 The Cell Cycle Integrates Intercellular and Intracellular Signaling and Links Development, Oncogenesis, and Neurodegeneration The high capacity of structural neuronal plasticity in the adult brain might

predispose neurons to tangle formation in AD (Arendt et al., 1995a, 1998a; Arendt 2001a). This high potential of neuroplasticity associated with the necessity of synaptic turnover and reorganization might require properties inherent to both growth cones and synaptic connections (Pfenninger et al., 1991). Highly plastic neurons might thus retain ‘‘immature’’ features and might not be ‘‘fully differentiated’’ or ‘‘truly postmitotic,’’ i.e., arrested in G0, an assumption supported by recent findings on the expression of cyclin B and E in hippocampal neurons of healthy elderly (Nagy et al., 1997a; Smith et al., 1999). It is, therefore, suggested that the reexpression of developmentally regulated genes, the induction of posttranslational modifications and the accumulation of gene products to an extent, which goes beyond that observed during regeneration, and the aborted attempt of ‘‘differentiated’’ neurons to activate the cell cycle, apparently is a critical event in the pathomechanism of AD (Arendt, 1993, 2001a; Arendt et al., 1998a, 1998c, 2000; Busser et al., 1998; McShea et al., 1997; Masliah et al., 1993c; Nagy et al., 1997a, b, 1998; Smith et al., 1999; Smith and Lippa, 1995; Vincent et al., 1996, 1997, 1998). This condition is associated with a re-expression of developmentally regulated genes, the induction of posttranslational modifications and accumulation of gene products to an extent which goes beyond that observed during regeneration and might, thus, be due to a loss of differentiation control that normally is involved in the regulation of

Neuronal cell‐cycle regulation during development and age‐related neurodegenerative disorders

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. Table 8-4 Evidence for cell-cycle activation in neurodegenerative disorders Alzheimer’s disease increased neuronal expression of cell-cycle regulators and proliferation associated proteins Cdk1 Cdk4 Cdk7 cyclin A cyclin B cyclin C cyclin D1 cyclin E cyclin G1 Cdk5/p35, p25 polo-like kinase CDC25A CDC25B neuronal CIP-1-associated regulator of cyclin B (CARB) p15INK4b p16INK4a p18INK4c p19INK4d p21Cip1 p27Kip1 p105 Ki-67 PCNA increased phosphorylation of Rb mitotic phosphoepitopes are absent in immature neurons of the human brain but reappear in potentially vulnerable neurons prior to neurofibrillary degeneration PHFs are associated with mitotic specific phosphoepitopes regulation of the phosphorylation and metabolism of APP is cell-cycle dependent partial tetraploidy of neurons abnormal mitotic regulation in peripheral cells such as lymphoblasts, peripheral lymphocytes, or skin fibroblasts Mild Cognitive Impairment (MCI) increased neuronal expression of cell-cycle regulators/proliferation associated proteins cyclin D cyclin B PCNA

Arendt et al. (1996, 1998d, e, 2000) Blalock et al. (2004) Busser et al. (1998) Ding et al. (2000) Dranovsky et al. (2001) Harris et al. (2000) Husseman et al. (2000) Illenberger et al. (1998) Jenkins et al. (1998) Jordan-Sciutto et al. (1999, 2002) Kondratick and Vandre´ (1996) Ledesma et al. (1992) Masliah et al. (1993) McShea et al. (1997) Nagy et al. (1997a, b, 2002) Ogawa et al. (2003) Pei et al. (2002) Pruess and Mandelkow (1998) Ranganathan et al. (2001) Smith and Lippa (1995) Smith et al. (1999) Stieler et al. (2001) Suzuki et al. (1994) Tsujioka et al. (1999) Ueberham et al. (2003) Urcelay et al. (2001) Vincent et al. (1996, 1997, 1998, 2001) Yang et al. (2001) Zhu et al. (2000, 2004)

Yang et al. (2003)

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. Table 8-4 (continued) Vascular dementia increased neuronal expression of cell-cycle regulators cyclin B Stroke increased neuronal expression of cell-cycle regulators Cdk4/ Cdk2 cyclin D1 Parkinson’s disease increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk5 ppRb Ki-67 Lewy body disease increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk5 Ki-67 Pick’s disease increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk1 cyclin B Ki-67 Frontotemporal dementia with Parkinsonism (FTDP-17) increased neuronal expression of cell-cycle regulators Cdk1 cyclin B Down’s syndrome increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk1 Ki-67 cyclin B higher incidence of leukemia in Down’s patients higher incidence of colo-rectal cancer in siblings of Down’s patients altered mitotic index in fibroblasts from Down’s patients maternal meiosis II errors in Down’s patients are associated with presenilin-1 polymorphism Temporal lobe epilepsy increased neuronal expression of cell-cycle regulators cyclin B Progressive supranuclear palsy increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk1/cyclin B Cdk5 Ki-67

Smith et al. (1999) Love (2003)

Brion and Couck (1995) Jordan-Sciutto et al. (2003) Smith and Lippa (1995)

Brion and Couck (1995) Smith and Lippa (1995)

Nagy et al. (1997-Acta 94) Husseman et al. (2000) Nagy et al. (1997-Acta 93) Smith and Lippa (1995) Husseman et al. (2000)

Hermon et al. (2001) Husseman et al. (2000) Nagy et al. (1997a, b) Petersen et al. (2000) Schneider and Epstein (1972) Smith and Lippa (1995) Vincent et al. (1997)

Nagy and Esiri (1998)

Husseman et al. (2000) Borghi et al. (2002) Smith and Lippa (1995)

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8

. Table 8-4 (continued) Corticobasal degeneration increased neuronal expression of cell-cycle regulators Cdk1 cyclin B Huntington’s disease increased neuronal expression of cell-cycle regulators Cdk1 cyclin B Amyotrophic lateral sclerosis increased neuronal expression of cell-cycle regulators cyclin D cyclin B1 Cdk5 phosphorylation of Rb Niemann–Pick’s disease type C (NPC) increased neuronal expression of cell-cycle regulators/proliferation associated proteins Cdk1 cyclin B Cdk5/p25 presence of mitotic τ phosphoepitopes: TG-3, MPM-2, H-5

Husseman et al. (2000)

Husseman et al. (2000)

Ranganathan and Bowser (2003) Husseman et al. (2000) Nakamura et al. (1997) Bajaj et al. (1998) Patzke and Tsai (2002) Ranganathan et al. (2001) Husseman et al. (2000) Bu et al. (2002 a, b)

. Figure 8-14 Neuroprotection by ectopic expression of the cyclin‐dependent kinase inhibitor p16INK4a. Microexplants prepared from rat embryonic day 17 were challenged with 10nM OA (24h), a strong inducer of apoptosis. Explants transfected with p16INK4a prior to OA treatment showed a much reduced rate of apoptosis (TUNEL, dUTP‐rhodamine)

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neuronal plasticity. A direct link between cell‐cycle reactivation and cell death is supported by observations on the neuroprotective action of overexpression of the cyclin‐dependent kinase inhibitor p16INK4a, which locks neurons in a differentiated stage and prevents cell‐cycle reentry (Arendt, 2003). Thus, it might be a ‘‘labile fixation’’ of plastic neurons in G0, which allows for ongoing morphoregulatory processes after development is completed. The delicate balance, however, between G0‐arrest and G1‐entry might be prone to a variety of potential disturbances during the lifetime of an individual. Morphodysregulation in AD, accompanied by aberrancies in intracellular mitogenic signaling, might thus be a slowly progressing dysfunction, which eventually overrides this differentiation control and results in de‐differentiation, a condition in conflict with the otherwise ‘‘mature’’ background of the nervous system. Cell cycle and differentiation control might thus provide the link between structural brain self‐ organization and neurodegeneration (Arendt, 1993; Heintz, 1993) both of which in the human brain have reached a phylogenetic level unique in nature.

4

Conclusions

Taken together, in multicellular organisms, the cell number is regulated spatially by extracellular signals through cell interactions controlling proliferation and survival in local neighborhood. Instructions from neighboring cells can induce cell proliferation, differentiation, or death. These stimuli include cell–cell and cell–ECM adhesion, growth factors, cytokines, neuropeptides, and mechanical factors. Signals from G‐ protein‐coupled receptors, tyrosine kinase receptors, and integrins cooperate to integrate information from multiple stimuli, which regulate cell‐cycle progression. To allow for a regulation of these processes, a tight link is necessary between cell attachment mechanisms and control of proliferation and differentiation, i.e., the cell‐cycle machinery. Contrary to many other cells that make up a multicellular organism, neurons use the molecular circuitry developed to sense their relationship with other cells to reorganize their connectivity according to the requirements for information processing within a cellular network. To form connections and to reshape them continuously, cell division needs to be shut down. The molecular machinery behind it is now used to regulate synaptic plasticity. This switch from proliferation control to plasticity control, which occurs during the process of differentiation of a neuron, implies the permanent risk of erroneously converting signals derived from plastic synaptic changes into positional cues that will activate the cell cycle (Arendt, 2003). This replay of developmental mechanisms, i.e., phylogenetic and ontogenetic regression in the adult nervous system, contrary to development, ultimately results in cell death. Maintaining neurons in a differentiated but still highly plastic phenotype will thus be the challenge to prevent neurodegeneration.

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mRNA Modulations in Stress and Aging

E. Meshorer . H. Soreq

1 1.1 1.2 1.3 1.4 1.5

The Mammalian Stress Response—Neurochemical Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 Defining Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 The HPA Axis: Neuroendocrine Response to Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Glucocorticoid Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Other Stress‐Related Hormones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 Stress and the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

2 Pre‐mRNA Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 2.1 The Splicing Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 2.2 SR Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 3 3.1 3.2 3.3

Splicing and Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Constitutive Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222 Alternative Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Stress‐Induced Changes in SR‐Related Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224

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Stress‐Associated Dendritic Translocation of mRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

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Defects in pre‐mRNA Splicing as Causes of and Predisposition to Diseases . . . . . . . . . . . . . . . . . . . 225

6 Splicing and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 6.1 Pre‐mRNA Splicing Modulations in Senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227 6.2 Pre‐mRNA Splicing Modulations in Aging‐Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 7 Candidate Gene Study: ACHE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 7.1 The Molecular Biology of AChE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 7.2 AChE in Multitissue Stress Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232 8

AChE’s 30 Alternative Splicing under Stress: Long‐Term Implications . . . . . . . . . . . . . . . . . . . . . . . . . . 233

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Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

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Abstract: Ample information suggests multileveled relationships between mRNA modulations and long‐ term stress responses, especially with regard to alternative splicing. The nature of these relationships is predictably complex and multileveled. Here, we describe the contribution of both hormonal and stress‐ induced changes to alternative splicing, and address the molecular pathways that lead to the long‐lasting stress‐associated modulation in alternative splicing as well as the cellular changes that follow. We cover the general areas of stress responses (both to psychological and chemical insults) and their long‐term consequences, the mechanism of splicing and alternative splicing, and the phenomenon of neuritic translocation of messenger RNA (mRNA). To present a more detailed example, we describe the alternative splicing patterns of acetylcholinesterase (AChE) pre‐mRNA, which is neuronally expressed, stress‐responsive, and yields dendritically translocated products, and therefore served as an adequate case study for this purpose. The ACHE gene and its variant AChE protein products are thus presented as both subjects to and putative mediators of long‐term consequences of stress and aging. We attempt to illustrate, citing from the current literature, how these diverse areas are nonetheless intertwined. List of Abbreviations: ACh, Acetylcholine; AChE, Acetylcholinesterase; ACTH, Adrenocorticotropic hormone; AD, Alzheimer’s disease; ALS, Amyotrophic lateral sclerosis; AP-1, Activator protein 1; ASAP, Apoptosis- and splicing-associated protein; ATF-3, Activating transcription factor 3; ATM, Ataxia-telangiectasia; BBB, Blood brain barrier; BDNF, Brain derived neurotrophic factor; CaMK, Calcium-calmodulin dependent protein kinase; CBG, Corticosteroid-binding globulin; CNS, Central nervous system; ColQ, Collagen Q; CRF, Corticotrophin-releasing factor; CSF, Cerebrospinal fluid; DFP, Diisopropylfluorophosphonate; ESE, Exonic splicing enhancer; ESS, Exonic splicing suppressor; FTDP-17, Frontotemporal dementia with parkinsonism linked to chromosome 17; GCs, Glucocorticoids; GPI, Glycosylphosphatidyl inositol; GR, Glucocorticoid receptor; HnRNP, Heterogeneous nuclear ribonucleoprotein; HPA, Hypothalamic-pituitary-adrenal; HRE, Hormone response element; HSP, Heat shock protein; ISE, Intronic splicing enhancer; ISS, intronic splicing suppressor; LTP, Long-term potentiation; MAP, Microtubule-associated protein; NCAM, Neural cell adhesion molecule; NFκB, Nuclear transcription factor κB; NO, Nitric oxide; PET, Positron emission tomography; PFC, Prefrontal cortex; PRiMA, Proline-rich membrane anchor; PS1, Presenilin 1; PTB, Polypyrimidine tract binding protein; PTSD, Post-traumatic stress disorder; PVN, Paraventricular nucleus; RRM, RNA recognition motif; Py, Polypyrimidine; SMaRT, Spliceosome-mediated RNA trans-splicing; SPECT, Single photon emission computed tomography; SnRNA, Small nuclear RNA; SnRNP, Small nuclear ribunucleoprotein particle; U2AF, U2 auxiliary factor; UTR, Untranslated region

1

The Mammalian Stress Response—Neurochemical Aspects

1.1 Defining Stress Adopted from the field of physics, in which stress is referred to as a physical pressure, biological stress, by nature, is a subjective idiom. It was hence neglected for a long time, even years after the fields’ pioneers Walter Cannon and Hans Selye advanced stress to the laboratory bench, coining famous terms such as ‘‘fight or flight’’ (Cannon, 1939), ‘‘homeostasis’’ (Cannon, 1939), or ‘‘general adaptation syndrome’’ (GAS) (Selye, 1978), referring to the collection of stress symptoms in an individual. Stress in biology is regarded today as the physiological and/or behavioral responses to a challenge (‘‘stressor’’) that involve adaptive, hormonal, or behavioral changes, which inevitably link to neurochemical processes. The myriad types of stresses that may lead to such responses are largely dictated by one’s own capacity to perceive and interpret the stressful signal(s). This results in hormonal and molecular responses influencing—mildly or strongly, acutely or chronically, and sometimes indefinitely—the individual’s responses. Early in life, Hans Selye (1936) regarded physical insults as the main culprit causing the diverse effects of stress, but his later reports highlight the power of psychological stress (Selye, 1978). Nowadays, it is widely accepted that different psychological challenges are among the most powerful stressors. For example, anticipation of punishment activates a much stronger stress response than the punishment itself (McEwen, 2002).

mRNA modulations in stress and aging

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1.2 The HPA Axis: Neuroendocrine Response to Stress Stressful experiences include, but are not limited to, physical and psychological traumatic events (grief, car accidents, war trauma, etc.), physical and psychological abuse, prolonged fatigue, and life threatening events (Gershuny and Thayer, 1999). In essence, therefore, life itself is a stressful experience, and the process of aging resembles the long‐term consequences of stressful events. In principle, stress responses are vital for surviving the immediate challenge; a fleeing animal or person turn on the immediate stress response (Sapolsky et al., 2000). This includes the release of catecholamines (epinephrine and norepinephrine) from the sympathetic nervous system, and the stimulation of the hypothalamic–pituitary–adrenal (HPA) axis (> Figure 9-1). HPA activation results in hypothalamic secretion of corticotrophin‐releasing factor (CRF)

. Figure 9-1 The HPA axis. The regions involved in the activation of the HPA axis are shown. The area enlarged is the hypothalamic region. The hypothalamus is shaded gray in the inset. See text for details. PVN, paraventricular nucleus; CRF, corticotrophin‐releasing factor, ACTH, adrenocorticotropic hormone; PFC, prefrontal cortex; GC, glucocorticoids

and vasopressin (AVP) from the cell bodies of the parvicellular hypophysiotropic neurons of the hypothalamic paraventricular nucleus (PVN) (Sapolsky et al., 2000). The axons of these neurons terminate at the hypothalamic median eminence, where CRF secreted from these neuronal processes diffuses into the hypophyseal portal circulation, and through the binding of CRF to its specific receptor, CRFR‐1, it triggers the anterior pituitary gland to release adrenocorticotropic hormone (ACTH) from specialized cells called corticotropes into the general circulation (Sapolsky et al., 2000). ACTH then reaches the kidney’s adrenal cortex, where it binds specific cell surface receptors on the adrenal cells in the zona fasciculata, and through cAMP‐dependent pathways stimulates the synthesis and secretion of glucocorticoids (GCs) (cortisol in humans and corticosterone in rodents) and mineralocorticoids (aldosterone) into the general circulation (Sapolsky et al., 2000). GCs, acting back on the hypothalamic PVN, activate a negative feedback loop, which inhibits CRF and hence ACTH production, consequently decreasing the synthesis and secretion of GCs from the adrenal gland.

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1.3 Glucocorticoid Actions About 95% of the GCs in the circulation are bound to proteins, mainly corticosteroid‐binding globulin (CBG), and also albumin, to a lesser extent. Free GCs, and only free GCs, can readily enter cells and bind their specific intracellular corticosteroid receptors (Beato et al., 1996). Bound GCs can also be functional, for example, in cases where the carrier protein interacts with cell surface receptors, or following enzymatic cleavage of CBG by serine proteases in inflamed tissues. Two distinct subtypes of intracellular corticosteroid receptors have been identified: the high‐affinity mineralocorticoid receptor (MR, type I) and the low‐ affinity glucocorticoid receptor (GR, type II). Although MR binds both GCs and mineralocorticoids with high and almost equal affinity (Kd
0.5–2 nM), GR displays weaker binding and is selective for GCs (Kd
10–30 nM) (Sapolsky et al., 2000). Following the binding of GCs to GRs, the GRs dimerize and the hormone–receptor complex subsequently translocates into the nucleus, where it recognizes and binds consensus DNA sequences known as hormone response elements (HRE) (Chandler et al., 1983; Beato et al., 1996). This binding usually activates transcription, but may also interfere with the activity of other transcription factors, such as activator protein 1 (AP‐1) (Heck et al., 1994) or may have dual functions of activation or repression, depending on the promoter context, such as with nuclear transcription factor kB (NFkB) (Hofmann and Schmitz, 2002). The GCs‐responding genes include cytokines (Arimura et al., 1994; Licinio and Wong, 1999), transcription factors (Persico et al., 1993; Jolly and Morimoto, 1999), synaptic proteins (Persico et al., 1995), brain receptors (Duman et al., 1999), and neurotrophins (Altar, 1999), to name a few. In addition to acting as transcription factors, GCs exert multiple effects on the organism. In general, the actions of GCs can be divided into permissive or proactive, and suppressive or protective (Buckingham, 2000). The permissive roles maintain the basal activity of the HPA axis, setting its threshold for stress responsiveness, and normalizing the body’s response to stress by priming defense mechanisms. The protective roles include anti‐inflammatory actions, redirection of metabolism to meet energy needs during the stress response, promotion of specific memory processes (Furey et al., 2000), and suppression of nonessential functions such as digestion, growth, and reproduction (Sapolsky et al., 2000).

1.4 Other Stress‐Related Hormones As evident from GCs’ actions, the stress response avoids wasting expensive energy on reproduction, digestion, and growth, while concentrating on stress ridding. Prolonged stress responses may, therefore, have harmful effects on the general maintenance of many different properties. However, GCs do not act alone. In order to obtain a regulated response, additional hormones, acting directly on specific target tissues, also participate in the mammalian stress response. Such hormones include prolactin (Kjaer et al., 1991), which suppresses the reproductive system; endorphins and enkephalines (Amir et al., 1980), which blunt pain receptors and promote alertness; and vasopressin (Robertson, 1976), which acts to accelerate heartbeat and to elevate blood pressure. Other hormones are suppressed. These include growth hormone (Brown et al., 1978), estrogen (Ballinger, 1990), testosterone (Jeong et al., 1999), progesterone (Magiakou et al., 1997), and insulin (Keltikangas‐Jarvinen et al., 1998). The picture that emerges is that of a complex response, which affects most, if not all, of the mammalian systems. The complexity can be specifically illustrated by the example of the immune response. Following stress, GCs induce granulocytosis (Stefanski, 2000), yet quench inflammatory responses in a long‐lasting manner. It was demonstrated, for example, that psychological stress renders individuals more susceptible to the common cold (Cohen et al., 1991). These immunosuppressive properties of GCs have made them desirable anti‐inflammatory agents in wide clinical use. However, whereas chronic stress clearly suppresses immune responses, it was shown that some stress conditions such as short‐duration stressors can have stimulating actions on immune functions (Dhabhar and McEwen, 1999). Different stressors can lead to differential activation of certain proinflammatory cytokines (e.g., IL‐1, IL‐6, and TNFa) while suppressing others (usually IL‐2, IL‐3, and IL‐5). An additional level of complexity is added by the ability of the immune system to act back on the HPA axis: for instance, it was demonstrated that proinflammatory cytokines such

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as IL‐1 and IL‐6, the same ones that are suppressed by GCs or conversely abundantly produced in response to immunological stimuli (microbes, lipopolysaccharides), can activate the HPA axis and exert additional effects on the central nervous system (Pruett, 2003).

1.5 Stress and the Brain The stress–brain connection was only made in 1968 with the discovery of GRs in the rat hippocampus (McEwen et al., 1968). A plethora of studies followed, establishing the hippocampus as a major stress‐ responding area having the highest concentration of GRs in the brain. As is the case with the immune system, stress can exert multiple, sometimes contrasting or even opposite effects on the central nervous system. For example, the release of epinephrine during stress is known to modulate memory consolidation, enhancing memory for many different training experiences (McGaugh, 2000). On the other hand, it was shown that stress can impair neurogenesis in the hippocampus (Gould et al., 1998), lead to hippocampal atrophy in primates (Uno et al., 1989), and significantly impair the hippocampus‐related declarative memory function (Lupien et al., 1998). Hippocampal long‐term potentiation (LTP) impairments, reflecting compromised memory, were also observed following various stress paradigms, especially chronic stress (Diamond and Rose, 1994; McEwen, 1999a, b). Stress‐induced hippocampal plasticity is a key factor in repeated and chronic stress responses (McEwen, 1999a, b). Chronic restraint stress was shown to alter synaptic terminal structures in the hippocampus, mainly in the CA3 region (Magarinos et al., 1997), and cause atrophy of apical dendrites in this region (Magarinos et al., 1996). Chronic stress also leads to impaired feedback mechanisms, which are associated with a decreased expression of hippocampal corticoid receptor genes. In addition, or in parallel, long‐term stress accelerates the accumulation of a number of biological markers of aging, increases the excitability of CA1 pyramidal neurons through a calcium‐ dependent mechanism, and causes loss of hippocampal pyramidal neurons. Also, the persistence of excitatory amino acid release after the termination of a stressful experience may render the aging hippocampus more vulnerable (McEwen, 1999a, b). In humans, atrophy of the hippocampus was reported as a result of both elevated GCs (Lupien et al., 1998) and severe traumatic stress (Bremner, 1999). A schematic diagram of the hippocampus is shown in > Figure 9-2. Related syndromes include Cushing’s disease (Starkman et al., 1992), major depression (Sheline et al., 1996), schizophrenia (Bogerts et al., 1993), aging‐related syndromes (Convit et al., 1995), Alzheimer’s disease (de Leon et al., 1993), and finally posttraumatic stress disorder (PTSD) (Bremner et al., 1995). It is tempting to speculate that GCs and the processes induced under their excess accumulation are responsible for the hippocampal atrophy associated with the syndromes listed here. Indeed, long‐lasting . Figure 9-2 The hippocampus and prefrontal cortex (PFC). Two of the major stress‐responding areas in the human brain are illustrated. The hippocampus is shown as a dark crescent and the PFC is marked with a circle.

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elevated glucocorticoid levels were shown to associate with diverse psychological syndromes such as anxiety, depression, and related mood disorders (Hoehn‐Saric et al., 1991; Yehuda et al., 1993; Calvo and Volosin 2001; Kalinichev et al., 2002). However, considerable controversy still exists regarding the specific contributions of the HPA axis to PTSD. Thus, PTSD patients may have normal to low levels of circulating GCs, while presenting features reminiscent of those of normal individuals with excess GCs (reviewed in (Yehuda, 2002). The contribution of the initial burst of GCs during trauma or stress leading to the PTSD phenotype, hence, remained enigmatic. An additional brain region central to the stress response is the prefrontal cortex (PFC, > Figure 9-2). Similar to the hippocampus, the PFC is rich in GRs and participates in the feedback regulation loop of the HPA (Lupien and Lepage, 2001). The PFC is known to serve cognitive and emotional functioning, both of which are disrupted by stress (Moghaddam, 2002), and chronic stress was shown to induce impairments of spatial working memory, a type of memory most associated with the PFC (Mizoguchi et al., 2000). Using in vivo brain imaging methods, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), the PFC was identified as a major area affected in a variety of depressive and mental states (Ebert and Ebmeier, 1996). Indeed, PFC malfunctioning was shown in patients with PTSD (Shin et al., 2001). PFC efferents and most of the afferents to the PFC, especially those that arrive from the thalamus and the hippocampus, are glutamatergic. Glutamate‐mediated neurotransmission is hence a major player in regulating PFC stress responses (Bagley and Moghaddam, 1997). Nevertheless, the PFC is by no means restricted to glutamatergic neurotransmission. Levels of other neuromodulators were found to be modified following stress in the PFC, attesting to the complex functionality of the PFC during stress. These include serotonin (Kawahara et al., 1993), dopamine (Hamamura and Fibiger, 1993), monoamines (Flugge et al., 1997), excitatory amino acids (Moghaddam, 1993), norepinephrine (Nakane et al., 1994), GABA‐A (Gruen et al., 1995), and acetylcholine (Mark et al., 1996). The latter poses an interesting and important link between the glutamatergic and the cholinergic systems in the brain. Cholinergic– glutamatergic interactions have been associated with higher brain functions such as LTP, memory, and behavior (Aigner, 1995), all of which are affected by stress.

2

Pre‐mRNA Splicing

In eukaryotic cells, especially in mammalian cells, the newly transcribed pre‐mRNA is subjected to a series of processing events before leaving the nucleus, reaching its subcellular location, and translating into protein. These include 50 capping, splicing, cleavage, and 30 polyadenylation (Rio, 1992). The precise regulation of these events is critical for ensuring the accuracy and the diversity of gene expression. Particularly interesting is the splicing machinery, which recognizes and removes introns to produce the mature mRNA (Hastings and Krainer, 2001). In up to 60% of the genes, the splicing machinery further generates different transcripts from the same transcription unit by selecting or excluding different exons, a process known as alternative splicing. The different transcripts created must then be spatially and temporally regulated. Alternative splicing is the key process responsible for the large proteomic diversity, estimated to produce hundreds of thousands of proteins (Hodges et al., 2002), from merely 20,000 to 30,000 genes by alternative splicing of the majority of the pre‐mRNAs (Lander et al., 2001). This estimation is based on the comparison of expressed sequence tags (ESTs) with genomic data. Therefore, the real number is likely to be higher, as numerous low‐abundance mRNAs are not taken into account, and because ESTs are biased toward the mRNA 30 ends (Lander et al., 2001). One extreme example includes the Drosophila Down syndrome cell adhesion molecule (Dscam), encoding an axon guidance receptor shown to possess more than 38,000 alternatively spliced transcripts (Schmucker et al., 2000).

2.1 The Splicing Machinery The splicing reaction involves two trans‐esterification steps. In the first reaction, the 50 end of the intron binds to the branchpoint—a conserved adenine upstream from the 30 splice site—to form an intron lariat

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intermediate. In the second reaction, the two exons are ligated and the lariat is released for nuclear degradation. In general, for an intron to be recognized, it must include, in addition to the conserved branchpoint sequence, conserved 50 and 30 splice sites (usually GU and AG, respectively), and a polypyrimidine (Py) tract, located close to the 30 end of the intron. These elements recruit the required molecules carrying out the splicing reaction. The splicing machinery is mainly governed by small nuclear ribonucleoprotein particles (snRNPs), which recognize and bind conserved elements within the pre‐mRNA. SnRNPs are RNA–protein complexes made out of five small nuclear RNAs (snRNAs) (U1, U2, U4, U5, and U6) and associated proteins, which form the core spliceosome (Hastings and Krainer, 2001). As shown in > Figure 9-3, the first steps of

. Figure 9-3 Spliceosome assembly. Early complex (E) involves binding of U1 snRNP to the 50 SS (GU) and the two subunits (65 and 35) of U2AF to the Py tract and 30 SS (AG), respectively. Recognition of the branch point (A) by U2 snRNP creates the A complex. Further binding of the U4/U6·U5 tri‐snRNP breaches the association of U1, which is released with U4, to form the B complex. The 50 splice site then juxtaposes near the branch point to form the C complex, and the lariat‐shaped intron is removed

spliceosome assembly involve 50 splice site recognition by U1 and binding of U2 auxiliary factor (U2AF) to the Py tract and 30 splice site. These early interactions form the E complex. Further base pairing of U2 with the branchpoint forms the A complex. Subsequent steps include association of the U4/U6·U5 tri‐snRNP with the spliceosome. Before the first catalytic step, the U4–U6 interaction is disrupted and an invariant motif in U6 replaces U1 snRNA at the 50 splice site (forming the B complex). A specific base pairing between U2 and U6 snRNAs then juxtaposes the branchpoint and the 50 splice site. U5 snRNP holds onto the 50 exon intermediate, which has been cut free, and the spliceosome is reconfigured to bring the 30 splice site into the catalytic core and align the exons for the second catalytic step (C complex). The spliceosome involves, apart from the above, some additional 150 spliceosome‐associated proteins (Zhou et al., 2002). Non‐snRNPs relevant for splicing catalysis include heterogeneous nuclear ribonucleoproteins (hnRNPs), additional RNA‐binding proteins, and most important, a family of SR proteins (Zahler et al., 1992), which promote splicing activity (Zahler et al., 1993) and affect alternative splicing patterns (Caceres et al., 1994).

2.2 SR Proteins SR proteins are characterized by a modular structure with a conserved C‐terminal arginine/serine‐rich domain (RS domain) that interacts with components of the basic splicing machinery, and one or two RNA‐recognition motifs (RRMs) (Tacke and Manley, 1999). The RRMs recognize loosely conserved RNA

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sequences, identified as either exonic or intronic splicing enhancers (ESE/ISE) or suppressors (ESS/ISS) (Cartegni et al., 2002). When SR proteins bind to their corresponding ESEs, they recruit the early splicing component, i.e., U1 snRNP and U2AF to their correct sites (> Figure 9-4). In alternative splicing, ESEs are

. Figure 9-4 ESE—SR protein interactions. A pre‐mRNA containing both introns (white) and exons (dark) is shown. Exons contain exonic splicing enhancers (ESE) recognized and bound by the RNA recognition motif (RRM) of SR proteins (ovals). SR proteins recruit U1 snRNP to the 50 splice site and U2AF to the Py tract and the 30 splice site. Subsequently, U2AF recruits U2 snRNP to the branchpoint (A). SR proteins can also bind intronic splicing enhancers (ISE) and mediate protein–protein interactions through their RS domain

usually found in the alternatively spliced exon, and function by recruiting splicing factors to a suboptimal 30 splice site, thus stimulating splicing of an upstream intron or inclusion of an alternative exon (Cartegni et al., 2002). It was demonstrated, in vitro, that SR proteins alone can influence splice site selection and hence regulate alternative splicing (Ge and Manley, 1990; Krainer et al., 1990). In vivo, it is likely that the fine‐tuned balance between SR proteins, hnRNPs, splice sites, and enhancer/silencer elements can be modulated to achieve a change in the exon usage of pre‐mRNA (Hastings and Krainer, 2001). The levels of SR proteins and hnRNPs vary among tissues (Hanamura et al., 1998) and can be further modulated by releasing proteins from intranuclear storage compartments, such as the speckle domains at the nuclear internal boundaries through protein phosphorylation (Mintz and Spector, 2000) and by tissue‐specific factors, especially in the nervous system (Dredge et al., 2001). Using computational algorithms together with in vivo and in vitro validation, several functional ESEs were identified (Schaal and Maniatis, 1999; Liu et al., 2000; Cartegni et al., 2002). These were found to be 6–8 bases long, purine‐rich sequences that are usually degenerate. In addition to serving in alternative splicing, these elements may have crucial roles in identifying correct exons for constitutive splicing, as the other relevant conserved sequences in exons (i.e., splice sites, Py tract, and branchpoint) are weak. If this is indeed the case, it would explain the somewhat paradoxically high occurrence of ESEs in exons rather than introns, and would support the model, which suggests that the recognition unit of the spliceosome is the exon itself rather than the intron (Robberson et al., 1990).

3

Splicing and Stress

3.1 Constitutive Splicing The initial observations that cellular stress leads to splicing modifications were made about two decades ago with the discovery that a brief severe heat‐shock impairs the splicing process, leading to the accumulation of unspliced RNA products. This inhibition of splicing could be partly avoided in cells that were previously exposed to a mild heat shock, an adaptation process that involved the cellular activation of heat‐shock proteins (HSPs) (Yost and Lindquist, 1986). This first set of experiments suggested a controllable association of heat‐shock‐related proteins with components of the splicing machinery. Interestingly, whereas the

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splicing of HSP27 was impeded during heat shock (Bond, 1988), the splicing of the small HSP21 remained unaffected but the length of its poly(A) tail, which usually confers longer half‐life of mammalian mRNAs (Soreq et al., 1974; Jacobson and Peltz, 1996), was increased (Osteryoung et al., 1993), demonstrating a finely regulated mechanism and suggesting stress‐induced stabilization of HSP21 mRNA. Later, two of the HSPs participating in the reactivation of splicing following heat shock were identified as HSP70 and HSP104 (Vogel et al., 1995). Additional factors contributing to stress‐induced splicing alterations include the family of hnRNP‐M proteins and the splicing‐related protein 2H9, shown to participate in basal splicing processes as well as in the early stages of stress‐induced splicing arrest (Gattoni et al., 1996; Mahe et al., 1997). Other reports showed disruption of the U4/U6·U5 tri‐snRNPs complex in HeLa cells under heat stress (Bond, 1988; Shukla et al., 1990), as well as inactivation of a splicing factor associated with U4/U6·U5 (Utans et al., 1992). Indeed, a heat‐shock‐induced shift from speckled to diffusive distribution of snRNP antigens was reported in the nucleus of mouse fibroblasts, whereas the splicing factor SC35 retained its normal distribution (Spector et al., 1991). Remarkably, spatial association between splicing factors and transcriptionally active genes in nuclear sites of transcription was shown to be intron‐independent, as evident by the association of both an intron‐rich, HSP90 alpha and the intron‐less HSP70 protein with nuclear speckles upon exposure of cells to heat shock (Jolly et al., 1999). These findings indicate an intrinsic, finely tuned targeting capacity for splicing proteins to potential splice sites during cellular stress.

3.2 Alternative Splicing In addition to splicing arrest, various stressful stimuli were shown to involve regulated, specific modulations in alternative splicing. Splicing arrest may result in the accumulation of intron‐containing transcripts, which under strict evolutionary pressure can be beneficial for the cell. Differential availability of splicing‐ related proteins could further result in a regulated‐like fashion of alternative splicing, because the fine balance between the splicing factors, more than the precise concentration of any specific protein, is the key regulatory element (Hastings and Krainer, 2001). This phenomenon may be viewed as a beneficial adaptation strategy that modifies a finely tuned regulation process; alternatively, or in addition, these changes might reflect adverse or neutral consequences of the uncontrolled processing of pre‐mRNA. In recent years, a growing body of evidence links diverse stress responses of cells and tissues to alternative splicing modulations. Although the cellular signaling pathways affecting alternative splicing under modified stimuli are poorly understood, recent efforts by several investigators highlight interesting possibilities. In one study, activation of the MKK3/6‐p38 cascade was shown to modify the subcellular distribution of hnRNP A1, resulting in modified alternative splicing of target pre‐mRNAs (van der Houven van Oordt et al., 2000). This demonstrated that the stress‐induced biochemical cascades can influence the patterning of alternative splicing through modifying the phosphorylation state of upstream molecules, thereby affecting their cellular compartmentalization. In another example, the stress‐activated ERK MAP‐ kinase pathway was shown to regulate the phosphorylation state, and subsequent cytoplasmic localization of hnRNP K, an RNA‐binding protein that silences mRNA translation (Habelhah et al., 2001). The same ERK/MAP‐kinase pathway was also shown to be involved, upon activation of T cell lymphocytes, in the alternative splicing of the cell membrane adhesion glycoprotein CD44 by retaining its exon v5 sequence in the mature mRNA (Weg‐Remers et al., 2001), demonstrating how kinase cascades can regulate different posttranscriptional events simultaneously by activating a single stress‐induced pathway. Stress‐induced changes in alternative splicing have been demonstrated for the transcripts of numerous genes. The GR serves as a most relevant example in our case. Perinatal manipulations and postnatal handling were both shown to selectively elevate GR mRNA containing a hippocampus‐specific exon, which facilitates adaptation to modified stimuli (McCormick et al., 2000). GCs also regulate the splicing pattern of the murine Slo gene encoding a brain potassium channel, which depends on neuronal depolarization (Xie and McCobb, 1998). Hypoxia was shown to induce the alternative splicing of the presenilin‐ 2 (PS2) gene, generating an isoform also found extensively in the brain of Alzheimer’s disease patients (Sato et al., 1999). A detailed study of Tra2b1 and additional SR proteins under ischemic conditions in the

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brain revealed that some of them translocate from the nucleus to the cytosol after an ischemic event, which could explain the observed changes in splice site selection (Daoud et al., 2002). Activating transcription factor 3 (ATF3) was shown to possess a specific stress‐associated splice variant, ATF3DeltaZip2, modulating the activity of the normal ATF3 protein during stress responses (Hashimoto et al., 2002). Splice site selection can hence serve both as a physiologic adaptation to a change in the external conditions and as a contribution to pathophysiologic events. Intriguingly, some of the evoked changes in alternative splicing produce transcripts that have antagonistic cellular actions. For example, the heat‐shock factor 4 transcript can generate either an activator or a repressor of downstream heat‐shock genes by alternative splicing (Tanabe et al., 1999). Also, alternative splicing of the apoptotic gene, bcl‐x, substitutes a large protein product, Bcl‐xL, which inhibits cell death, for a smaller one, Bcl‐xS, which antagonizes this property under certain conditions (Boise et al., 1993). The gene encoding neural cell adhesion molecule (NCAM) was shown to undergo alternative splicing during nitric oxide (NO)‐induced apoptosis and during neuronal differentiation, producing a smaller protein product than the normal one, through the action of c‐Jun/AP‐1 (Feng et al., 2002). This smaller species, NCAM‐140, protects cells against NO‐induced apoptosis, illustrating the significance of alternative splicing regulation during stress responses. Most important, a direct link between splicing and apoptosis was recently shown with the isolation of a protein complex termed the apoptosis‐ and splicing‐associated protein (ASAP). ASAP was shown to disassemble during apoptosis, releasing the splicing activator RNPS1, which could possibly participate in the splicing and/or alternative splicing of target genes that are activated during apoptosis (Schwerk et al., 2003), strengthening the stress/apoptosis and splicing relationship. An examination of the cases studied so far (e.g., GR, Slo, PS2, ATF3, HSF4, Bcl‐x, NCAM, and AChE, see later) supports the notion that alternative splicing serves as an adaptation strategy. Thus, alternative splicing in these cases yields specific splice variants, which facilitate beneficial stress responses, at least in the short range. For example, the alternative products of GR, Slo, ATF3, HSF4, Bcl‐x, and NCAM actively participate in adapting to the novel conditions, either by generating an antagonizer to the action of its normally spliced counterpart (e.g., ATF3, HSF4, Bcl‐x) or by generating a protective protein (e.g., GR, NCAM). In the case of the potassium ion channel, Slo, a calcium‐calmodulin dependent protein kinase (CaMK) IV responsive RNA element (CaRRE) was identified. To demonstrate its function, this element was experimentally transferred from the 30 splice site upstream of STREX, the alternatively spliced product of Slo, to a heterologous gene. The otherwise constitutive acceptor sequence became sensitive to CaMK IV (Xie and Black, 2001). This sets an intriguing example for the role of the pre‐mRNA sequence itself in stress responses, which in this particular case, introduces long‐term adaptive changes to the neuronal electrophysiological properties.

3.3 Stress‐Induced Changes in SR‐Related Proteins As stated earlier, several proteins emerge as particularly relevant to stress responses. These include, but are not limited to, the splicing activator RNPS1, the splicing‐associated complex ASAP, and the SR‐related protein Tra2b1. Another potentially involved member of the SR protein family worth mentioning is SC35. SC35 possesses an RRM and an RS domain, and was shown to affect both splice site selection and alternative splicing (Wang et al., 2001). In addition, the SC35 pre‐mRNA itself undergoes alternative splicing (Sureau et al., 1997) and the SC35 protein further promotes splicing events that destabilize its own mRNA, exercising a self‐control mechanism that limits its durability (Sureau et al., 2001). This, in turn, suggests that to maintain its long‐lasting high levels, SC35 mRNA should be continuously overproduced in the poststress brain. ASF/SF2, the first SR protein to be identified (Ge and Manley, 1990; Krainer et al., 1990), was also shown to affect both alternative splicing and choice of splice sites (Mayeda and Krainer, 1992; Sun et al., 1993; Wang et al., 1998). Interestingly, the 32 kD subunit of the splicing factor ASF/SF2, termed SF2p32 inhibits ASF/SF2 activity through inhibition of ASF/SF2 phosphorylation even in cells that maintain unchanged levels of the ASF/SF2 protein (Petersen‐Mahrt et al., 1999). As ASF/SF2 antagonizes the splicing activity of SC35 (Gallego et al., 1997), SF2p32 increases should potentially facilitate increases in the ratio

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between the alternative RNA targets of ASF/SF2 and SC35. Both proteins should hence be tested in the poststress brain.

4

Stress‐Associated Dendritic Translocation of mRNAs

Identified more than 15 years ago (Davis et al., 1987), dendritic transport of mRNAs is known today to be a feature of over 400 different transcripts, comprising about 5% of the total expressed neuronal mRNAs (Eberwine et al., 2001). Dendritic targeting of mRNAs and local protein synthesis were both shown to be associated with hippocampal synaptic plasticity (Kang and Schuman, 1996), as well as with memory storage (Mayford et al., 1996), predicting involvement with aging and stress. As expected from a process regulating synaptic function and plasticity, targeting of mRNAs to dendrites was shown to be stimulus‐induced (Tongiorgi et al., 1997; Steward et al., 1998). Yet more specifically, potassium depolarization was demonstrated to induce calcium‐dependent transport of the mRNAs for brain derived neurotrophic factor (BDNF) and the tropomyosin‐related kinase B (TrkB) BDNF receptor into dendrites. This translocation did not require newly synthesized RNA, yet was followed by local protein synthesis (Tongiorgi et al., 1997). In another study, the mRNA for the immediate‐early gene activity‐ regulated cytoskeleton‐associated protein (Arc) was shown to translocate selectively to activated dendritic segments in the dentate gyrus. In this case as well, mRNA translocation was followed by new synthesis of the Arc protein (Steward et al., 1998). Activity‐dependent mRNA targeting may thus provide a mechanism for synaptic modulation, which requires local protein translation of specific transcripts. The recent generation of mice lacking dendritic transport ability and local protein synthesis of calcium‐ and calmodulin‐ dependent protein kinase II alpha (CaMKIIa) provided a direct proof that local translation is required for LTP and memory consolidation (Miller et al., 2002). The translocation of mRNAs into neuronal processes is generally considered to be associated with cis‐ acting elements, primarily within the 30 untranslated region (UTR) of the mRNA (Steward and Schuman, 2001). However, the length of the sequence required for dendritic targeting varies considerably. For example, a 21 nucleotide long sequence in the 30 UTR of the myelin basic protein (MBP) mRNA was shown to be sufficient for dendritic translocation (Ainger et al., 1997), whereas in another study, 640 nucleotides from the 30 UTR of the microtubule‐associated protein 2 (MAP2) were shown to be essential for mRNA targeting (Blichenberg et al., 1999). For a‐CaMKII, 94 nucleotides from the 30 ‐UTR were shown to mediate its dendritic localization in cultured hippocampal neurons (Mori et al., 2000), whereas in a different study, a 1,230 nucleotide region of the 30 ‐UTR was shown to be responsible (Blichenberg et al., 2001). These apparent discrepancies could be settled if the secondary structure, rather than primary sequence, was responsible for targeting. Indeed, careful analyses demonstrated that dendritically localized mRNAs do not share a common consensus sequence (Tiedge et al., 1999). It is therefore likely that RNA folding into secondary structures might act as the driving mechanism (Ainger et al., 1997; Blichenberg et al., 1999).

5

Defects in pre‐mRNA Splicing as Causes of and Predisposition to Diseases

Alternative splicing of a growing number of gene products can be found in numerous types of cancerous cells, although not abundant in the corresponding normal cells (Herrera‐Gayol and Jothy, 1999; Caballero et al., 2001; Perry et al., 2002), probably reflecting a general modification of the processing machinery during tumorogenesis (Scorilas et al., 2001; Stoilov et al., 2002). In humans, SC35 expression is altered under other diseases as well, e.g., HIV infection (Maldarelli et al., 1998) and also during pregnancy (Nie et al., 2000). ASF/SF2 too is spatio‐temporally regulated in the uterine myometrium during pregnancy (Pollard et al., 2000), again, probably insinuating a more general phenomenon for the regulation of splicing‐related genes during various stresses or under adaptation to altered conditions. A striking example of a splicing‐related protein, which directly affects the severity of disease state, is illustrated by the putative splicing factor SCNM1 (sodium channel modifier 1). A single point mutation changes a donor splice site in

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the sodium channel gene Scn8a leading to chronic movement disorder. In one mouse strain, C57BL/6J, an additional modifier nonsense mutation in SCNM1 exacerbates the Scn8a disorder into a lethal neurological disease. The effect of the modifier mutation is a reduction of the abundance of correctly spliced sodium channel transcripts below the threshold that is essential for survival (Buchner et al., 2003). This example demonstrates how genetic variation in a splicing factor influences disease susceptibility. A large number of mutations lead to aberrant splicing (Stoilov et al., 2002). These can directly disrupt or create acceptor or donor splice sites, or alternatively, disrupt exonic or intronic splicing enhancers or suppressors. Intriguingly, in a few cases, disease‐associated alternative or aberrant splicing was reported, but the culprit gene was found to be devoid of any splicing‐associated mutations. These are particularly relevant examples for this discussion, because a splicing regulatory protein is likely to be at fault, causing the abnormal phenotype. We present several examples to illustrate each of these cases. Familial hypercholesterolemia is an autosomal dominant genetic lipoprotein disorder caused by defects within the low‐density lipoprotein receptor gene. Some of its many mutations were shown to disrupt acceptor splice sites: an A to G substitution in the penultimate nucleotide of intron 16 (Lombardi et al., 1993) and a G to C transposition at the last nucleotide of intron 7 (Yu et al., 1999). In both cases, a cryptic splice site was activated, leading to the formation of a mutated receptor protein. Another example of a splice site mutation that leads directly to a disease phenotype is molybdenum cofactor deficiency (MoCoD), an inherited autosomal recessive disease that leads to early childhood death. Two bicistronic genes, MOCS1 and MOCS2, are responsible for the generation of the molybdo‐enzymes, sulfite oxidase, xanthine dehydrogenase, and aldehydeoxidase. An MOCS1 splice site mutation leads to the deficiency of all these molybdenum cofactor‐related enzymes (Reiss et al., 1999). An increasing number of alterations, both in normal and in alternative splicing, are being linked to defects in enhancer/silencer sequences (Philips and Cooper, 2000; Cartegni et al., 2002). A number of these mutations are present in an exon, but do not change the protein sequence. Because they cause aberrant splice site selection, they can cause a disease, although they do not modify mRNA translation. Examples of these mutations include a T to C (L284L) mutation in tau exon 10 that disrupts an exonic splicing enhancer, causing frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP‐17) (D’Souza et al., 1999), a C to G (R28R) mutation in porphobilinogen deaminase that results in exon 3 skipping, and that causes one of the many forms of porphyria (Llewellyn et al., 1996) and an A to G mutation in pyruvate dehydrogenase resulting in Leigh’s encephalomyelopathy (De Meirleir et al., 1994). Mutations that alter the splicing process can occur outside of both splice sites and enhancer/silencer elements. An example is the mutation described in a patient with ataxia‐telangiectasia (ATM), which represents a deletion in an intron‐splicing processing element crucial for accurate intron removal (Pagani et al., 2002). The deletion of four nucleotides in this element abolishes a binding site for U1 snRNP and leads to activation of a cryptic exon, thus producing an abnormal mRNA transcript. Changes in splicing factors can also be phenotypic. The appearance of splicing‐associated diseases is often correlated with impaired plasticity and longevity of the affected cells. For example, mutations in the human homologs of the splicing factors PRP31 or PRPC8 may cause retinitis pigmentosa, a progressive loss of rods and cones, which causes loss of over 90% of vision during childhood (McKie et al., 2001; Vithana et al., 2001). An example of a disease with unexplained aberrant splicing includes sporadic amyotrophic lateral sclerosis (ALS), which was shown to be associated with a change in the splicing patterns of the mRNAs for the glutamate transporter EAAT2 (Lin et al., 1998), as well as nitric oxide synthase (NOS) (Catania et al., 2001). This indicates a basic defect in the pre‐mRNA processing machinery, which gives rise to altered isoforms associated with the disease. To a certain extent, pre‐mRNA processing thus appears to have the potential to adapt the information stored in the genome to the physiologic requirements of circumstance, place, and time. The failure of such adaptation is frequently traced to defects in processing. These defects can manifest themselves directly in a disease or may remain silent until some internal or environmental stimulus (e.g., stress) or time (e.g., old age) allows the mutation to become apparent. Furthermore, the sequential nature of pre‐mRNA processing raises the interesting possibility that pleiotropic diseases with a variable phenotype may be caused by specific compositions of trans‐acting factors.

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Splicing and Aging

6.1 Pre‐mRNA Splicing Modulations in Senescence Splicing alterations are particularly important during prolonged altered conditions associated with chronic cellular stress states, which include aging (Meshorer and Soreq, 2002). From the viewpoint of a molecular biologist, aging reflects gradual deterioration of the molecular components, checkpoints, and/or events, the concerted functioning of which is vital for cell viability and proliferation. The complexity of alternative splicing makes this process particularly vulnerable to senescence, leading to both transient changes and chronic aging‐related diseases. Available examples of modified alternative splicing events during aging predict a generally modified state of pre‐mRNA processing machinery in senescent cells, leading to altered expression levels of pre‐mRNA processing factors and their downstream target mRNAs and corresponding proteins during aging. With the emergence of microarray technologies, changes in gene expression profiles during aging have recently become the focus of extensive research. However, relatively little is yet known about changes in alternative splicing, a key pre‐mRNA processing mechanism during cellular and organismal senescence. The gradual aging process is often ascribed to passive accumulation of late‐onset mutations due to lack of natural selection beyond the age of reproduction. Alternatively, aging is viewed as the outcome of active selection of traits that are beneficial at an early stage of life but have deleterious effects later on (reviewed in Partridge and Gems, 2002). The idea that aging is a genetically programmed mechanism was also subject to some debate. Planned or sporadic, passive or active, aging also involves changes in the pathway for gene expression that are not necessarily associated with mutations. This is exemplified in that most aging‐related diseases, representing the best‐studied cases of aging, involve aberration in the alternative splicing of pre‐mRNA. Aging‐related modifications in the alternative splicing of specific gene products were sought for by several researchers, primarily as means to search for the molecular origin(s) of aging‐related deterioration in the processes they were studying. Examples include neural adhesion processes, dopaminergic neurotransmission, insulin responses, and gastric cytoprotection (> Table 9-1). Together, the picture that

. Table 9-1 Aging‐associated changes in alternative splicing Gene APP, APLPs Dopamine D2 receptor N‐CAM Insulin receptor MGF COX‐1 NF1

Organism rat rat rat rat rat rat Human

Tissue Brain Neostriatal subregions Heart Liver, muscle, heart Skeletal muscle Stomach Blood

Affected process amyloid plaque dopaminergic neurotransmission neural adhesion insulin responses insulin responses gastric cytoprotection rRas signaling, neural architecture

Reference (Sandbrink et al., 1997) (Merchant et al., 1993) (Andersson et al., 1993) (Vidal et al., 1995) (Owino et al., 2001) (Vogiagis et al., 2000) (Wimmer et al., 2000)

emerges from these studies is of a general control switch that causes multiple aging‐induced impairments in alternative splicing. For example, such impairments could all reflect one or a few changes in regulatory upstream factor(s). Support for this concept can be found in more recent microarray screens for changes in gene expression during aging. Significant age‐related tissue‐specific changes were found in the expression levels of several RNA processing genes in aged, compared to young, gastrocnemius muscle, neocortex, and cerebellum of C57BL/6 mice (Lee et al., 1999). Changes spanned SR proteins, hnRNPs, as well as 30 ‐end

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. Table 9-2 Aging‐associated alterations in the expression of murine pre‐mRNA processing genes Fold changea 3.4

Tissue Cerebellum

3.2 2.4 2.5

Gastrocnemius muscle gastrocnemius muscle neocortex

PTB

2.3

cerebellum

PolyAþ RNA export protein hnRNP H3 (hnRNP 2H9) CStF DDX18

2.1

gastrocnemius muscle

2.0

cerebellum

0.7 0.7

neocortex neocortex

PRP16

0.6

cerebellum

PABI PRP22

0.4 0.4

cerebellum cerebellum

Gene SF3A2 (SAP62, PRP11) U2AF65 Sox17 PRP22 (HRH1)

Affected pre‐mRNA processing step U2 snRNP binding

Reference to reported function (Ruby et al., 1993)

Splice site recognition, splicing Transcription, Splicing Spliceosome disassembly, mRNA release Alternative splicing

(Wang et al., 1995) (Ohe et al., 2002) (Company et al., 1991) (Wagner and Garcia‐Blanco 2001)

30 end processing, mRNA export Splicing, heat‐shock splicing arrest Transcript cleavage DEAD‐box protein, specific function unknown Aberrant lariat discard mRNA stability Spliceosome disassembly, mRNA release

(Mahe et al., 1997)

(Burgess and Guthrie 1993) (Company et al., 1991)

a

From Lee et al. (1999)

processing factors (> Table 9-2). Because both SR proteins and hnRNPs influence splice site selection, misdirected regulation of their expression levels can lead to alternative or aberrant splicing of many downstream target sequences. One of the first essential splicing factors to be identified was the 65 kD subunit of the U2 auxiliary factor (U2AF65). U2AF65 binding to the conserved Py tract at the 30 end of introns is required for the subsequent binding of U2 snRNA to the 50 splice site. Another subunit protein of this complex, U2AF35, binds the 30 splice site. U2AF65 mRNA levels were found to increase by over threefold in the gastrocnemius muscle of aged mice (> Table 9-2). As U2AF65 contains both the RNA recognition motif and the RS domain of SR‐proteins, it can regulate splicing in a concentration‐dependent manner. Uncontrolled regulation of the expression level of U2AF65 may therefore lead to aberrant regulation of splicing activity and alternative splicing of numerous target genes (Wang et al., 1995) (> Figure 9-5). The Py tract‐binding protein (PTB, hnRNP I) also binds intronic Py tracts. PTB was shown to possess a direct role in pre‐mRNA alternative splicing (Wagner and Garcia‐Blanco, 2001), and its expression levels were reported to increase over twofold in the cerebellum of aged mice, which can potentially harm finely regulated alternative splicing of many pre‐mRNAs (> Figure 9-5). Other factors that may have a direct or indirect role in influencing splicing during aging are PRP22, PRP16, SF3A2, DDX18, and hnRNP H3 (Table 1-2). The expression level of the splicing factor PRP22, for example, is 2.5‐fold increased in the neocortex of aged C57BL/6 mice, whereas the same factor displays 2.7‐fold decrease in the cerebellum of these mice. Although the function of several of these factors is still largely obscure, this expanded list points to a central involvement of splicing changes in the aging process (> Figure 9-5). Another checkpoint, which may affect aging‐related changes in pre‐mRNA splicing, lies upstream, at the earlier phases controlling the initiation and efficacy of transcription. This involves the expression levels

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. Figure 9-5 Misdirected regulation of pre‐mRNA processing during aging. Factors that were reported to change during aging are depicted in the figure, along with the different steps of pre‐mRNA processing, in which they are involved in normal and aged cells. a: The ‘‘naked’’ pre‐mRNA transcript. Exons are dark and introns are marked white. The 50 (GU) and the 30 (AG) splice sites, the branchpoint (A), and the polypyrimidine tract (Py) are shown. The 50 end of the pre‐mRNA is capped shortly after initiation of transcription, b: Initial snRNP complex formation. U2AF65 and PTB were shown to be significantly overproduced in the gastrocnemius muscle and cerebellum, respectively, of aged C57BL/6 mice, thus, potentially affecting splicing, c: Spliceosome assembly and attraction of SR proteins. The splicing‐related factor/RNA helicase PRP16 was shown to be twofold decreased in the cerebellum, d: Intron release and exon joining. Altered expression level of the different factors shown earlier may lead to the alternatively spliced transcript shown, e: 30 end processing. The cleavage and polyadenylation complex, which consists of CPSF, which recognizes and binds the poly(A) site (AAUAAA), CStF, CFI, CFII, and PAP, which catalyze the first, slow, polyadenylation step are shown. PABII then binds the short, newly formed, poly(A) tail and rapidly adds additional 200–250 adenines. CStF and PAP were both about 1.5‐fold decreased in the neocortex of C57BL/6 aged mice, potentially harming the 30 ‐end processing pathway and the splicing pathway. Abbreviations: Py, polypyrimidine tract; snRNP, small nuclear ribonucleoprotein particle; U2AF, U2 auxiliary factor; PTB, polypyrimidine tract‐binding protein; SR‐proteins, serine, arginine‐rich proteins; CPSF, cleavage and polyadenylation specificity factor; CStF, cleavage stimulation factor; CF, cleavage factor; PAP, poly(A) polymerase; PAB, poly(A)‐binding protein.

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of a large number of transcription factors, also shown to be modified in aged tissues. As transcription and splicing are tightly coupled processes, such changes are likely to affect the splicing machinery as well. The transcription factor SOX17, for example, with a direct role in pre‐mRNA splicing (Ohe et al., 2002), demonstrated 2.4‐fold overexpression in the aged gastrocnemius muscle. Altered expression of transcription‐associated genes during aging thus adds further support to the concept of pre‐mRNA splicing as an aging‐affected process. Indeed, aging‐related diseases often reflect abnormal upstream factors, impaired regulation, or aberrant fine‐tuning of mRNA processing in general and pre‐mRNA splicing in particular.

6.2 Pre‐mRNA Splicing Modulations in Aging‐Related Diseases Age‐related, late‐onset diseases are especially vulnerable to splicing variations, of several different origins. These include, for example, mutations affecting the correct splicing of a particular gene. Unlike diseases of early onset, the phenotype associated with these mutations displays a delayed onset. Age‐related macular degeneration (AMD) serves as an example of an aging‐related disease that is associated with a late‐onset splicing mutation. AMD is the most common cause of acquired visual impairments in the elderly. The occurrence of AMD is often associated with mutations within the Stargardt disease gene (STGD1 or ABCR), coding for a photoreceptor‐specific member of the ATP‐binding cassette (ABC) superfamily of transporter proteins. One of these mutations, a G for A substitution at position 5196, was found to be a donor splice site mutation (Allikmets et al., 1997). The late onset of the disease phenotype, in this case, may be explained by age‐related modifications in the functioning efficacy and/or the composition of pre‐mRNA processing factors. Alzheimer’s disease (AD) is the most common neurodegenerative disorder of aging, characterized by progressive memory loss and cognitive deterioration. AD involves premature death of cholinergic neurons, associated with the formation of amyloid plaques the appearance of which in the brain of patients is facilitated by mutations in several different genes. Misdirected splicing regulation of relevant gene products due to specific mutations and aberrant processing of such products with no known mutations were both demonstrated for AD. Presenilins provide examples for both cases: mutations within the fourth intron of presenilin 1 (PS1) were shown to impair PS1 splicing and cause early onset AD; moreover, an exon 5‐lacking splice variant of apparently normal PS2, which accumulates following hypoxia in cultured neuroblastoma cells, was found to be prevalent in the brains of AD patients, indicating that this unique splice variant is inducible, rather than inherent in the genome (Sato et al., 1999). Tauopathies are a family of late‐onset neurodegenerative diseases associated with mutations within the microtubule‐associated protein (MAP) tau gene (Lee et al., 2001). Progressive accumulation of filamentous tau inclusions causes neural degeneration in specific brain regions of patients with tauopathies. The late onset of this phenotype suggests that an age‐related molecular change is responsible. Tau is alternatively spliced in the adult human brain (Lee et al., 2001) and mice overexpressing the human shortest tau variant display age‐dependent CNS deterioration reminiscent of human tauopathies (Ishihara et al., 1999). Thus, impairments in pre‐mRNA processing and/or splicing might be the cause. As shown earlier, FTDP‐17 serves as an example of a tauopathy associated with changes in alternative splicing. In this case, as well, the mutated genotype leads to an abnormal phenotype of a delayed onset, hinting at a change in a yet unidentified age‐related factor involved in pre‐mRNA processing.

7

Candidate Gene Study: ACHE

Transcriptional and posttranscriptional poststress responses induce a cascade of changes in the levels and properties of certain brain proteins that may play major roles in the subsequent events. This particularly relates to proteins involved in relevant neurotransmission pathways. For example, acetylcholine (ACh) levels were shown to be transiently elevated in the mammalian brain during stress responses (Imperato et al., 1991). Similar increases in ACh were also reported following exposure to cholinesterase inhibitors

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(Dazzi et al., 1995). Indeed, psychological stress elicits strikingly similar neuropsychological effects to those observed after acute or chronic exposure to anticholinesterases (Rosenstock et al., 1991). In addition, acute stress and pharmacological inhibition of acetylcholinesterase (AChE), the ACh hydrolyzing enzyme (> Figure 9-6), suppress the production of the ACh‐synthesizing enzyme choline acetyltransferase (ChAT)

. Figure 9-6 Acetylcholine hydrolysis by AChE. AChE cleaves acetylcholine to acetate and choline at a rate of 300,000 molecules per minute, making it one of the most efficient enzymes in nature. Its active gorge includes a serine residue responsible for the enzyme’s catalytic activity (Soreq and Seidman, 2001)

(Kaufer et al., 1998; Anguelova et al., 2000), while inducing rapid increases in AChE mRNA and protein levels in response to forced swim stress in the mouse brain (Kaufer et al., 1998). This suggests a bimodal mechanism for suppressing the cholinergic excitation, which follows stress insults.

7.1 The Molecular Biology of AChE The single AChE gene in vertebrates generates at least three different pre‐mRNAs with distinct 30 regions due to alternative splicing (Soreq and Seidman, 2001). These include the ‘‘synaptic,’’ AChE‐S (also known as ‘‘tailed’’ AChE‐T), the ‘‘erythrocytic’’ AChE‐E (also known as ‘‘hydrophobic,’’ AChE‐H), and the ‘‘readthrough,’’ AChE‐R (> Figure 9-7). As mentioned earlier, the latter is the stress‐induced form, rarely found in naı¨ve adult tissues. Conversely, AChE‐S mRNA is generally ubiquitously expressed and is subject to transcriptional and posttranscriptional development‐related regulation, whereas AChE‐E mRNA is primarily expressed in red blood cell progenitors. The three different 30 transcripts give rise to three protein

. Figure 9-7 30 alternative splicing of AChE. The distal enhancer glucocorticoid response element (GRE), the human AChE gene, and the three 30 ‐splicing products (S, E, R) are shown. Linkage of exons 2, 3, and 4 is common to all variants (a). The R transcript includes at its 30 terminus pseudointron 4 (bottom) and exon 5; The E transcript is generated by splicing out intron 4, and option b yields the S transcript by connecting exon 4 to 6 (top).

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isoforms, which differ at their C‐termini, dictating different cell adherence and noncatalytic properties for each of the three. The C‐terminus of human AChE‐S consists of a 40 residue peptide, which includes a cysteine 4 residues upstream from the C‐terminus. This cysteine enables the formation of dimers and tetramers, which are able to further bind a cholinesterase‐specific collagen tail (ColQ) unique to AChE in neuromuscular junctions (Massoulie, 2002) or a proline‐rich membrane anchor (PRiMA) in the central nervous system (Perrier et al., 2002). The former generates ‘‘asymmetric’’ AChE forms through interactions between the ColQ tails of two or three different tetrameric units, and the two structural subunits anchor AChE‐S to synapses and neuromuscular junctions (Massoulie, 2002). AChE‐E forms dimers exclusively, through a cysteine residue at its 43 amino acid C‐terminus (position 8 of 43). This peptide is subsequently cleaved after amino acid 14 (557 from the N‐terminus) to enable linkage of the remaining C‐terminus through a glycosylphosphatidyl inositol (GPI) to the erythrocytic membrane. AChE‐R has a shorter, 26 amino acids long, C‐terminal peptide, which lacks a cysteine residue and hence remains monomeric and soluble (Soreq and Seidman, 2001). The core sequence of human and rodent AChE, common to all variants, consists of 543 amino acids encoded by exons E2, E3, and E4. Exon 2 codes for the three amino acids responsible for the catalytic activity of AChE inside the active gorge. This ‘‘catalytic triad’’ consists (in humans) of Ser203, Glu334, and His447. Thus, AChE‐S and AChE‐R share the same capacities to hydrolyze acetylcholine (Soreq and Seidman, 2001); however, the catalytic function of AChE‐R in vivo should be extrasynaptic because it does not attach to the synaptic membrane. This variant is hence more likely to tune the modulatory roles of acetylcholine, presumed to reside outside synapses (Legay et al., 1995). Several lines of evidence suggest that AChE‐S and AChE‐R have distinct roles in the normal brain, as well as in the poststress processes (Seidman et al., 1995; Karpel et al., 1996; Shohami et al., 2000; Sternfeld et al., 2000; Birikh et al., 2003; Brenner et al., 2003). Transgenic mice overexpressing one of the corresponding human AChE isoforms (Beeri et al., 1995; Sternfeld et al., 1998) display distinct characteristics. Mice overexpressing AChE‐S show accelerated stress‐related pathology (Sternfeld et al., 2000) including loss of dendritic arborizations and spines, leading to progressive deterioration (Beeri et al., 1997), neuromotor malfunctions (Andres et al., 1997), hypersensitivity to anti‐cholinesterases (Shapira et al., 2000), and to closed‐head injury (Shohami et al., 2000), and vulnerability to a switched day‐and‐night cycle (Cohen et al., 2002). Intriguingly, transgenic mice expressing human AChE‐S, which display changes in the balance of their cholinergic neurotransmission (Erb et al., 2001) possess high levels of HSP70 (Sternfeld et al., 2000) associated with neuronal splicing abnormalities, reflected in high levels of their murine AChE‐R mRNA variant (Cohen et al., 2002). Further research will be required to explore the possibility of an involvement of cholinergic signaling pathways in the maintenance of neuronal mRNA splicing. AChE‐R‐expressing mice display normal neuromuscular function and their brains are relatively protected from the stress‐associated hallmarks of pathology, which predict age‐dependent neurodeterioration (Sternfeld et al., 2000). They further display intensified LTP, parallel to the stress‐induced LTP enhancement that associates with AChE‐R mRNA accumulation in the hippocampus of nontransgenic FVB/N mice (Nijholt et al., 2003). Although it is not yet clear whether this stress‐induced alternative splicing of AChE occurs as a cause or an effect, this phenotype may predict physiological relevance for the exclusive overexpression of AChE‐R following stress in the consolidation of fearful memories. Apart from this apparent protective function, AChE‐R may mediate at least some of the adverse cellular changes associated with delayed stress responses. Intriguingly, AChE‐R mRNA was found in apical dendrites of neurons from all cortical layers following exposure to cholinesterase inhibitors (Kaufer et al., 1998), suggesting possible local regulation and translation of AChE in the synapse (Steward and Schuman, 2001) also in noncholinergic neurons.

7.2 AChE in Multitissue Stress Responses The apparent link between stress and AChE was investigated in a variety of other tissues and systems. AChE mRNA overexpression was observed in the mouse intestinal epithelium and the intestinal gland 2 h following intra‐peritoneal (i.p.) injection of the anti‐AChE diisopropylfluorophosphonate (DFP) (Shapira

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9

et al., 2000). In addition, repeated forced swim stress induced AChE mRNA overproduction in spermatozoa during early spermatogenesis and elevated AChE protein levels at later stages of sperm formation (Mor et al., 2001). In mice subjected to forced swim stress, serum AChE was subject to C‐terminal proteolysis whereas human CD34þ hematopoietic progenitor cells presented cortisol‐induced AChE mRNA synthesis (Grisaru et al., 2001). Also in humans, increased levels of AChE were reported in the cerebrospinal fluid (CSF) of hospitalized patients, but not in age‐matched controls. These increases were associated with blood brain barrier (BBB) disruption, increased CSF albumin levels, and elevated serum cortisol values (Tomkins et al., 2001). Interestingly, all the above‐mentioned instances of AChE overexpression involve elevations of a single isoform of this enzyme, AChE‐R, produced by 30 alternative splicing of the AChE pre‐mRNA. The stress‐induced changes in the alternative splicing of AChE pre‐mRNA and the overproduction of AChE‐R were shown to be beneficial under acute stress responses, when AChE‐R tunes down the stress‐ induced hyperarousal (Kaufer and Soreq, 1999). However, prolonged stress responses likely present a different case. Numerous physiological functions are adversely suppressed under prolonged stress responses, with critical consequences for the affected organism sometimes. Likewise, transgenic animal studies (Cohen et al., 2002; Birikh et al., 2003), animal disease models (Shohami et al., 2000; Brenner et al., 2003), and human clinical trials with an antisense agent destroying AChE‐R mRNA (Argov et al., 2005) suggest that prolonged overproduction of AChE‐R may take its toll.

8

AChE’s 30 Alternative Splicing under Stress: Long‐Term Implications

The major product of AChE’s 30 alternative splicing, AChE‐S, was found to be abundantly expressed in numerous tissues and developmental states. As the main variant responsible for terminating cholinergic transmission, AChE‐S forms tetramers through its unique C‐terminus, allowing increased density of the anchored enzyme in the synaptic cleft (2500–3000 molecules·mm–2, (Anglister et al., 1994). This involves interaction with structural proteins, such as ColQ (Krejci et al., 1997) or PRiMA (Perrier et al., 2002). AChE‐R expression, on the other hand, was found to increase during brain development, under poststress situations (Soreq and Seidman, 2001; Massoulie, 2002), and in glial tumors (Perry et al., 2002). The overexpression of AChE‐R following stress could possibly be beneficial for short‐term responses, ridding of excess ACh secreted now at higher rates. However, the long‐term excess of this protein is likely to be causally involved in the long‐term hypersensitization associated with prolonged stress responses (Meshorer et al., 2002). AChE is also modified in AD, shifting its protein product from tetramers to monomers (Talesa, 2001), and AD patients treated with anticholinesterases display increased levels of AChE‐R in their CSF (Darreh‐Shori et al., 2004). Studies in our laboratory demonstrated rapid, yet long‐lasting stress‐induced shift from neuronal AChE‐S to AChE‐R mRNA transcripts, which was associated with the accumulation of their protein products in neurites, diverting the composition of neuritic AChE from the C‐terminally adhered synaptic membrane isoform to AChE‐R, the variant equipped with a hydrophilic nonadhered C‐terminus (Meshorer et al., 2002). This shift was common to different neurons and stressors, suggesting that stimulus‐induced changes in alternative splicing and dendritic mRNA translocation—both considered major contributors to neuronal plasticity—are intimately interrelated for AChE. In vivo, such changes were associated with long‐ term cholinergic hypersensitivity in both nonstressed controls and transgenics overproducing human AChE‐S and murine AChE‐R (Meshorer et al., 2002), indicating that they may contribute toward the long‐known neuronal hypersensitivity that follows stressful experiences (Antelman et al., 1980) for which there was no mechanistic explanation. Several steps along the pathway for gene expression may contribute toward the neuritic AChE‐R overexpression under stressful insults. First, elevated glucocorticoid levels activate transcription through GRE interactions (Sapolsky et al., 2000). That corticosterone induces AChE overproduction in cultured PC12 cells and primary cerebellar neurons suggests their participation in the initiation of neuronal AChE‐R mRNA overexpression. Second, stress‐induced alternative splicing [e.g., through Nova, Buckanovich et al. (1993)] might regulate the production of AChE‐R by binding to the Nova short consensus intronic motif, UCAUY (Jensen

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et al., 2000), whereas the brain‐enriched polypyrimidine tract‐binding protein (brPTB) can antagonize this Nova‐generated alternative splicing (Polydorides et al., 2000). AChE pre‐mRNA possesses both the UCAUY motif and a possible polypyrimidine tract in conserved 30 intronic locations of both the human (positions 7,242–7,246 and 7,220–7,245 respectively in GeneBank accession number AF002993) (> Figure 9-8) and mouse ACHE genes (positions 13,242–13,246 and 13,210–13,244, AF312033), suggesting their possible relevance to this process.

. Figure 9-8 Nova‐related sequences on AChE pre‐mRNA. Two Nova recognition sites (UCAUY) and a possible polypyrimidine (Py) tract, which can potentially be recognized by the brain‐enriched polypyrimidine tract‐binding protein (brPTB), which antagonizes Nova‐induced alternative splicing, are shown. Note that among the variants of AChE, these elements are absent in AChE‐S mRNA

Third, stress‐induced AChE‐R mRNA accumulation is accompanied by neuritic translocation and most likely, local AChE‐R synthesis. Translocation into neuronal processes presumably depends on cis‐acting elements, primarily within the 30 untranslated region (Wallace et al., 1998). Dendritic targeting sequences, however, do not share common consensus motifs (Tiedge et al., 1999) and the 30 ‐UTR of AChE‐R mRNA includes none of the known dendritic targeting sequences. Rather, RNA secondary structures were proposed to direct targeting (Ainger et al., 1997; Blichenberg et al., 1999). The differential regulation of AChE‐R mRNA targeting into dendrites may thus be associated with its unique 30 sequence and secondary structure. Alternatively, or additionally, AChE‐R mRNA translocation may reflect the stress‐induced accumulation of many more nascent AChE‐R mRNA transcripts. This is compatible with the intense nuclear labeling of AChE‐R mRNA, reflecting a higher pre‐mRNA:mRNA ratio for this transcript. Fourth, neuritic stabilization of AChE‐R mRNA may be involved. The normally short half‐life characteristic of AChE‐R mRNA (Chan et al., 1998) is reminiscent of that of cytokines, where an AUUUA motif or a longer U‐rich element in the 30 ‐UTR is responsible for mRNA destabilization through the binding of trans‐acting proteins [reviewed by (Guhaniyogi and Brewer, 2001)]. A candidate U‐rich element (UUUAUUUUUUUUUU) is present in the 30 ‐UTR of mouse AChE‐R mRNA (positions 13,020–13,007 in GeneBank accession number AF312033) but not in AChE‐S mRNA; however, its importance to the stability of these transcripts remains to be tested. Neuritic AChE‐R mRNA is further resistant to antisense oligonucleotide‐catalyzed degradation (Meshorer et al., 2002). This further supports the notion of its site‐dependent stabilization, and is probably due to the low nuclease levels (especially RNase H, which targets DNA–mRNA hybrids) in neurites. The subcellular distribution of RNase H in neurons is not yet known, but tagged overexpressed chimeric RNase H displayed in the protozoa Crithidia fasciculata a gradient pattern, from high levels in the nucleus to low levels in the cytoplasm (Engel et al., 2001), which may extrapolate to low RNase H levels in neurites. Cholinergic–glutamatergic interactions were demonstrated in several brain regions (Giovannini et al., 1994) to affect CNS functions such as LTP, memory, and behavior (Aigner, 1995), functions that show hyperexcitation following stress. Electrophysiological recordings in the hippocampal CA1 region (Meshorer et al., 2002) demonstrated weeks‐long hypersensitivity of prestressed mice to anticholinesterases, attributing a role for these changes in the cholinergic impact over poststress sensitization (Bell et al., 1994).

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Stress‐induced neuronal hypersensitivity was shown to occur through intensification of cholinergic– glutamatergic interactions (Meshorer et al., 2002), and intensified LTP was found in AChE‐R overexpressing brain (Nijholt et al., 2004). This sheds new light on the well‐known phenomenon of sensitization following stress (Orsini et al., 2001). The glutamatergic nature of the hypersensitized response may be relevant to the additional functions attributed to neuritic AChE‐R, especially noncatalytic capacities to compete with and mediate cell–cell and cell–matrix interactions, based on AChE’s electrotactin properties (Botti et al., 1998). Neuroligin 1, for example, is a postsynaptic cell adhesion molecule of excitatory synapses (Song et al., 1999), which includes an extracellular catalytically inactive AChE‐homologous domain. When overexpressed, AChE‐R shares the neuritogenic properties of neuroligin 1 (Grifman et al., 1998), possibly through interaction with its partner protein neurexin b2 (Nguyen and Sudhof, 1997). However, the distinct Ca2þ‐binding domains in AChE and neuroligin (Tsigelny et al., 2000) predict differences in their adherence properties, suggesting that excess AChE‐R may modify glutamatergic functions by impairing neurexin–neuroligin interactions. The experiments given here suggest that stress‐induced increases in ACh and neuronal sensitization are tightly linked with AChE splice variations, implying a novel mechanism for the stress‐induced neuronal hypersensitivity (Meshorer et al., 2002). This involves neuritic replacement of the membrane‐bound AChE‐ S protein, an indispensable enzyme continuously needed to maintain cholinergic neurotransmission, with its C‐terminally hydrophilic and relatively unstable counterpart, AChE‐R. At the short term, the increased diameter allowing local AChE‐R production and release would dampen more inhibitor molecules than in the nonstressed brain, leading to an initial phase of apparent protection (Kaufer et al., 1998). However, AChE‐R overproducing neurites may fail to promote local AChE‐S synthesis under repeated stimulus due to limited translational capacity and the shifted AChE‐R:AChE‐S mRNA ratio. Neurites of a previously stressed or exposed organism would therefore produce C‐terminally hydrophilic AChE‐R monomers, rather than possess C‐terminally membrane‐adhered AChE multimers, impairing the capacity of dendritic synapses to respond to repeated cholinergic stimuli. AChE‐R properties are likely to be, at least partially, species‐specific. Nevertheless, when expressed in transgenic mice, human AChE‐R acts as a cross‐species protective agent, preventing the accumulation of pathological stress hallmarks (Sternfeld et al., 2000). In contrast, transgenics expressing human AChE‐S respond by overexpressing endogenous murine AChE‐R and display progressive dendrite and spine loss (Beeri et al., 1997), compensatory increases in hippocampal ACh release (Erb et al., 2001), and anticholinesterase hypersensitivity (Shapira et al., 2000). Their antisense‐suppressible vulnerability to closed‐head injury (Shohami et al., 2000) and working memory failure (Cohen et al., 2002) further support the notion that AChE‐R is causally involved with the failure to address cholinergic stimuli. Altogether, this may indicate that the stimulus‐induced choice between the splicing decisions, which lead to production of these two AChE isoforms, is at fault.

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Conclusions

The realm of pre‐mRNA splicing modulations in aging, disease, and stress is now beginning to emerge. Although accumulating evidence creates a clear association between splicing aberrations and disease states, the biochemical, cellular, and molecular pathways are far from being understood. Harnessing the use of high‐throughput techniques, such as splicing‐specific microarrays (Clark et al., 2002) and fiber‐optic arrays (Yeakley et al., 2002) over the coming years promises to bring about novel and exciting examples to add to this growing list, and to shed new light on the intracellular pathways that generate alternatively spliced products during cellular crisis. Studies aimed at the production of abnormal transcripts will pave the way for novel RNA‐targeted therapies that are already being tested in various disease models. These include antisense oligonucleotides, ribozymes, RNAi, or spliceosome‐mediated RNA trans‐splicing (SMaRT) (Stoilov et al., 2002). The use of such technologies will hopefully enable delicate manipulations of gene expression in disease and aging, suppressing the alternatively spliced culprits, while maintaining accurate levels of the normal proteins.

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Molecular Mechanisms of Dendritic Spine Plasticity in Development and Aging

M. R. Kreutz . I. Ko¨nig . M. Mikhaylova . C. Spilker . W. Zuschratter

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 2 The Structure of Dendritic Spine Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246 3 The Postsynaptic Density of the Spinous Synapse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 4 Synaptogenesis and the Formation of Dendritic Spine Synapses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 5 Molecular Dynamics of the Plastic Spine or What are Spines Good for . . . . . . . . . . . . . . . . . . . . . . . . . 251 6 Molecular Mechanisms of Spine Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 7 Structural Alterations of Spine Number and Formation in Brain Disease States . . . . . . . . . . . . . . . 254 8 The Dendritic Spine in the Aging Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

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Abstract: The postsynaptic density (PSD) of spinous excitatory synapses is characterized by an electron‐ dense filamentous meshwork of cytoskeletal proteins that serve three major functions: (1) the organization of glutamate receptors, (2) the clustering of synaptic adhesion molecules, and (3) the coupling of synaptic membrane proteins to intracellular signaling cascades. Consequently, in recent years a plethora of protein– protein interactions of PSD scaffolding proteins has been mapped and their possible function for the formation and integrity of the synapse delineated. It is assumed that structural alterations in the PSD precede morphological alterations of the spine. Detailed studies of such processes are of particular significance because they will probably lead to a new conceptual framework in our appreciation of how changes in the protein composition and protein–protein interactions in the PSD can regulate spine morphogenesis. This should provide novel answers to the long‐standing question—what molecular mechanisms underlie synaptic plasticity and in turn learning and memory processes? List of Abbreviations: AKAP79, A‐kinase anchor protein 79; CaMKII, calcium/calmodulin‐dependent protein kinase II; CNS, central nervous system; GAP, GTPase‐activating protein; GEF, guanine nucleotide exchange factors; LTD, long‐term depression; LTP, long‐term potentiation; NCAM, neural cell adhesion molecule; PAK, p21‐activated kinase; PSD, postsynaptic density; SER, smooth endoplasmic reticulum; SynCAM, synaptic cell adhesion molecule

1 Introduction Synapses are sites of a specialized cell–cell contact between neuronal cells and represent the major structure involved in chemical neurotransmission in the nervous system. In higher mammals, the majority of brain excitatory synapses using the neurotransmitter glutamate is found on the principal neuron of the cortex and hippocampus, the pyramidal cell. Pyramidal cells are characterized by a complex dendritic cytoarchitecture harboring approximately 104–105 synaptic contact sites with other neurons. It is estimated that only 1% of all synaptic contacts of cortical pyramids is concerned with the wiring to subcortical areas (Creutzfeldt, 1995), implying that the predominant synapse of the mammalian telencephalon is concerned with input from a closely related neuron in terms of cell lineage, morphology, and functional characteristics. This fact is mainly emphasized because our knowledge about synaptic plasticity is to a large degree based on this type of synaptic input. Within the central nervous system (CNS), glutamatergic synapses are the major means by which excitatory signals are conveyed from one neuron to another. As such, glutamatergic synapses are crucially important for CNS function. Furthermore, it is widely believed that glutamatergic synapses are important loci for modifying the functional properties of CNS networks, possibly providing the basis for phenomena collectively referred to as ‘‘learning and memory.’’ Given their importance, it is not surprising that enormous efforts are being made to understand the formation, structure, function, and regulation of glutamatergic synapses. To date, significant progress has been made in our understanding of their ultrastructure, molecular composition, and physiological properties, as well as the principles of how these synapses are initially assembled. The term synaptic plasticity covers many different aspects of use‐dependent synaptic modifications. Synaptic plasticity is therefore used in this chapter in a broader sense covering aspects of synaptic signal transmission as well as structural alterations in the molecular makeup of the synapse related to synaptic signaling events. The scope of this chapter is therefore (1) to introduce to current concepts on the molecular mechanisms in the formation of spine synapses, (2) to survey current concepts on activity‐dependent remodeling of the synaptic cytoskeleton, and (3) to summarize the present knowledge about changes in spine synapses in brain disease states and during aging. Given that the topic is rather broad, extensive reference to other recent reviews that cover certain aspects in more detail is given.

2 The Structure of Dendritic Spine Synapses Spines are dendritic membrane protrusions that build synaptic contacts to the presynaptic membrane compartment. The spine density of a typical pyramidal neuron varies between 4 and 10 spines/10 mm dendritic length. As outlined above, gross calculations suggest that an average principal neuron harbors up

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to 10,000 synaptic contacts. > Figure 10-1 depicts a reconstruction of such a pyramidal neuron with its spine protrusions in two dimensions to give an impression of the morphological complexity that is imposed by the existence of spinous synaptic contacts.

. Figure 10-1 A Lucifer yellow‐filled neuron from the cortex of a gerbil (Meriones unguiculatus) shows numerous dendritic spines along its dendric arborization. Note the variable shape and size of the dendritic spines in the inset. Imaging of the neuron and dendrite was done by a confocal laser scanning microscope, with which approximately 80 focal planes were taken from the specimen, and subsequently volume rendered by a 3D imaging program

Spines have different shapes and their three‐dimensional structure varies substantially. Mature spines are typically 0.5–2 mm in length but can also reach up to 5–6 mm. Accordingly, the spine volume ranges from less than 0.01 mm3 to 0.8 mm3. Large spines contain mitochondria and also smooth endoplasmic reticulum (SER), an internal store of Ca2þ (> Figure 10-2). Many attempts have been made in the past to assign spines into discrete groups. At present, spine synapses are classified commonly into one of the following: filopodia‐ like protrusions, thin, stubby, mushroom‐shaped, or cup‐shaped spines (Hering and Sheng, 2001) (see > Figure 10-3). This classification scheme, however, should be treated with some caution. Spines can change their morphology rather rapidly (see also below) and in many cases it is unclear whether certain spine types exist only as a developmental transition state. Moreover, not all spines can be clearly assigned to one group, and it is also unclear whether this classification reflects differences in spine function. Accordingly, recent measurements of spine dimension do support the existence of distinct spine categories (Wallace and Bear, 2004). Despite these limitations, the obvious question that arises is how the different shape and the molecular dynamics of dendritic spines are brought about.

3 The Postsynaptic Density of the Spinous Synapse An important role for the three‐dimensional organization of the spine synapse is played by an electron‐ dense structure beneath the postsynaptic membrane termed postsynaptic density (PSD) (> Figure 10-3). The PSD of the spinous synapse consists of a specialized cytoskeleton, which is crucially involved in the

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. Figure 10-2 Dendritic spine and shaft synapse from human hippocampus. Left: A dendritic spine (S) forms an asymmetric contact with a presynaptic terminal (Pre), while another synaptic bouton contacts the dendritic shaft (D). Note, both synaptic terminals are filled with numerous clear synaptic vesicles. In addition, the spine synapse contains some mitochondria (Mi). Scale bar: 0.5 mm. Right: A dendritic shaft synapse, which contains a huge number of clear synaptic vesicles and mitochondria (Mi), forms multiple contacts with a dendrite (D). Arrowheads indicate postsynaptic contact zones. Scale bar: 0.5 mm

. Figure 10-3 Different morphologies of dendritic spines depending on type of neurons, developmental stage, and activity status. From left to right: filopodium, thin, stubby, mushroom‐shaped, and cup‐shaped spines

organization of the neurotransmitter receptive apparatus and the adhesion of the postsynapse to presynaptic terminals (Sheng, 2001) (see > Figure 10-4). Furthermore, this structure contains specialized and elaborate molecular scaffolds that link synaptic neurotransmission to various signaling cascades and the cytoskeleton (McGee and Bredt, 2003; Kennedy et al., 2005). This is of particular importance since the spine head is highly enriched in actin filaments (Matus et al., 1982), which mediate spine shape changes and motility (Fischer et al., 1998). Fluoresance‐recovery after photobleaching (FRAP) experiments of EGFP‐ actin in dendritic spines indicate that 85% of the actin in dendritic spines is dynamic, with an average cycle

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. Figure 10-4 Molecular structure of the postsynaptic density (PSD) of excitatory synapses. Since excitatory synapses contain thousands of proteins that form a complex, highly ordered functional meshwork, only a subset of molecules and protein interactions are shown, focusing on the PSD. Among the key components of the PSD are on one hand proteins of the MAGUK family‐like SAP‐90/PSD‐95, Chapsyn110/PSD‐93, SAP‐97, and SAP‐102, which form complexes with NMDA and AMPA‐type glutamate receptors, kainate receptors, cell adhesion proteins like neuroligin, or signaling proteins like AKAP79 (A‐kinase anchor protein 79) and kalirin‐7. AKAP79 is able to target protein kinase A (not shown) to specific substrates in the PSD. Kalirin‐7 is a GDP/GTP exchange factor (GEF) for the small GTPase Rac1, which belongs to the Rho subfamily of small GTPases. Additional signaling proteins illustrated here are calcium/calmodulin‐dependent protein kinase II (CaMKII), which can interact with NMDA receptors, and caldendrin, which was shown to bind to L‐type calcium channels. On the other hand, proteins of the ProSAP/Shank family are important scaffolding molecules that connect neurotransmitter receptors in the postsynaptic membrane via direct or indirect protein interactions with the actin cytoskeleton and are therefore believed to play a crucial role in the functional organization of the PSD during synaptogenesis, synaptic plasticity, and also in spine morphology. Among the proteins that interact directly with ProSAPs/ Shanks are, for example, Homer proteins, which provide a connection to metabotropic glutamate receptors (mGluR1a) in the postsynaptic membrane and to inositol‐1,4,5‐trisphosphate receptors (InsP3R) in the membrane of the SER. Cortactin and a‐fodrin both connect ProSAPs/Shanks directly to the actin cytoskeleton. GKAP/ SAPAPs provide the link between MAGUK proteins and ProSAPs/Shanks. Recently, it was shown that ProSAPiP, a novel ProSAP‐interacting protein, binds to SPAR (spine‐associated Rap GTPase‐activating protein). This interaction provides an additional link for ProSAPs/Shanks to the actin cytoskeleton via SPAR and also to MAGUKs, since SPAR was shown to be directly associated with PSD‐95 or PSD‐93. Additionally, it is assumed that SPAR can bind to cell‐surface proteins like ephrins or neuroligins

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time of 44 s (Star et al. 2002). The actin cytoskeleton is linked to the PSD via a number of protein interactions including the actin‐binding proteins, cortactin and a‐fodrin, and it is thought that this interface will be responsible for the molecular dynamics of the spine (Oertner and Matus, 2005). At present it is not yet entirely clear which regulatory events within the PSD control these events (but see also below). Molecular and ultrastructural analysis of the PSD has lead to the identification of several hundred PSD constituents (see Li et al., 2004) and to the notion that the PSD is a multilayered complex, composed of membrane molecules (such as glutamate receptors), primary scaffolding molecules (such as SAP90/PSD‐95), and higher‐order scaffolding molecules (such as ProSAPs/Shanks) that bind subcomplexes and cytoskeletal components into even more elaborate structures (> Figure 10-4). These scaffolding molecules harbor a variety of domains for protein–protein interactions, including ankyrin repeats, a GUK domain, a SH3 domain, a PDZ domain, a SAM domain, and a proline‐rich region (> Figure 10-4). Accordingly, a large number of protein–protein interactions have been ascribed to these domains supporting the view that their multidomain structure underlies their supposed function as scaffolders of the PSD (see > Figure 10-4 for more details). Part of this function is to integrate signaling molecules like kinases or GTPases into the complex network of the synaptic cytoskeleton. Such signaling components will eventually connect synaptic activation to its downstream effectors and can also feedback to the structural organization of the PSD and the morphology of the spine.

4 Synaptogenesis and the Formation of Dendritic Spine Synapses Much progress has been made in recent years in understanding the mechanisms that underlie synaptogenesis of excitatory brain synapses (for reviews see Garner et al., 2002; Ziv and Garner, 2004). With the identification of the protein components of the synapse, their functional characterization with molecular and genetic approaches, and with recent advances in optical imaging of living neurons it has become increasingly clear that synapse assembly is a multistep process. In most cases, the development of dendritic spines occurs concurrently with the growth of the presynaptic terminals, suggesting that cell–cell interactions and extrinsic cues likely induce the formation of dendritic spines. Thus, spines and filopodia actively contact presynaptic afferents (Ziv and Smith, 1996) and can subsequently induce the formation of the release‐competent presynaptic membrane, but also the opposite, i.e., ingrowing axons initiating the emergence of dendritic protrusions, which has been also described (Jontes et al., 2000). Moreover, it was also suggested that dendritic filopodia participate in the formation of shaft synapses by recruitment of the presynaptic contact to the dendrite (Sorra and Harris, 2000). Unexpectedly, it was shown that synaptogenesis in brain as well as in culture can proceed in the complete absence of synaptic transmission (Verhage et al., 2000; Varoqueaux et al., 2002). This implies that synaptic activity is not required for the establishment of synaptic contacts. In any case, however, the question remains how the initial contact site between the axon and the dendrite is specified. The molecular mechanisms of synapse formation in the brain are not yet completely understood. For the formation of the presynaptic membrane, synaptic precursor vesicles have been described in the axon that are 80 nm in diameter and characterized by a dense core appearance. They contain the large presynaptic multidomain proteins Piccolo and Bassoon, which participate in the formation of the cytoskeletal matrix at the active zone of mature synapses (Ziv and Garner, 2004). Additionally, other core components of the active zone are present on these transport vesicles indicating that larger parts of the presynapse will already arrive as one complex at the initial site of synaptic contact. Interestingly, no corresponding precursor transport vesicles have been found for the postsynapse (Bresler et al., 2004). Whether a particular molecule determines the precise site on a dendrite where an axon will form a synapse remains also a largely unresolved issue. An important role, however, seems to be played by the cell recognition molecules neurexins and neuroligins (Dean et al., 2003). The most convincing evidence for an instructive role of the postsynaptic compartment comes from the work of Scheiffele and coworkers (2000), who showed that expression of the postsynaptic cell adhesion molecule neuroligin in nonneuronal cells is sufficient to trigger the development of presynaptic structures in contacting axons. An induction of synapse formation after

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overexpression of neuroligin by activation of its binding partner neurexin was also found in cultured hippocampal neurons (Dean et al., 2003). Many other cell recognition molecules accumulate at synapses during development, which indirectly suggests that they also might be involved in synaptogenesis or synapse stabilization through their adhesive properties. These molecules include N‐cadherin, which is initially associated with all types of synapses in cell culture but then becomes restricted to excitatory synapses (Benson and Tanaka, 1998), and members of the immunoglobulin superfamily, such as synaptic cell adhesion molecule (SynCAM) (Biederer et al., 2002), nectins (Mizoguchi et al., 2002), and neural cell adhesion molecule (NCAM) (Sytnyk et al., 2002). Like neuroligin, overexpression of SynCAM in nonneuronal cells induces formation of synapses on the transfected cells by axons of co‐cultured neurons (Biederer et al., 2002), whereas inhibition of nectin‐based adhesion results in a decrease of synapse size and a concomitant increase in synapse number (Mizoguchi et al., 2002). Finally, the ephrin‐B and EphB tyrosine kinase receptor system has also been implicated in the development of excitatory synapses through phosphorylation of the cell‐surface‐exposed heparin sulphate proteoglycan syndecan (Ethell et al., 2001) or interaction with the NMDA receptor (Dalva et al., 2000). These findings suggest that EphB receptors participate not only during the initial formation of synapses but also in their maturation.

5 Molecular Dynamics of the Plastic Spine or What are Spines Good for Computer modeling as well as experimental evidence supports the idea that the dendritic spine serves as a biochemical microcompartment. Ca2þ transients can be restricted to single spines, thus isolating the effect of activation of specific synapses (Yuste et al., 2000; Sabatini et al., 2002). Under physiological conditions, Ca2þ diffusion across the spine neck is negligible, and the spine head functions as a separate compartment on long timescales, allowing localized Ca2þ buildup during trains of synaptic stimuli (Sabatini et al., 2002). Along these lines it has been proposed by many authors that morphology of synapses and dendritic spines affects postsynaptic integration of signals, for example, allowing neurons to compensate for the attenuation of signals from synapses distant from the soma (reviewed by Hausser, 2001). It was also reported that thick spines (mushroom type) whose head is fully developed remain mostly stable (reviewed by Hering and Sheng, 2001). Therefore, it was proposed that memory in the cerebral neuronal circuit is possibly stored in the spine morphology (Oertner and Matus, 2005) and that the spine separation is a specialization for regulating anatomical plasticity. A stunning observation made with the invention of novel in vivo imaging techniques in the last decade is, however, that the structure of dendritic spines is plastic, in the sense that spines can take various shapes both during neuronal activity and at rest. Spine movements based on alterations in the cytoskeleton have been postulated on theoretical grounds first by Crick (1982). This hypothesis was based on the idea that spine structure–stability–function relationships are well suited for a molecular account of use‐dependent modifications in the brain largely referred to as learning and memory. The first observation that spines are very mobile structures that seem to constantly change their shape and size at least in culture was made by the pioneering work of Andrew Matus and coworkers (Fischer et al., 1998, 2000). Furthermore, de novo spine formation was shown to occur after induction of hippocampal long‐term potentiation (LTP) (Engert and Bonhoeffer, 1999; Toni et al., 1999; but see also Fiala et al., 2002a for contradictory results). More recent work showed that synapse formation and elimination occur throughout life, although the magnitude of such changes at distinct developmental stages seems to be quite different in vivo. Interestingly, thin spines appear and disappear over a few days, while most thick spines persist for months (Grutzendler et al., 2002; Holtmaat et al., 2005; Zuo et al., 2005). Utilizing transcranial two‐photon microscopy, it was shown that during postnatal week two to four spine retractions exceed additions, resulting in a net loss of spines (Grutzendler et al., 2002). The fraction of persistent spines grows gradually during development and into adulthood providing evidence that synaptic circuits continue to stabilize even in the adult brain, long after the closure of known critical periods (Grutzendler et al., 2002; Holtmaat et al., 2005). Moreover, filopodia‐like dendritic protrusions, extending and retracting over hours, are abundant in young animals

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but virtually absent in the adults (Trachtenberg et al., 2002; Holtmaat et al., 2005; Zuo et al., 2005) (> Figure 10-5). In summary, the published data suggest that spines, initially plastic during development, become remarkably stable in the adult with the majority of them lasting throughout life, and providing a potential structural basis for long‐term information storage. This, however, does not seem to preclude synapse formation in the adult cortex in the context of experience‐dependent plasticity. Thus, experience‐ dependent plasticity of cortical receptive fields was reported to be accompanied by increased synapse turnover (Trachtenberg et al., 2002). . Figure 10-5 Time lapse 3D imaging of a GFP‐transfected hippocampal neuron grown in vitro for 10 days. The 3D image sequence shows ejecting and retracting filopodia as well as highly motile spines along a dendrite during an observation period of approximately 2 h

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6 Molecular Mechanisms of Spine Plasticity To this end, it is largely unknown how synaptic signaling is transferred to the underlying alterations in the spinous actin cytoskeleton and how altered spine morphology may feedback onto the efficacy of neurotransmission. It is assumed that structural alterations in the PSD precede the morphological alterations and some important mechanisms have been investigated. To this end, it was shown that activity‐dependent remodeling of the PSD is accompanied by altered protein turnover, which is remarkably rapid and robust (Ehlers, 2003). In turn, this remodeling occurs with corresponding increases or decreases in ubiquitin conjugation of synaptic proteins and requires proteasome‐mediated degradation (Colledge et al., 2003; Ehlers, 2003). Functionally, these modifications alter synaptic signaling to major downstream effectors (Pak et al., 2001, Pak and Sheng, 2003). In addition, a long‐lasting modification of synaptic strength requires protein synthesis (Kang and Schuman, 1996; Steward and Worley, 2002), suggesting that newly synthesized proteins contribute to the modification of synaptic efficacy. The detection of polyribosomes beneath synapses at the base of dendritic spines (Steward and Levy, 1982) indicates that protein synthesis may occur locally in dendrites under the control of the synapse itself. This concept implies that mRNAs are present in dendrites, and that dendritically synthesized proteins can mediate activity‐dependent structural and functional modifications of the postsynaptic apparatus. Therefore, active protein synthesis at or in spines has been proposed to function as a synaptic tag for transcription‐dependent memory formation (Frey and Morris, 1997). A variety of experimental evidence has underscored the involvement of local protein synthesis in paradigms of synaptic plasticity. Ostroff and coworkers (2002) could show that polyribosomes redistribute from dendritic shafts to synapses during LTP in rat hippocampal slices. The importance of dendritic protein translation for synaptic plasticity has been demonstrated for calcium/ calmodulin‐dependent protein kinase II‐a (CaMKII‐a) (Miller et al., 2002). Another important mechanism of synaptic plasticity is through movement of neurotransmitter receptors and regulatory proteins to and from the synapse (Malinow and Malenka, 2002; Kasai et al., 2003). Several activity‐triggered biochemical signaling mechanisms control these movements and their outcome can therefore be quite diverse (Sheng and Kim, 2002; Kennedy et al., 2005). Activation of glutamate receptors can cause an increase or decrease of spine size depending upon the activation scheme. These differing responses may be due to activation of diverse downstream molecules. For example, spine collapse requires calcineurin activation (Halpain et al., 1998), whereas an increase in size, which is likely due to an increased recruitment of AMPA receptors to the postsynaptic membrane, requires CaMKII (Jourdain et al., 2003; Matsuzaki et al., 2004). Interestingly, this pattern is reminiscent of long‐term depression (LTD) and LTP regulation and their morphological correlates in the regulation of spine size (Matsuzaki et al., 2004; Zhou et al., 2004). LTD has been shown to be accompanied by shrinkage of spine size while induction of NMDA receptor‐dependent LTP had the opposite effect (Matsuzaki et al., 2004; Zhou et al., 2004). The preferential sites of LTP induction are small spines, which express a small number of AMPA receptors. In accord, it was hypothesized that they might correspond to the postsynaptic structures of so‐called silent synapses (Matsuzaki et al., 2004). During LTP‐induction, NMDA receptor activation might also engage molecules that directly affect actin redistribution. For instance, profilin, an actin‐binding protein, moves to the spine head following NMDA receptor activation and suppresses actin dynamics (Ackermann and Matus, 2003), which leads to spine stabilization. Another actin‐binding protein, cortactin, shows a similar movement to the dendrite after NMDA activation (Hering and Sheng, 2003). A knockdown of cortactin, however, leads to a decrease in spine density, whereas overexpression results in an elongation of spines (Hering and Sheng, 2003). In addition to its binding site for actin, cortactin also binds to ProSAPs/Shanks, thereby providing a physical link between the NMDA receptor and the actin cytoskeleton that could potentially control spine morphology (Naisbitt et al., 1999). Accordingly, the protein levels of ProSAPs/ Shanks at the synapse control spine shape (Naisbitt et al., 1999). Other components of the PSD, like SAP90/PSD‐95, also indirectly link glutamate receptors to the actin cytoskeleton. This is brought about by its interaction with the small GTPase Ras, through the GTPase‐ activating protein (GAP) SynGAP (Chen et al., 1998), and Rap, through another GAP, SPAR (Pak et al., 2001). Other small GTPases, specifically those of the Rho family, play a role in the regulation of spine

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motility and morphogenesis (Tashiro et al., 2000). They are activated by guanine nucleotide exchange factors (GEFs). For one of these GEFs, kalirin‐7, it was shown that its overexpression can increase spine density (Penzes et al., 2001), whereas reduced kalirin‐7 levels are accompanied by shortened dendrites and significantly lower numbers of dendritic spines (Ma et al., 2003). The putative downstream effectors of small GTPases that control spine morphology include the p21‐activated kinase (PAK). Phosphorylated PAK colocalizes with SAP90/PSD‐95 in dendritic spines, and transgenic mice expressing a dominant negative form of PAK show a decreased spine density, morphological spine abnormalities, as well as enhanced LTP and reduced LTD (Hayashi et al., 2004). Moreover, recent studies show that the interaction of kalirin‐7 with Eph tyrosine kinase receptor has important consequences for spine morphology and incidence. These effects are most likely mediated by the downstream activation of PAK and small GTPases. Transsynaptic binding of the ligand ephrin‐B to the EphB receptor leads to a recruitment of kalirin‐7 to the activated spine. Kalirin‐7 subsequently activates Rac1 via its GEF activity, which then results in enhanced PAK activity and actin remodeling (Penzes et al., 2003), leading to an increased spine density and size. In summary, these studies are especially important because they will probably lead to a new conceptual framework in our understanding of how changes in the protein composition of the PSD can regulate spine morphogenesis. Further work will provide novel answers for the old question—which molecular mechanisms underlie synaptic plasticity and thereby indirectly learning and memory processes?

7 Structural Alterations of Spine Number and Formation in Brain Disease States First accounts of altered spine morphology in brain diseases date back to the 1980s. Given the delicate balance between the supply and breakdown of PSD molecules, it is likely that seemingly minor genetically or environmentally related perturbations of postsynaptic maintenance mechanisms may eventually lead to synaptic dysfunction, disintegration, and ultimately elimination, resulting in pathological synaptic loss and degenerative conditions. Indeed, PSD and dendritic spine pathologies have been associated with a rapidly increasing number of CNS diseases, ranging from neurodegenerative disease states to epilepsy and schizophrenia (> Figure 10-6). For example, the loss of synaptic contacts is an early hallmark of both Alzheimer and Huntington disease (Selkoe, 2002; Smith et al., 2005). The major hereditary mental retardation syndromes, fragile X and Down, are accompanied by changes in spine morphology, in particular a decrease in mature dendritic spines with well‐ formed PSDs and an increase in filopodia‐like protrusions that lack PSDs (Kaufmann and Moser, 2000; Irwin et al., 2000). The development of epileptic seizures is associated with rather drastic changes in the protein composition of the PSD (Wyneken et al., 2001, 2003) and the loss of dendritic spines (Mizrahi et al., 2004), whereas chronic administration of the psychostimulants cocaine and amphetamine produce increased spine density in the nucleus accumbens (Robinson and Kolb, 1997, 1999). In schizophrenia, one of the most prevalent psychiatric diseases, dysplastic events at the dendritic spine level not only are a common observation but also are thought to be causally related to the onset of the disease (Costa et al., 2001; Blanpied and Ehlers, 2004). More recently, downregulation of the expression of the synaptic scaffolding protein SAP90/PSD95 has been causally linked to synaptic dysfunction in cocaine‐induced drug addiction (Yao et al., 2004). This list could be extended further, although a question arises whether such alterations are really the cause or merely a consequence of a particular disease (Fiala et al., 2002b).

8 The Dendritic Spine in the Aging Brain At present, our knowledge about alterations in dendritic spines of the aged brain is still sparse. Most reports are concerned with the morphology and number of spines in the aged brain. But very little is known about the underlying mechanisms of such alterations. Aging is often accompanied by cognitive impairments like memory and spatial‐learning deficits in senescent humans, nonhuman primates, and rodents. In contrast to initial studies of neuronal densities, more recent studies based on stereological methods and counting of

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. Figure 10-6 Spine pathologies. (b) A reduction of the density of dendritic spines as compared to (a) the spines occur in cases of deafferentation, most mental retardation syndromes, severe alcohol abuse, epilepsy, Alzheimer’s disease, and others. (c) An increase in dendritic spine density was shown for some types of deafferentation syndromes, the fragile X syndrome, sudden infant death syndrome, and the abuse of psychostimulants. (d) Sensory deprivation leads to a reduction of spine size. The same spine phenotype is described for Down’s syndrome and schizophrenia. Morphological changes that result in a (e) distortion of spine shape are also typical for most mental retardation symptoms, epilepsy, severe alcohol abuse, poisoning with neurotoxicants, and spongiform encephalitis

total neuron number of brain material of several species have established that only little, if any, loss of excitatory and inhibitory neurons occurs in different brain regions in normal aging and that this loss cannot account for the observed age‐related functional deficits in multiple neurotransmitter systems. Studies have demonstrated subregional specificities in sensitivity to the effects of advancing age, and it has become increasingly clear that the mechanisms underlying age‐related changes are of a more subtle nature and sometimes compensatory rather than detrimental to cognitive functioning (Morrison and Hof, 1997). Disruption and age‐related structural alterations of the myelin sheaths of neocortical axons have been described and correlated with age and degree of impairment in monkeys (Peters et al, 2000). In both rodents and humans, changes have been reported in dendritic arbor, spines, and synapse morphology (Morrison and Hof, 1997). Spine number and density seem to decrease with age (Duan et al., 2003), and dendritic length seems to decline in cortical neurons (Jacobs et al., 1997). Rather than changes in spine number, reduction in size of perforated postsynaptic densities, whose formation is postulated to be a structural correlate of enhanced efficacy of synaptic transmission, has been reported for hippocampal axospinous synapses (Nicholson et al., 2004). Studies in the monkey revealed decreased synaptic excitation and increased synaptic inhibition of layer 2 and 3 pyramidal cells in the prefrontal cortex (Luebke et al., 2004). Thus, alterations first appear on the level of synaptic connectivity and further manifest themselves on the subcellular and molecular level. Bertoni‐Freddari and coworkers (2003, 2004, 2005) recently described age‐induced changes in cellular energy metabolism. They observed an increase in the number of enlarged synaptic mitochondria with generally decreased metabolic competence in cerebellar cortex of adult rats, as well as an age‐related decline

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in metabolic competence of small and medium‐sized synaptic mitochondria. Age also affects Ca2þ homeostasis and changes in regulation of intracellular Ca2þ concentrations underlie some age‐related deficits in plasticity and cognition (reviewed in Foster, 1999; Foster and Kumar, 2002; Rosenzweig and Barnes, 2003; Toescu and Verkhratsky, 2004). A number of mechanisms for handling Ca2þ are thought to be modified during aging, including intracellular buffering, extrusion, and influx of Ca2þ. Consequently, hippocampal synaptic function is altered as seen by a shift in synaptic plasticity thresholds with LTP requiring a higher stimulation threshold for induction and more induction sessions to saturate LTP mechanisms in aged animals, and LTD showing a reduction in threshold, such that robust LTD is observed for lower frequency stimulation patterns in aged compared with young animals. Thus, aged animals acquire new information only slowly and with more repetition and forget rapidly. In this context, it is hypothesized that a postsynaptic increase in Ca2þ influx through L‐type VDCCs and reduced Ca2þ influx through NMDARs with age not only modifies the threshold for induction of Ca2þ‐dependent synaptic plasticity in favor of LTD induction but also increases the duration and amplitude of afterhypolarization following cell discharge activity and with that alters information processing. The effects seem to be mediated by age‐related changes in the Ca2þ‐signaling pathways, including the activity of protein phosphatases and kinases involved in the expression of synaptic plasticity. The Ca2þ‐ and CaM‐dependent phosphatase calcineurin and protein phosphatase 1, which is regulated by calcineurin, are enzymes critical for LTD and according to Foster and coworkers (2001), activity of both increases with advanced age in an L‐channel related manner. They showed that the phosphorylation state of calcineurin substrates (e.g., of CREB) was reduced and that increased calcineurin activity correlated with memory function. This finding is challenged by a more recent study by Agbas and coworkers (2005) who showed that calcineurin activity decreases in the aging brain due to increased aggregation of the catalytic subunit of calcineurin caused by oxidation. Other components of the synaptic transmission machinery have also been analyzed for age‐related changes. Whereas several groups examined the expression of membrane channel subtypes and receptor subunits in cortical and hippocampal neurons of different species and documented regional and subtype/ subunit and sometimes species specific alterations (Tanaka and Ando, 2001; Hof et al., 2002; Bai et al., 2004; Iwamoto et al., 2004), levels of presynaptic proteins like synaptotagmin‐1, synaptophysin, and syntaxin in rat cerebral cortex were not significantly reduced with age (Iwamoto et al., 2004). Interestingly, in animal models some of the described age‐related changes and effects on cognitive function can be reversed either directly or in a complementary way by the administration of trophic factors that otherwise decline with age, like insulin‐like growth factor‐1 and estrogen (Sonntag et al., 2000; Adams and Morrison, 2003; Adams et al., 2004; Shi et al., 2005).

Acknowledgments The work of M.R.K. is supported by the BMBF, DAAD, DFG, the Land Sachsen‐Anhalt, and the Schram Foundation.

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S. Roßner . S. F. Lichtenthaler

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Introduction to Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262

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The Discovery of BACE Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264

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BACE Gene Structure and Alternative Splicing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265

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Transcription and Translation of BACE1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268

5 Posttranslational Modifications, Intracellular Transport, and Binding Partners of BACE1 . . . 271 5.1 Posttranslational Modifications and Intracellular Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 5.2 BACE1 Interacting Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 272 6 6.1 6.2 6.3 6.4 6.5

BACE1 Expression in Aging and AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 Localized BACE1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 APP Transgenic Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 BACE1 Expression in AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 Astrocytic BACE1 Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275

7 Biological Function and Substrate Spectrum of BACE1 and BACE2 . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 7.1 Substrates of BACE Proteases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 276 7.2 BACE1 Deficient Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278

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Abstract: The aberrant proteolytical processing of the amyloid precursor protein (APP) gives rise to b‐amyloid peptides, which accumulate as perivascular or parenchymal deposits in brains of Alzheimer’s disease (AD) patients. The generation of b‐amyloid peptides is initiated by APP cleavage by an aspartyl protease named beta‐site APP‐cleaving enzyme 1 (BACE1), followed by g‐secretase cleavage. The discovery of BACE1 some years ago and subsequent studies on the regulation of its expression and enzymatic activity allowed for a better understanding of APP processing, b‐amyloid generation and the pathogenesis of AD in general. Some of the unique features of BACE1, such as regulatory elements in the 50 region of BACE1 mRNA and factors that contribute to its cell type‐specific activation helped to explain epidemiological observations and opened new rational therapeutic opportunities. However, although BACE1 has been shown to be the only protease in brain with significant b‐secretase activity, there are a number of alternative substrates in addition to APP, indicating that a careful estimation of possible side effects of BACE1 inhibitors is required. Nevertheless, experimental data from transgenic mouse models of AD, BACE1 knockout mice and pharmacological studies corroborate the potential usefulness of drugs that interfere with BACE1 expression and/or enzymatic activity for the treatment of AD. List of Abbreviations: Ab, b‐amyloid peptide; AD, Alzheimer’s disease; ADAM, a disintegrin and metalloprotease; APLP, amyloid precursor‐like protein; APP, amyloid precursor protein; BACE, beta‐site APP‐ cleaving enzyme; CCS, copper chaperone for superoxide dismutase‐1; ChAT, choline acetyltransferase; DS, Down’s syndrome; ER, endoplasmatic reticulum; FAD, familial forms of Alzheimer’s disease; GFAP, glial fibrillary acidic protein; GGA, Golgi‐localized g‐ear‐containing ARF binding; HEK, human embryonic kidney; MDCK, Madin‐Darby canine kidney; NSAIDs, nonsteroidal anti‐inflammatory drugs; PLSCR 1, phospholipid scramblase 1; PPARg, peroxisome proliferator‐activated receptor‐g; PPRE, PPARg responsive element; pro, prodomain; RTN3, reticulon 3; RTN4b, reticulon 4b; TGN, trans‐Golgi network; SP, signal peptide; TACE, tumor necrosis factor a‐converting enzyme; TMD, transmembrane domain; 50 UTR, 50 untranslated region; uORF, upstream open reading frame

1

Introduction to Alzheimer’s Disease

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease, affecting about 20 million people worldwide [for an overview see Selkoe and Schenk (2003)]. It is characterized by progressive loss of memory, declining cognitive function and, ultimately, leads to decreasing physical functions and death. The neuropathological hallmarks of AD are the senile plaques and the neurofibrillary tangles, which are protein aggregates deposited in the brain. The neurofibrillary tangles represent intraneuronal bundles of paired helical filaments, which mainly consist of the microtubule‐associated protein tau in an abnormally phosphorylated form [(for a review see Iqbal et al. (2005)]. The extracellular amyloid plaques mainly consist of the 42 residue long amyloid b‐peptide (Ab, bA4) (Glenner and Wong, 1984; Masters et al., 1985), which is proteolytically derived from the much larger amyloid precursor protein (APP, bAPP) (Kang et al., 1987). The generation and subsequent aggregation of Ab seem to be at the origin of the disease and are believed to trigger a complex pathological cascade that causes neuronal dysfunction, the appearance of the neurofibrillary tangles, inflammatory processes, neuronal loss, cognitive impairment, and finally the onset of the disease. This pathological cascade, which is now widely accepted, is also referred to as the amyloid hypothesis of AD [for overview see Hardy and Selkoe (2002), Citron (2004)]. Although drugs are currently available that may ameliorate late‐stage symptoms like the cognitive deficits for a short time, no drugs are on the market that specifically target the cellular mechanisms of the disease, namely the proteolytic generation of the Ab peptide from APP. APP is a type I membrane protein with unclear biological function. Suggested functions for APP include a role in cell adhesion and copper homeostasis [for a review see De Strooper and Annaert (2000)], cellular motility (Sabo et al., 2001) and being a cell surface receptor (Cao and Sudhof, 2001) or a receptor for the motor protein kinesin (Kamal et al., 2001). The proteolytic processing of APP involves protease activities that are referred to as a‐, b‐, and g‐secretase because their molecular identity was initially not known. The three proteases process APP in two different pathways (> Figure 11-1a), one of which is called anti‐amyloidogenic because it prevents Ab generation. The other one is termed amyloidogenic as it leads to the generation of the Ab peptide. Both

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. Figure 11-1 Proteolytic processing pathways of APP. (a) Amyloidogenic pathway: Cleavage of APP by the protease b‐secretase (BACE1) occurs at the N‐terminus of the Ab‐domain and yields the secreted sAPPb and a C‐terminal fragment of APP of 99 amino acids (C99). C99 is further cleaved within its transmembrane domain by g‐secretase, leading to the secretion of the Ab peptide and the generation of the APP intracellular domain (AICD). The consecutive cleavage of APP by b‐ and g‐secretase constitutes the amyloidogenic pathway as it generates Ab. Anti‐amyloidogenic pathway: Cleavage of APP by a‐secretase or potentially BACE2 within the Ab domain yields the neurotrophic and neuroprotective sAPPa as well as the C‐terminal fragment C83. The a‐secretase is a member of the ADAM family of metalloproteases. Similar to the processing of C99, C83 is further processed by g‐secretase within its transmembrane domain, leading to the generation of AICD and to the secretion of the p3 peptide that is not deposited in amyloid plaques. (b) Amino acid sequence of APP within and around the Ab peptide domain. Indicated are the major cleavage sites of the different protease activities and the double mutation directly preceding the b‐secretase cleavage site. This mutation is responsible for a familial form of AD found in a Swedish family. Amino acids are shown in the one‐letter code and numbered according to the Ab sequence

pathways and their cleavage products (including Ab) are part of the normal proteolytic processing of APP [for an overview see Selkoe and Schenk (2003)], which occurs in most cells. In the amyloidogenic pathway, APP is first cleaved by the b‐secretase at the N‐terminus of the Ab domain (> Figure 11-1a). This cleavage generates the soluble sAPPb and a C‐terminal fragment, which undergoes a second cleavage by a protease called g‐secretase. The b‐secretase was recently identified as the membrane‐bound aspartyl protease BACE1 (also named memapsin2, Asp2). g‐Secretase also belongs to the aspartyl family of proteases but is an unusual protease activity in that it cleaves APP within its hydrophobic transmembrane domain. g‐Secretase is a hetero‐tetrameric protein complex consisting of presenilin, nicastrin, PEN‐2, and APH‐1 [for a review see Haass (2004)]. g‐Secretase cleavage occurs mainly after residue 40 and to a lower extent after residue 42 of the Ab sequence, thereby generating the 40 and 42 amino acid long Ab40 and Ab42 peptides (> Figure 11-1b). Due to its more hydrophobic nature and its higher tendency to aggregate, it is the Ab42 peptide but not the Ab40 peptide, which is assumed to be the key player in the pathogenesis of AD. In fact, the amyloid plaques mainly consist of Ab42. The physiological relevance of b‐ and g‐secretase in Ab generation is clearly established. Mice deficient in the b‐secretase or the g‐secretase activity do not generate Ab, making both proteases suitable drug targets for AD [reviewed in Haass (2004)].

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In contrast, the anti‐amyloidogenic pathway starts with APP cleavage by a‐secretase, which cuts within the Ab domain (> Figure 11-1a, b). Thus, a‐secretase cleavage precludes Ab generation. Following a‐cleavage the C‐terminal APP fragment undergoes intramembrane cleavage by the g‐secretase complex. This leads to the generation of the p3 peptide (Haass et al., 1993) (> Figure 11-1a), which is assumed to be benign as it is not found in the AD amyloid plaques. a‐Secretase is a member of the ADAM‐family of proteases (A disintegrin and metalloprotease) [for a review see Allinson et al. (2003)], and is either ADAM10 (Lammich et al., 1999), ADAM17/tumor necrosis factor a‐converting enzyme (TACE) (Buxbaum et al., 1998) or even ADAM9 (Koike et al., 1999). At present, it is unclear, whether only one of them or all three together constitute the physiologically relevant a‐secretase. An a‐secretase‐like proteolytic cleavage at residues 19 and 20 of Ab (> Figure 11-1b) is catalyzed by the protease BACE2 (Farzan et al., 2000; Yan et al., 2001), which is a close homolog of the b‐secretase BACE1. Because BACE2 cleaves within the Ab sequence, it does not contribute to Ab formation, which is consistent with the finding, that a BACE1 knockout alone in mice is sufficient to completely suppress Ab generation (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001). The essential role of the secretases in the generation or prevention of the Ab peptide is underlined by genetic evidence. Mutations in the APP gene linked to familial forms of Alzheimer’s disease (FAD) are found at or very close to the secretase cleavage sites and generally increase Ab generation. The Swedish double mutation, which was found in a Swedish FAD family, is located directly N‐terminally to the b‐secretase cleavage site (> Figure 11-1b) and allows a much more efficient b‐secretase cleavage of APP and turnover to Ab. Mutations close to the g‐secretase site do not increase the total amount of Ab being generated but increase the amount of the longer Ab42 species relative to the Ab40 peptide. Additionally, FAD mutations were found close to the a‐secretase cleavage site, and appear to reduce a‐secretase cleavage, resulting in an increased b‐secretase cleavage of APP and thus in more Ab generation. The following chapters highlight various aspects of BACE1 and BACE2 protease biology, starting with a description of the discovery of both proteases.

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The Discovery of BACE Proteases

Early on in the research of the APP secretases, cell biological and pharmacological studies revealed that b‐secretase is an enzyme different from a‐ and g‐secretase. It became clear that the b‐secretase cleavage requires an acidic environment and is localized both in endosomes and in the secretory pathway (Koo and Squazzo, 1994; Haass et al., 1995). Moreover, it only cleaved membrane‐bound APP, suggesting that b‐secretase itself is a membrane protein. Additionally, b‐secretase activity was found in most tissues analyzed and showed a particularly strong activity in brain. Moreover, it cleaved Swedish mutant APP much more efficiently than wild‐type APP (Citron et al., 1992, 1995). Over the years, different candidate b‐secretases were suggested, but none fulfilled all the criteria for b‐secretase. All three APP secretases were identified within a short time. After the identification of ADAM17 and ADAM10 as a‐secretases (Buxbaum et al., 1998; Lammich et al., 1999), the presenilins were shown in early 1999 to be the catalytic subunit of g‐secretase (Wolfe et al., 1999). At the end of the same year, b‐secretase was the last secretase to be identified. Vassar and colleagues were the first ones to report the identification of the b‐secretase enzyme, which they named b‐site APP cleaving enzyme (BACE1, also called memapsin2 or Asp2) (Vassar et al., 1999). Four subsequent reports by other groups identified the same enzyme (Hussain et al., 1999; Sinha et al., 1999; Yan et al., 1999; Lin et al., 2000). The fact that all groups used different experimental approaches to identify BACE1 made a strong case for BACE1 being b‐secretase. Vassar and colleagues used an expression cloning approach to identify b‐secretase from a cDNA library derived from human embryonic kidney 293 cells (HEK293) (Vassar et al., 1999). As a reporter cell line they used HEK293 cells stably expressing Swedish mutant APP and transiently transfected them with pools of cDNAs. Pools that increased Ab secretion into the conditioned medium were further subdivided by the sib selection approach until they finally identified the individual cDNA that increased Ab generation. This cDNA turned out to be BACE1.

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Another group used an affinity‐purification strategy and identified b‐secretase from human brain using a peptide‐based transition‐state analog of the APP sequence (Sinha et al., 1999). This compound inhibits b‐secretase activity and binds tightly to the protein. After several enrichment steps the enzyme was N‐terminally sequenced. The obtained amino acid sequence was then used to clone the cDNA of b‐secretase. Three additional groups hypothesized that b‐secretase is an aspartyl protease and applied a genomics approach searching EST databases to identify BACE1 (Hussain et al., 1999; Yan et al., 1999; Lin et al., 2000). BACE1 is a type I membrane protein with 501 amino acids (> Figure 11-2). Its gene is encoded on chromosome 11. The N‐terminal signal peptide is followed by a prodomain, a catalytic domain comprising the two catalytic aspartic acid residues [amino acids 93–96 (DSGT) and amino acids 289–292 (DTGS)] characteristic for aspartyl proteases, a transmembrane domain, and a short cytoplasmic tail. Several experiments show that BACE1 fulfills all criteria for being b‐secretase (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et al., 1999; Lin et al., 2000). Mutation of the catalytic active site aspartic acid . Figure 11-2 Domain structures of BACE1 and BACE2. N‐ and C‐termini (N, C) of both proteins are indicated as well as the transmembrane domain (TMD) and the cleavage sites of the signal peptide (SP) and the prodomain (pro). Also shown are the two catalytic aspartic acid residues (D) and the C‐terminal sequences of both proteins

residues abolishes its activity, providing evidence that BACE1 is indeed an aspartyl protease. Moreover, BACE1 acts as a protease in vitro assays and cleaves APP at the correct peptide bond. The catalytic residues are in the luminal domain of BACE1, and thus have the correct topology for cleaving within the luminal domain of APP. Overexpression of BACE1 increases b‐secretase cleavage of APP and Ab generation. Conversely, BACE1 knockout mice do not generate Ab anymore (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001). BACE1 is ubiquitously expressed, with higher expression levels observed in brain and particularly in neurons, which is in agreement with the increased b‐secretase activity found in this cell type. Additionally, BACE1 has an acidic pH‐optimum of 4.5, and localizes to the Golgi, TGN, and the endosomes, where b‐secretase activity is found. Most important, BACE1 cleaves the Swedish mutant APP much more efficiently than wild‐type APP (Lin et al., 2000; Gruninger‐Leitch et al., 2002). Taken together, BACE1 fulfills the criteria for being b‐secretase. Shortly after the discovery of BACE1, a homolog was identified by EST database searches and named BACE2 (also referred to as memapsin1, Asp1, or DRAP) (Saunders et al., 1999; Yan et al., 1999; Acquati et al., 2000; Lin et al., 2000; Solans et al., 2000). The BACE2 protein contains 518 amino acids, and its sequence shares 46% identity and 62% similarity with the corresponding sequence of BACE1. Both proteases are evolutionarily highly conserved. The sequence identity between the human and the mouse protein is 95% for BACE1 and 89% for BACE2. BACE1 and BACE2 belong to the pepsin family of aspartyl proteases, and are the only membrane‐bound family members. BACE2 shares the same domain structure as BACE1 (> Figure 11-2).

3

BACE Gene Structure and Alternative Splicing

Initial important information on the regulation of BACE1/BACE2 expression and APP processing might be obtained from the BACEs gene structure and alternative splicing events. Because BACE1 is the predominant b‐secretase in brain, and BACE2 is expressed peripherally under basal conditions, most studies performed so far focused on BACE1 only.

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BACE1 mRNA encoding the full‐length, 501 aa BACE1 protein is expressed at high levels in brain and in pancreas, but significant concentrations of enzymatically active BACE1 proteins are found exclusively in brain (Hussain et al., 1999; Sinha et al., 1999). BACE1 mRNAs are synthesized as nine exons and eight introns from a 30.6 kb region of the human chromosome 11q23.2–11q23.3 (Saunders et al., 1999) and the presence of multiple mRNAs that terminate at different sites has been attributed to variable polyadenylation (Sambamurti et al., 2004). Additionally, full‐length BACE1 mRNA may undergo alternative splicing to give rise to three truncated BACE1 mRNAs with the deletion of 132, 75, and 207 nucleotides, respectively (Tanahashi and Tabira, 2001; see also > Figure 11-3). Removal of 132 bp of exon 3 results in the expression . Figure 11-3 Alternative splicing of BACE1 mRNA. Schematic representation of BACE1 coding exons and alternatively spliced transcripts. Alternative splicing leads to the removal of 132 (44), 75 (25) or 207 (69) nucleotides (amino acids) and results in the expression of BACE1‐457; BACE1‐476, and BACE1‐432 isoforms. In the transcript BACE1‐457 two‐thirds of exon 3 are spliced, resulting in the removal of two glycosylation sites (arrows) of the corresponding protein. In the transcript BACE1‐476, 75 nucleotides of exon 4 are spliced and in the BACE1‐ 432 mRNA both mentioned nucleotide sequences of exon 3 and exon 4 are removed. Asterisks, active‐site motifs; SP, signal peptide sequence; pro, prodomain; TMD, transmembrane domain

of a 457 aa BACE1 protein lacking two glycosylation sites between the two active site motifs (> Figure 11-3). This BACE1‐457 mRNA was detected at high levels not only in pancreas (Bodendorf et al., 2001) but also at much lower concentrations in brain (Tanahashi and Tabira, 2001). The resultant BACE1‐457 protein is retained in a proenzymatic and endoglycosidase H‐sensitive state in the endoplasmatic reticulum (ER; Bodendorf et al., 2001; Tanahashi and Tabira, 2001). Similar observations were made for the BACE1‐476 isoform lacking 75 nucleotides of exon 4 (Tanahasi and Tabira, 2001). Because the BACE1‐501 isoform is resistant to endoglycosidase H treatment (Capell et al., 2000; Huse et al., 2000), different posttranslational modifications of the distinct BACE1 isoforms are discussed (see also > Section 5). Most importantly, the deletion of 25 or more aa between the two active‐site motifs of BACE1 impairs APP processing at the b‐secretase site. This might partly explain the discrepancy between high levels of BACE1 mRNA but low BACE1 enzymatic activity with regard to APP cleavage in pancreas. However, despite the existence of truncated BACE1 mRNAs in pancreas, the full‐length BACE1 isoform has been shown by RNAse protection assays to be the major pancreatic BACE1 transcript (Ehehalt et al., 2002). On the other hand, the presence of BACE1 isoforms with reduced or lacking b‐secretase activity might be interpreted as a first hint for the subsistence of alternative BACE1 substrates, which are processed by tissue‐specific BACE1 isoforms in distinct organs.

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What is the function of BACE2? Although it cleaves APP more efficiently within the b‐amyloid sequence than at the b‐secretase site (see also > Figure 11-1), it might play a role in specific forms of AD. When comparing by database analysis the chromosomal localization of the BACE2 gene in different species it came to our attention that BACE2 (but not BACE1) and APP map to the same chromosome in mice, rats, and humans (> Figure 11-4). The localization of the APP gene on human chromosome 21 (Kang et al.,

. Figure 11-4 Chromosomal localization of BACE1, BACE2, and APP in mouse, rat, and human. BACE2, but not BACE1, and its substrate APP map to the same chromosome in all three species

1987; Tanzi et al., 1987) and the finding that virtually all patients more than the age of 40 suffering Down’s syndrome (DS; trisomy 21) invariantly develop the clinical and pathological characteristics of AD, support the hypothesis that increased APP expression plays a significant role in the progression of the disease [for review see Kola and Hertzog (1997)]. In addition to APP, its secretase BACE2 is localized on human chromosome 21 and maps to the ‘‘Down critical region’’ in 21q22.3 (Acquati et al., 2000; Solans et al., 2000) and, therefore, the increased expression of both, substrate and its processing secretase might increase the generation of Ab peptides and contribute to the pathogenesis of AD. Indeed, the increased secretion of BACE2 from fibroblasts of patients suffering from DS has been reported (Barbiero et al., 2003). BACE2 overexpression was also observed in the DS fetal brains and in human neural embryonic DS stem cells in which conditioned media BACE2 was secreted (Barbieri et al., 2003). Moreover, changes in the expression of BACE2 were observed immunohistochemically in the frontal cortex of DS patients. The immunoreactivity for BACE2 was particularly detected in neurofibrillary tangle‐bearing neurons from the elderly DS brains with AD‐type neuropathology, but was not detected in those of DS brains without AD‐type neuropathology or in those of control brains of any age (Motonaga et al., 2002). This suggests the possibility that the elevated expression of BACE2 is involved in the AD‐type neuropathology of DS. However, recent studies using transfection and knockdown strategies revealed that overexpression of BACE2 reduces amyloidogenic APP processing and increases the generation of nonamyloidogenic APP fragments whereas elimination of BACE2 produced opposite effects (Sun et al., 2005, 2006a). It has to be established in transgenic mice how BACE2 functions in brain in vivo. One way to address that question is to overexpress BACE2 neuronally on the background of hAPPtg/BACE1
/
mice and to quantify Ab generation and Ab plaque formation. An alternative mechanism possibly contributing to AD pathology in DS patients was revealed recently. DS brains displayed an higher ratio of mature to immature forms of BACE1 in the Golgi, resulting in increased b‐secretase activity (Sun et al., 2006b). This indicates that overproduction of Ab in DS might be caused by abnormal BACE1 protein trafficking and maturation.

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The transcriptional regulation of BACE2 appears to be quite different from that of BACE1. The BACE2 gene is controlled by a TATA‐less promoter and although SP1 can regulate BACE1 and BACE2 genes there is very little similarity between the two promoters as revealed by comparative sequence analysis and transcription factor prediction (Sun et al., 2005). Moreover, these authors identified a neuron‐specific repressive element between
652 bp to
200 bp of the BACE2 promoter. Although it is not known yet under which conditions the neuronal BACE2 repression can be overruled, such information might be important with regard to disease‐related APP processing (see earlier) and a potential upregulation of neuronal BACE2 expression as an AD treatment strategy.

4

Transcription and Translation of BACE1

Observations on altered BACE1 protein concentrations in brains of sporadic AD patients (see later) indicate transcriptional and/or translational regulation of BACE1 expression in brain. The BACE1 promoter is highly conserved between rat, mouse, and human (> Figure 11-5) and contains a number of putative transcription factor binding sites (Lange‐Dohna et al., 2003). This would suggest that regulatory mechanisms of BACE1 expression are common between these species and that mice and rats represent valuable animal models to reveal such mechanisms and to test therapeutic strategies aimed at reducing BACE1 expression in AD. Interestingly, the BACE1 promoter contains several putative transcription factor binding sites that are known to be involved in the expression of other AD‐related proteins. For example, NF‐kB sites are present in the promoters of APP (Grilli et al., 1995), choline acetyltransferase (ChAT; Toliver‐Kinsky et al., 2000) and presenilins [for review see Glasgow and Perez‐Polo (2000)]. Likewise, SP‐1 sites have been reported for the APP (Lahiri and Robakis, 1991; Pollwein, 1993; Lukiw et al., 1994; Hoffman and Chernak, 1995), ChAT (Hahn et al., 1992; Inoue et al., 1993), presenilin‐1 (Mitsuda et al., 1997), and presenilin‐ 2 (Pennypacker et al., 1998) promoters. Based on results derived from luciferase reporter assays it was suggested that an upstream NF‐kB site might act as a repressor of BACE1 transcription (Lange‐Dohna et al., 2003). A detailed analysis using BACE1 promoter constructs carrying mutations of the NF‐kB site revealed a unique cell type‐specific regulation of BACE1 promoter activity. In neuron‐like cells, such as retinoic acid‐ differentiated SH‐SY5Y cells or nerve growth factor‐differentiated PC12 cells, the NF‐kB site indeed acts as repressor of the BACE1 promoter whereas in C6 glioma cells it acts as activator (Bourne et al., 2005). This has important therapeutic implications and might explain in part the cell type‐specific upregulation BACE1 in reactive astrocytes of APP transgenic mice with amyloid plaque formation (Roßner et al., 2001), in chronic lesion paradigms (Hartlage‐Ru¨bsamen et al., 2003) and in brains of AD patients (Hartlage‐ Ru¨bsamen et al., 2003; Leuba et al., 2005). These results are also consistent with observations demonstrating that nonsteroidal anti‐inflammatory drugs (NSAIDs) decreased the BACE1 mRNA levels, protein expression, and also BACE1 enzymatic activity (Sastre et al., 2003). Pharmacological studies suggested that this effect is mediated through activation of the peroxisome proliferator‐activated receptor‐g (PPARg; Sastre et al., 2003). In line with that, the absence of PPARg potentiates BACE1 mRNA levels by increasing BACE1 promoter activity. Conversely, overexpression of PPARg as well as NSAIDs and pioglitazone, a PPARg activator, reduced BACE1 promoter activity (Sastre et al., 2006). These effects appear to be mediated by a PPARg responsive element (PPRE) within the BACE1 promoter. Mutagenesis of this PPRE element abolished the binding of PPARg to the PPRE and increased BACE1 gene promoter activity (Sastre et al., 2006). Furthermore, pro‐inflammatory cytokines decreased the transcription of the PPARg gene, an effect that was suppressed by NSAIDs. Interestingly, brain extracts from AD patients showed decreased PPARg expression and reduced binding to PPRE in the BACE1 promoter. From these results, it was concluded that the protective mechanism of NSAIDs in AD is exerted through activation of PPARg and decreased BACE1 gene transcription (Sastre et al., 2006). This conclusion is supported by data from a transgenic mouse model of AD, in which treatment with NSAIDs or pioglitazone suppressed pro‐inflammatory markers, reduced BACE1 mRNA and protein levels and lowered Ab plaque load (Heneka et al., 2005). Transcriptional regulation of BACE1 expression occurs also via a functional SP1 element 850 nucleotides upstream of the transcription start site (Christensen et al., 2004). In HEK293T cells and in PC12 cells this SP1‐ site appears to act as an activator of BACE1 expression as shown by reduced BACE1 expression after

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. Figure 11-5 Rat, mouse, and human BACE1 promoter sequences. Comparison of the rat BACE1 promoter sequence with that of its mouse and human homologs. There is 92% similarity between rat and mouse and 81% similarity between rat and human within the region from bp
1 to
600. The similarity between rat and mouse by about 86% extends between bp
600 and
1000, but the similarity between the rat and the human sequence is only 39% in this fragment. At a greater distance than bp
1000, this similarity is reduced significantly (after Lange‐Dohna et al., 2003; modified)

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targeted disruption of the SP1 gene and by the potentiated BACE1 expression after overexpression of SP1 (Christensen et al., 2004). In addition to a possible regulation of BACE1 expression by transcriptional mechanisms, there is ample evidence for posttranscriptional modulation of BACE1 enzymatic activity. For example, BACE1 protein levels are significantly upregulated up to 2.7‐fold in the brains of sporadic AD patients compared with nonAD controls (Fukumoto et al., 2002; Holsinger et al., 2002; Yang et al., 2003), whereas mRNA levels were unchanged (Yasojima et al., 2001; Holsinger et al., 2002; Preece et al., 2003). This indicates that posttranscriptional mechanisms, such as translational control or altered BACE1 protein degradation, regulate BACE1 protein levels. In fact, tissue culture experiments have provided evidence for both mechanisms in controlling BACE1 protein levels. An altered BACE1 degradation has been described by Kovacs and coworkers (Puglielli et al., 2003). They reported that the lipid second messenger ceramide, which is elevated in the brain of AD patients, increases the half‐life of BACE1 and thereby increases Ab generation (Puglielli et al., 2003). Translational control of BACE1 expression has been observed independently by different groups (De Pietri Tonelli et al., 2004; Lammich et al., 2004; Rogers et al., 2004). The studies were based on the observation that the 50 untranslated region (50 UTR) of the BACE1 mRNA contains unusual sequence features that are often found in mRNAs showing tight translational control. In most vertebrate mRNAs, the 50 UTR is 10–200 nucleotides long, is unstructured, not very GC‐rich and does not contain upstream open reading frames (uORFs). In contrast, less than 10% of vertebrate genes, including many regulatory proteins like proto‐oncogenes, have 50 UTRs that are longer than 200 nucleotides and are often GC‐rich (70–90%). This indicates a high degree of secondary structure, which may impede the efficient scanning of the ribosome (Kozak, 1987; Willis, 1999). Additionally, these long 50 UTRs may contain uORFs [for a review see Clemens and Bommer (1999)] that can inhibit the translation of the main ORF. The 50 UTR of BACE1, which is highly conserved in the human, rat, and murine sequence, belongs to the class of the long 50 UTRs similar to many regulatory genes. It is 446 nucleotides long, has a GC‐content of 77%, and contains 3 uORFs (> Figure 11-6). Importantly, all three different BACE1 mRNA species detected by Northern Blot analysis

. Figure 11-6 The mRNA domain structure of BACE1. The BACE1 mRNA consists of the 50 untranslated region (50 UTR), followed by the coding region (open reading frame with the start codon AUG) and the 30 UTR. The 50 UTR has the indicated high GC‐content and contains three upstream open reading frames (uORFs), represented as boxes

contain the 50 UTR (Lammich et al., 2004). The role of the 50 UTR in a possible translational control of BACE1 mRNA was tested in different cell lines as well as in vitro translation experiments (De Pietri Tonelli et al., 2004; Lammich et al., 2004; Rogers et al., 2004). The use of BACE1 constructs lacking part or all the 50 UTR revealed that the presence of the 50 UTR decreased BACE1 translation by up to 90%. Moreover, when fused to luciferase, the 50 UTR of BACE1 strongly suppressed luciferase translation and activity, revealing that the translation repressing activity must be encoded within the 50 UTR. The uORFs within the 50 UTR were not the major reason for the inhibition of translation. Instead, the experiments suggest that due to its high GC‐content the 50 UTR forms a constitutive translation barrier that prevents the ribosomes from efficiently translating the BACE1 mRNA. Additionally, Zacchetti and colleagues showed that the 50 UTR dependent translational repression may be alleviated in activated astrocytes (De Pietri Tonelli et al., 2004), leading to an increased expression of BACE1 protein. In fact, activated astrocytes expressing elevated levels of BACE1 are found around amyloid plaques in an animal model of AD and in the AD brain (Roßner et al.,

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2001; Hartlage‐Ru¨bsamen et al., 2003) (see also > Section 6). Thus, pathophysiological conditions leading to the activation of astrocytes may increase BACE1 expression and thereby Ab generation and could exacerbate AD pathogenesis.

5

Posttranslational Modifications, Intracellular Transport, and Binding Partners of BACE1

A variety of cell biological studies have provided important insights into the posttranslational modifications of BACE1 and the control of its intracellular transport. Like in the studies addressing transcriptional and translational control of BACE1 expression (> Section 4), an elucidation of BACE1 modifications or transport may allow to find new therapeutic approaches for AD. They may be used to lower BACE1 activity or shift the APP cleavage away from the Ab generating b‐secretase toward an increased a‐secretase cleavage that prevents Ab generation.

5.1 Posttranslational Modifications and Intracellular Transport On cotranslational integration of BACE1 into the ER the signal peptide is cleaved after amino acid 21 (> Figure 11-2). The immature form of BACE1 (pro‐BACE1) in the ER is N‐glycosylated and has an apparent molecular weight of 60 kDa. On rapid transport through the Golgi, BACE1 undergoes maturation. Complex sugars are added (Charlwood et al., 2001), which become sulfated (Benjannet et al., 2001), and BACE1 becomes endoglycosidase H resistant (Capell et al., 2000). Furin or a furin‐like prohormone convertase removes the prodomain by cleaving after residue 45 (> Figure 11-2) (Bennett et al., 2000; Capell et al., 2000; Benjannet et al., 2001). The maturation of BACE2 follows a similar pattern as BACE1. However, its prodomain cleavage is autocatalytic (Hussain et al., 2001). The prodomain of many proteases, such as ADAM10 and ADAM17, inhibit the proteolytic activity until they are removed. In this regard, the prodomain of BACE1 is unusual in that it does not strictly inhibit BACE1 activity (Shi et al., 2001). In fact, overexpressed pro‐BACE1 can cleave APP in the ER (Huse et al., 2002). The role of the prodomain of BACE1 seems to be the facilitation of the correct folding of BACE1 (Shi et al., 2001). O‐glycosylation has not been observed for BACE1 (Haniu et al., 2000). BACE1 contains four N‐glycosylation sites (> Figure 11-3), which all carry sugar moieties (Haniu et al., 2000; Charlwood et al., 2001). The N‐glycosylation affects BACE1 activity, since a BACE1 mutant engineered to contain only two out of the four sites shows reduced BACE1 activity (Charlwood et al., 2001). It remains unclear, though, whether this is a direct effect on BACE1 activity or a more indirect effect due to alterations in folding, stability, or solubility of the enzyme. BACE1 contains six cysteine residues in its ectodomain, which form disulfide bridges in a pattern that is conserved in its homolog BACE2, but that is not found in the other members of the pepsin family of aspartyl proteases (Haniu et al., 2000). At the boundary between transmembrane and cytoplasmic domain BACE1 has three cysteine residues, which are palmitoylated (Benjannet et al., 2001). This modification reduces the ectodomain shedding of BACE1, but does not seem to have a major effect on BACE1 activity, as revealed by the analysis of BACE1 mutants lacking these cysteine residues (Benjannet et al., 2001). Mature BACE1 with an apparent molecular weight of 70 kDa is a relatively stable enzyme with a half‐ life of > 9 h in HEK293 cells (Haniu et al., 2000; Huse et al., 2000). Another form of posttranslational modification of BACE1 is ectodomain shedding. On overexpression of BACE1, secreted BACE1 lacking its transmembrane and cytoplasmic domain has been detected in the conditioned medium of HEK293 cells (Benjannet et al., 2001). The secretion could be reduced by inhibitors of metalloproteases, suggesting that members of the ADAM‐family or matrix metalloproteases might mediate the shedding of BACE1 (Hussain et al., 2003). Whether this shedding also occurs under conditions not involving BACE1 overexpression, remains to be analyzed. In contrast to BACE1 shedding, full‐length BACE1 has been detected in the cerebrospinal fluid of AD patients and controls, with the BACE1 levels being higher in the AD patients (Holsinger et al., 2004). How full‐length BACE1 including its transmembrane and cytoplasmic domain can be released into the CSF remains unknown. An additional proteolytic cleavage on a surface exposed helix

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within the ectodomain of BACE1 has been described between residues Leu228 and Ala299. This leads to two BACE1 fragments, which remain bound to each other and which retain BACE1 activity. This cleavage was mainly observed in liver, pancreas, and muscle, but not in brain (Huse et al., 2003). Like APP, mature BACE1 cycles between the TGN, the plasma membrane, and the endocytic pathway, particularly early and late endosomes (Huse et al., 2000; Walter et al., 2001). In fact, APP and BACE colocalize at the plasma membrane and in endosomes (Kinoshita et al., 2003). BACE2 shows a similar cellular distribution as BACE1, but a larger proportion of BACE2 is found at the plasma membrane (Hussain et al., 2000; Yan et al., 2001). Close to its C‐terminus BACE1 contains an evolutionarily conserved di‐leucine amino acid motif (> Figure 11-2), which conforms to endocytic trafficking signals found in other proteins, such as mannose‐6 phosphate receptor (Johnson and Kornfeld, 1992). Deletion of this motif alters BACE1 trafficking, such that more BACE1 is localized at the cell surface and less is found in endosomes (Huse et al., 2000). Phosphorylation of BACE1 by casein kinase 1 at Ser498 seems to be another way of regulating BACE1 sorting in the cell. Replacing Ser498 by aspartic acid (Ser498Asp), which mimics phosphorylated BACE1 is predominantly found in the TGN and in late endosomes, whereas an alanine at position 498 (Ser498Ala) mimics nonphosphorylated BACE1 and is mainly localized in early endosomes (Walter et al., 2001; Pastorino et al., 2002). Despite the differences in cellular sorting, both BACE1 mutants have similar effects on APP processing when transfected into HEK293 cells, revealing that the phosphorylation of BACE1 does not have a major effect on its catalytic activity (Pastorino et al., 2002). In cultured cells, overexpressed APP and BACE1 have been detected partly within and partly outside cholesterol‐rich membrane domains, so‐called detergent‐resistant membrane microdomains or rafts. The question whether BACE1 cleavage of APP occurs within or outside the cholesterol‐rich domains is of particular interest, since cholesterol lowering drugs have been shown to reduce Ab generation. In contrast to initial studies suggesting that APP cleavage by BACE1 occurs within the detergent‐resistant membranes (Riddell et al., 2001; Tun et al., 2002; Cordy et al., 2003; Ehehalt et al., 2003), a recent report suggests that this cleavage event takes place outside the detergent‐resistant membranes, at least under endogenous expression levels (Abad‐Rodriguez et al., 2004). Further studies are needed to fully understand the role of cholesterol and cholesterol‐rich membrane domains in BACE1 activity and AD pathogenesis.

5.2 BACE1 Interacting Proteins An increasing number of proteins have been reported to interact with BACE1. The two g‐secretase complex subunits nicastrin and presenilin coimmunoprecipitate with BACE1 when overexpressed (Hattori et al., 2002; Hebert et al., 2003). Whether the interaction is also seen at endogenous expression levels, remains to be analyzed. Another binding partner of BACE1, the phospholipid scramblase 1 (PLSCR 1), was identified in a yeast two hybrid screen using the short, 24 amino acid long cytoplasmic tail of BACE1 as the bait (Kametaka et al., 2003). The type II membrane protein PLSCR 1 colocalizes with BACE1 in the Golgi and in endosomes and was shown to coimmunoprecipitate with BACE1 at endogenous expression levels. The interaction between both proteins requires the C‐terminal di‐leucine trafficking motif of BACE1, suggesting that PLSCR may be involved in the intracellular distribution of BACE1. Other proteins binding to the di‐leucine trafficking motif of BACE1 are members of the GGA (Golgi‐ localized g‐ear‐containing ARF binding) family, namely GGA1 and GGA2. The interaction was shown in pull‐down experiments and using peptides corresponding to the sequence of the BACE1 C‐terminus (He et al., 2002). The interaction was confirmed in cultured cells using a fluorescence resonance energy transfer assay (von Arnim et al., 2004). Because GGAs are intracellular trafficking proteins that also interact with mannose‐6‐phosphate receptors, the authors suggested that the interaction with GGAs may influence BACE1 intracellular transport. Phosphorylation of the serine residue directly preceding the di‐leucine motif seems to enhance the interaction between BACE1 and GGAs (He et al., 2003; Shiba et al., 2004). Experiments knocking‐down GGA expression resulted in accumulation of BACE1 in early endosomes,

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suggesting that the GGA–BACE1 interaction is required for the recycling of endocytosed BACE1 to the cell surface (He et al., 2005). Another binding partner of BACE1 that was identified in a yeast two hybrid screen is the brain specific type II membrane protein BRI3, which colocalizes with BACE1 in HEK293 cells but also in brain (Wickham et al., 2005). Moreover, BRI3 coimmunoprecipitates with endogenous BACE1 in HEK293 cells. The maximal interaction between both proteins requires the cytoplasmic domain of BACE1 to be palmitoylated. The functional consequences of this interaction and the function of BRI3 itself are as yet unknown. Given the link of copper to the pathogenesis of AD [for an overview see Multhaup et al. (2002), Cuajungco et al. (2005)], the identification of the copper chaperone for superoxide dismutase‐1 (CCS) is of particular interest. CCS binds to the cytoplasmic tail of BACE in rat brain extracts, and both proteins are cotransported through axons. Surprisingly, the cysteine residues in the cytoplasmic domain of BACE1 bind a single Cu(I) atom (Angeletti et al., 2005). Whether the binding of copper or CCS to BACE1 has an effect on APP processing remains to be studied. Proteins that alter BACE1 activity by binding to BACE1 are reticulon 3 (RTN3) and its homolog RTN4b (NoGo) and heparan sulfate. Heparan sulfate was found to be a natural extracellular ligand for BACE1 (Scholefield et al., 2003). This interaction inhibits BACE1 cleavage of APP but not the a‐secretase cleavage of APP. The underlying molecular mechanism is not yet known. Heparan sulfate and BACE1 colocalize in the Golgi and at the plasma membrane and coimmunoprecipitate at endogenous expression levels in HEK 293 cells. RTN3 binds BACE1 in neurons, whereas RTN4b binds BACE1 in oligodendrocytes. The interaction of BACE1 with RTN3 was not only found in cells overexpressing both proteins but also at endogenous expression levels in brain. Most important, increasing the expression of RTN3 inhibited the secretion of Ab, whereas lowering the expression of RTN3 by RNA interference increased the secretion of Ab, suggesting that reticulon proteins are negative modulators of BACE1 by blocking access of BACE1 to APP (He et al., 2004). Yet another binding partner of BACE1 is BACE1 itself (Schmechel et al., 2003; Westmeyer et al., 2004). The homo‐dimerization of BACE1 was described to occur already in the ER and to increase BACE1 proteolytic activity (Westmeyer et al., 2004). The dimerization takes place between the ectodomains and requires BACE1 to be membrane‐anchored. Homo‐dimerization of BACE1 was observed both in cultured cells overexpressing BACE1 and in brain tissue. Taken together as shown in > Table 11‐1, several BACE1 binding proteins have been identified. Some of them seem to influence BACE1 intracellular trafficking (PLSCR 1, GGAs), whereas other ones affect BACE1 . Table 11-1 Posttranslational modifications and binding partners of BACE1 Posttranslational modifications of BACE1 Removal of signal peptide in the ER N‐glycosylation at four sites Removal of prodomain Three disulfide bridges Ectodomain shedding Palmitoylation Phosphorylation Sulfated at mature N‐sugars Proteolytic cleavage within its ectodomain in liver, pancreas and muscle

Binding partners of BACE1 g‐secretase (presenilin and nicastrin) Phospholipid scramblase 1 (PLSCR 1) GGA (Golgi‐localized g‐ear‐containing ARF binding) BRI3 Copper chaperone for superoxide dismutase 1 (CCS) Heparan sulfate Reticulon 3 and 4b (NoGo) BACE1 (homodimer formation)

activity (heparan sulfate, RTNs, BACE1 homo‐dimerization). Yet another group consists of proteins with poorly understood function with respect to their binding to BACE1 (g‐secretase subunits nicastrin and presenilin, CCS, BRI3).

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BACE1 Expression in Aging and AD

6.1 Localized BACE1 Expression It is widely accepted that neurons are the primary source of Ab peptides in brain. Therefore, it was believed for a long time that the b‐secretase crucial for Ab generation is expressed by neurons. After BACE1 was identified in 1999 as the long searched for b‐secretase, its cell type‐specific expression and localization in brain was investigated. In mouse brain, BACE1 is primarily expressed by neurons as shown by in situ hybridization (Vassar et al., 1999; Bigl et al., 2000; Marcinkiewicz and Seidah, 2000) and by immunohistochemistry (Roßner et al., 2001). As expected, BACE1 expression is robust in neocortical and hippocampal brain regions characterized by high expression levels of its major substrate — APP — and by the susceptibility to Ab plaque formation. However, BACE1 mRNA (Bigl et al., 2000; Marcinkiewicz and Seidah, 2000) and protein (Roßner et al., 2001) are also detected at high quantities in brain areas that are almost devoid of APP and which barely develop any Ab plaques, namely thalamus and striatum. This might be a first hint for the existence of alternative BACE1 substrates in different brain regions.

6.2 Aging During aging BACE1 mRNA (Bigl et al., 2000; Irizarry et al., 2001; Apelt et al., 2004) and protein levels (Roßner et al., 2001; Apelt et al., 2004; Fukumoto et al., 2004) are stable in brains of mice and humans. BACE1 enzymatic activity, in contrast, is increased with aging in mouse, monkey, and human brain (Fukumoto et al., 2004). In the light of aging being the most stringent risk factor for developing AD and the concentrations of Ab peptides in brain strongly correlating with age, this is an important observation and indirect evidence for the contribution of posttranslational mechanisms in the regulation of BACE1 activity and the pathogenesis of AD (see also > Section 5).

6.3 APP Transgenic Mice In the recent decade several strains of APP transgenic mice were developed and widely used to study the consequences of APP overexpression and/or Ab plaque formation on the generation of other histological and biochemical AD markers. The Tg2576 mouse strain is characterized by a five‐ to sevenfold overexpression of hAPP carrying the Swedish double mutation and by an age‐dependent formation of Ab plaques (Hsiao et al., 1996). Despite the APP overexpression, the BACE1 mRNA levels (Bigl et al., 2000; Irizarry et al., 2001; Apelt et al., 2004), BACE1 protein levels (Roßner et al., 2001; Gau et al., 2002; Fukumoto et al., 2004), and BACE1 enzymatic activities (Fukumoto et al., 2004) were not different between wild‐type and Tg2576 mice. This indicates that BACE1 is not the rate‐limiting enzyme for Ab peptide generation. As a consequence, a partial reduction of BACE1 enzymatic activity in the AD brain is unlikely to result in a significant depletion of Ab peptides.

6.4 BACE1 Expression in AD In the AD brain, increases in both BACE1 protein concentrations and enzymatic activities have been reported (Fukumoto et al., 2002, 2004; Holsinger et al., 2002; Sun et al., 2002; Yang et al., 2003). Interestingly, the soma of BACE1 expressing neurons did not directly associate with Ab plaques in hippocampal and neocortical areas characterized by robust Ab deposition (Sun et al., 2002), a finding which is consistent with observations from brains of APP transgenic mice (Roßner et al., 2001). The increased b‐secretase activity in AD brain is accompanied by a reduction of a‐secretase activity (Tyler et al., 2002). Interestingly, when plotting cortical a‐ or b‐secretase activities against ChAT activity in cortex of AD patients a strong positive (to a‐secretase) or negative (to b‐secretase) correlation can be established. Of course, this observation leaves open the possibility that a primary cholinergic deficit induces alterations in secretase activities. On the other

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hand, it is well established that a quantitative shift APP processing toward the generation of Ab peptides interferes with cholinergic neurotransmission. For example, solubilized Ab peptides strongly inhibit the potassium‐stimulated release of acetylcholine from hippocampal slices (Kar et al., 1996) and decreases ChAT activity but not acetylcholinesterase activity in the cholinergic SN56 cell line (Pedersen et al., 1996). Ab peptides also decrease the intracellular acetylcholine concentration (Hoshi et al., 1997) and impair M1‐associated signaling (Kelly et al., 1996) in primary septal or cortical cultures. All these effects of Ab on markers of cholinergic neurotransmission were observed at pM to nM concentrations and without obvious evidence of neurotoxicity. The effects of Ab on reduction of pyruvate dehydroxygenase activity (Hoshi et al., 1997), the key enzyme for the generation of acetylcholine used for neurotransmitter synthesis and citrate cycle, might explain both, cholinergic hypoactivity and metabolic dysfunction of cholinergic neurons after exposure to Ab at low concentrations.

6.5 Astrocytic BACE1 Expression In addition to quantitative changes of BACE1 activity in AD, there are also remarkable alterations with regard to the cell type‐specific expression of BACE1. As stated earlier and consistent with the notion that neurons are the primary source of Ab peptides in brain, BACE1 is exclusively expressed by neurons under normal conditions. However, reactive astrocytes in proximity to Ab plaques also display BACE1 immunoreactivity indicating that astrocytes — in their activated state — might contribute to Ab plaque formation (Hartlage‐Ru¨bsamen et al., 2003; Leuba et al., 2005). This astrocytic BACE1 expression is not only limited to the brains of AD patients, but was also observed in brains of APP transgenic Tg2576 mice with Ab plaque pathology. This was not found in transgenic mice before the onset of Ab plaque formation (> Figure 11-7) (Roßner et al., 2001). Furthermore, in a number of chronic but not acute lesion models, reactive astrocytes express BACE1 (Hartlage‐Ru¨bsamen et al., 2003) indicating that augmented BACE1 expression by chronically activated astrocytes may contribute to a localized increase in amyloidogenic APP fragments or plaque formation. This scenario is supported by studies using different experimental paradigms [for review see Roßner et al. (2005)]. For example, a single intrahippocampal LPS‐injection fails to increase the Ab load in APP/presenilin‐1 transgenic mice (DiCarlo et al., 2001), but the chronic intracerebroventricular infusion of . Figure 11-7 Astrocytic BACE1 expression in Tg2576 mice and AD. Confocal images of double immunofluorescence labeling for BACE1 and GFAP in the parietal cortex of 17‐month‐old Tg2576 mice and in Area 22 of the AD cortex. BACE1‐ immunoreactivity is shown by the green fluorescence; GFAP‐immunoreactivity is encoded by red fluorescence. In the overlay channel, colocalization of BACE1 and GFAP is indicated by yellow/orange color. Reactive astrocytes expressing BACE1 are indicated by arrowheads; the asterisks mark the position of Ab plaques (after Hartlage‐Ru¨bsamen et al., 2003; modified)

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LPS accelerates Ab deposition in APPV717F transgenic mice (Qiao et al., 2001). However, at the present stage it is not clear to what extent reactive astrocytes contribute to the production of Ab peptides and to Ab plaque formation. First, although activity measurements indicate that reactive astrocytes do express enzymatically active BACE1 (Roßner et al., 2005), it still has to be shown that the full‐length, nontruncated BACE1 isoform is expressed. Second, to contribute to Ab plaque formation, the substrate APP has to be available to astrocytic BACE1. It is known that primary astrocytes themselves do express APP and generate significant amounts of Ab peptides (Gray and Patel, 1993; Amara et al., 1999; Beck et al., 2000; Blasko et al., 2000; Docagne et al., 2004). Additionally, APP is also expressed by reactive astrocytes in experimental models of chronic gliosis [see, e.g., Martins et al. (2001)] and this induced astrocytic APP expression results in the increased generation of Ab peptides and BACE1 cleavage‐derived A4‐CT fragments (Bates et al., 2002; Lesne´ et al., 2003). Alternatively, astrocytic BACE1 might be secreted and cleave membranous APP at the neuronal surface. Such a shedding of BACE1 has been reported recently (Benjannet et al., 2001; Hussain et al., 2003).

7

Biological Function and Substrate Spectrum of BACE1 and BACE2

7.1 Substrates of BACE Proteases What is the biological function of BACE1? Knowing the answer to this question will allow not only a better evaluation of the therapeutic potential but also of the liabilities of BACE1 inhibition. In case that BACE1 cleaves a number of different substrates, the inhibition of BACE1 may cause side effects by interfering with the function of these proteins. Do BACE1 and BACE2 only cleave APP or do they contribute to the proteolytic cleavage of a larger number of membrane proteins, similar to a‐ and g‐secretase? The a‐secretases ADAM10 and ADAM17 mediate the proteolytic conversion of a variety of membrane proteins to their soluble counterparts [for a review see Hooper et al. (1997), Blobel (2002)]. This process is referred to as ectodomain shedding and has been described for growth factors, cytokines and their receptors, cell adhesion proteins, and proteins of unknown biological function, such as APP. Likewise, g‐secretase mediates the intramembrane proteolysis of an increasing number of type I membrane proteins [reviewed in Selkoe and Kopan (2003)]. In contrast to a‐ and g‐secretase, very few substrates have been identified for BACE1 and even less for BACE2. Both proteases were initially identified as APP cleaving enzymes. Although additional substrates were not immediately identified, many scientists assumed that more substrates for BACE1 and possibly BACE2 should exist. This assumption was based on the following theoretical considerations. In the polarized Madin– Darby canine kidney cell line (MDCK), BACE1 is predominantly found on the apical side (Capell et al., 2002), whereas APP is mainly transported to the basolateral side (Haass et al., 1994; Lo et al., 1994; De Strooper et al., 1995). Given this differential localization of APP and BACE1, it seemed reasonable to assume that APP is not the main substrate for BACE1 and that BACE1 may have one or several other substrates, which should also be found on the basolateral side. Additionally, in vitro experiments addressing the substrate specificity of BACE1 revealed that wild‐type APP is a much poorer substrate for BACE1 than APP carrying the so‐called Swedish double mutation, which is found in a large Swedish pedigree affected by an inherited form of Alzheimer’s disease. Carriers of this genetic mutation have the two amino acids lysine and methionine (KM) preceding the BACE1 cleavage site replaced by asparagine and leucine (NL) (> Figure 11-1b), allowing a much more efficient cleavage by BACE1 in vitro experiments using short peptides (Lin et al., 2000; Gruninger‐Leitch et al., 2002). The prediction that BACE1 should have additional substrates in addition to APP turned out to be true. A number of additional substrates have been identified over the past few years, namely the APP‐homologs APLP1 and APLP2 (Li and Su¨dhof, 2004; Pastorino et al., 2004), the sialyltransferase ST6Gal I (Kitazume et al., 2001, 2003, 2005), the P‐selectin glycoprotein ligand‐1 (PSGL‐1; Lichtenthaler et al., 2003), beta subunits of voltage‐gated sodium channels (VGSCb) (Wong et al., 2005), and the LDL receptor‐related protein (von Arnim et al., 2005). Both PSGL‐1 and ST6Gal I function in the immune system, where BACE1 is expressed. The type I membrane protein PSGL‐1 is involved in the inflammatory response, and

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contributes to the rolling of leukocytes along the vessel wall before they transmigrate through the endothelium. The type II membrane protein ST6Gal I is involved in B cell expansion and is required for the correct glycosylation of CD22 ligands. VGSCs are an abundant type of ion channels that are responsible for the initiation and propagation of action potentials in neurons. ST6Gal I, PSGL‐1, and VGSCb meet three basic criteria for BACE1 substrates: (a) their cleavage occurs under conditions of endogenous BACE1 expression, (b) exogenous expression of BACE1 strongly increases their cleavage and, importantly, (c) their cleavage is reduced or abolished in BACE1
/
cells or animals (Lichtenthaler et al., 2003; Kitazume et al., 2005; Wong et al., 2005), which establishes the physiological relevance of their cleavage. Interestingly, both ST6Gal I and PSGL‐1 are cleaved C‐terminally of a leucine residue, which is in good agreement with the in vitro substrate specificity of BACE1 and with the fact that BACE1 cleavage of the Swedish mutant APP also occurs after a leucine residue. Thus, additional substrates for BACE1 to be identified may also be cleaved preferentially after a leucine residue. The exact cleavage sites of BACE1 in APLP1 and APLP2 remain to be determined. BACE1 is not only able to cleave type I membrane proteins (APP, PSGL‐1, APLP1, and APLP2) but also a type II membrane protein (ST6Gal I), suggesting that, similar to the ADAM proteases, BACE1 may contribute to the ectodomain shedding of a larger number of membrane proteins of type I or type II membrane orientation. However, BACE1 is likely to have a more restricted set of substrate proteins, because it is not involved in the shedding of two other ADAM protease substrates, the TNFa receptor 2 and the cell adhesion protein L‐selectin (Lichtenthaler et al., 2003). The proteolytic processing of APP and PSGL‐1 reveals additional insights into cleavage preferences of BACE1. APP and PSGL‐1 are both cleaved by BACE1, but also by an ADAM metalloprotease. For both proteins most of the cleavage seems to occur by the ADAM proteases, whereas BACE1 cleavage constitutes only a smaller fraction of the total cleavage. An additional similarity is the localization of the BACE1 cleavage site in the membrane‐proximal domain. In contrast to APP, it is the BACE1 cleavage site in PSGL‐1 that is located more closely to the membrane than the putative ADAM protease cleavage site (Lichtenthaler et al., 2003). This is in opposite order compared with the proteolytic processing of APP. However, this finding agrees well with the known properties of BACE1 and ADAM proteases. The ADAM protease cleavage of APP occurs without a strict sequence specificity but at a fixed distance from the membrane (Sisodia, 1992), whereas the BACE1 cleavage seems to be more sequence‐ specific but not strongly dependent on the distance of the cleavage site from the membrane (Citron et al., 1995). Taken together, it is clear now, that BACE1 does not exclusively cleave APP, but has a wider substrate spectrum. Much less is known about BACE2 substrates. It is not known whether the three BACE1 substrates ST6Gal I, APLP1, and APLP2 can be cleaved by BACE2, but BACE2 clearly cleaves two other BACE1 substrates, namely APP and PSGL‐1. This raises the possibility, that BACE1 and BACE2 may have similar or identical substrates. However, both proteases do not necessarily cleave their substrates at identical peptide bonds. For example, BACE1 cleaves APP at the N‐terminus of Ab, whereas BACE2 cleavage occurs within the Ab domain and thus functions as an alternative a‐secretase, precluding Ab formation. However, BACE2 cleavage of APP may not reduce Ab levels in the brain, given the low expression level of BACE2 in this organ. At present, the specific function of BACE1 in the cleavage of its substrates remains unclear, but different possibilities are plausible. Given that several BACE1 substrates are also substrates for ADAM metalloproteases, it is possible, that both proteases cleave the substrates for different purposes. BACE1 seems to be constitutively active, whereas ADAM proteases are tightly regulated in their activity by different cellular signal transduction pathways (Allinson et al., 2003). Therefore, BACE1 cleavage, which partly occurs in the secretory pathway, could provide a low‐level constitutive cleavage and secretion of membrane proteins. In contrast, under conditions where a high level of secretion is needed (e.g., of a growth factor or cytokine) the activity of ADAM proteases might be upregulated. Another function of BACE1 could be the initiation of substrate protein degradation. In this scenario, BACE1 would cleave off most of the ectodomain of its substrate and leave the remaining membrane‐bound stub for additional degradation by the g‐secretase omplex. This idea is in agreement with the finding that BACE1 cleavage occurs after APP endocytosis in the endosomes and that the resulting secreted ectodomain of APP (sAPPb) does not have any known biological functions. In contrast, sAPPa has neuroprotective and neurotrophic properties (Furukawa et al., 1996; Meziane et al., 1998) and is generated by ADAM proteases at or close to the plasma membrane.

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7.2 BACE1 Deficient Mice For many proteins, the use of knockout mice has strongly contributed to elucidating the protein’s function. This approach was very successful in understanding the in vivo function and the main in vivo substrates of g‐secretase and of the two a‐secretases ADAM10 and ADAM17. Deficiencies in ADAM10 (Hartmann et al., 2002), ADAM17 (TACE) (Peschon et al., 1998) or in subunits of g‐secretase (De Strooper et al., 1998) resulted in severe phenotypic changes and embryonic or perinatal lethality in mice, revealing an essential function of these proteases during development. A more detailed analysis showed that the phenotype of these mice strongly correlated with the lack of cleavage of the major protease substrate during development. Thus, g‐secretase and ADAM10‐deficient mice show a phenotype characteristic for reduced or abolished proteolytic processing of Notch, whereas ADAM17 knockout phenotype was caused by the lack of TGF‐a cleavage and the resulting impaired EGFR signaling. In contrast to mice deficient in a‐ or g‐secretase activity, BACE1 and BACE2 deficient mice show a very mild phenotype (Cai et al., 2001; Luo et al., 2001; Roberds et al., 2001). BACE1
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mice are viable and fertile, ruling out an essential function for BACE1 during embryonic development. BACE1
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mice do not generate Ab, clearly implicating BACE1 as the physiological b‐secretase. Despite the lack of overt neurological or physiological defects, even in older mice (Luo et al., 2003), BACE1‐deficient mice seem to have more subtle defects. Harrison and colleagues recently reported that BACE1
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mice are more timid and anxious than control mice and show a less exploratory behavior correlating with increased 5‐HT turnover in the hippocampus (Harrison et al., 2003). Whether BACE1 has a direct role in neurotransmitter metabolism remains to be analyzed. Another study found that BACE1 deficiency rescued memory deficits and cholinergic dysfunctions observed in an animal model of AD, the APP transgenic Tg2576 mouse (Ohno et al., 2004). The same authors also found differences in spatial working memory between wild‐type and BACE1
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mice. Clearly, more studies are needed to fully understand the BACE1 knockout phenotype, in particular with regard to memory, cognition, and behavior. An additional field, where BACE1
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mice may have deficiencies, is the immune system, given that the two BACE1 substrates PSGL‐1 and ST6Gal I are proteins with immune function. Because these mice have not yet been immunologically challenged extensively, an immunological phenotype may not have been detected. Interestingly, BACE1 is only found in vertebrates but not in C. elegans and D. melanogaster, suggesting that BACE1 may have special functions in cellular processes developed later in evolution such as the elaborate immune system or complex brain functions of vertebrates.

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Protein Misfolding, a Common Mechanism in the Pathogenesis of Neurodegenerative Diseases

L. Vergara . K. Abid . C. Soto

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286

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Role of Protein Misfolding and Aggregation in Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287

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Clinical, Neuropathological, and Molecular Features of Neurodegenerative Diseases . . . . . . . . . 288 Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 Parkinson Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Huntington Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 Amyotrophic Lateral Sclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 Prion Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292

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Animal Models of Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293

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Structural and Mechanistic Basis of Protein Misfolding and Aggregation . . . . . . . . . . . . . . . . . . . . . 294

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Mechanism of Brain Degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

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Targets for Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296

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Conclusions and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298

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Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

Abstract: Neurodegenerative diseases are some of the most debilitating disorders, affecting abstract thinking, skilled movements, emotional feelings, cognition, and memory. Recent and compelling evidence indicates that the misfolding, aggregation, and accumulation of proteins in the brain may be the cause of these diseases. The aim of this chapter is to review the literature around the molecular mechanism and role of misfolded protein aggregates in neurodegeneration and the possibilities for therapeutic intervention. List of Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis; APP, amyloidprecursor protein; BACE, b-amyloid cleaving enzyme 1; BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; CWD, chronic wasting disease; fCJD, Familial CJD; GSS, Gerstmann-StrausslerScheinker; HD, Huntington disease; iCJD, Iatrogenic CJD; ND, neurodegenerative diseases; NFT, Neurofibrillary tangles; PD, Parkinson disease; RNAi, RNA interference; SCA, spinocerebellar ataxia; sCJD, Sporadic CJD; SOD1, superoxide dismutase 1; TSE, transmissible spongiform encephalopathies; vCJD, Variant CJD

1

Introduction

As a consequence of the longer life expectancy brought by the advances in medical care, the last decade has observed an increase in the incidence of a number of neurodegenerative diseases (ND) including Alzheimer’s disease (AD), Parkinson disease (PD), Huntington disease (HD) (and related polyglutamine disorders including several forms of spinocerebellar ataxia or SCA), transmissible spongiform encephalopathies (TSEs, which include several human and animal diseases), and amyotrophic lateral sclerosis (ALS). This diverse group of diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory, and other abilities (Martin, 1999). Despite their differences in clinical symptoms and disease progression, these disorders do share some common features: most of them (except HD and SCA) have both sporadic and inherited origins, all of them appear later in life (usually after the fourth or fifth decade), and their pathology is characterized by neuronal loss and synaptic abnormalities (Martin, 1999). Recent, but compelling evidences suggest that protein misfolding and aggregation is the most likely cause of ND (Soto, 2003). The physiology of cells, tissues, and organisms depends on the correct activity of a network of thousands of proteins. The function of a protein depends on its three-dimensional structure, which is determined by its amino acid sequence. In ND, some normal proteins become improperly folded and begin a process of polymerization to form large protein aggregates, commonly referred to as amyloidlike deposits (Soto, 2003). Amyloid was the name originally given to extracellular protein deposits found in AD and systemic amyloid disorders, but it has been recently used to refer also to some of the intracellular aggregates. Amyloid is a generic term that refers to aggregates organized in a cross-b structure, which contain specific morphological and tinctorial characteristics, such as binding to congo red and thioflavin S, higher resistance to proteolytic degradation, and a fibrillar appearance under electron microscopy (straight, unbranched, 10-nm-wide fibrils) (> Figure 12-1) (Glenner, 1980; Sipe, 1992). Misfolding and aggregation of different proteins is responsible for distinct diseases, and no structural homology or similar amino acid sequences have been demonstrated among the proteins implicated in different ND. Protein misfolding and aggregation not only occurs in the brain, and indeed several systemic disorders have also this molecular mechanism of pathogenesis, including diabetes type II, serpin deficiency disorders, hemolytic anemia, amyloid polyneuropathy, cystic fibrosis, dialysis-related amyloidosis, secondary or reactive amyloidosis, and more than 15 other diseases (Carrell and Gooptu, 1998; Soto, 2001). In this chapter we will review the characteristics and molecular mechanisms implicated in the pathogenesis of ND. We will also outline some of the strategies under development for the treatment of these yet incurable disorders.

Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

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. Figure 12-1 Morphological, tinctorial, and structural characteristic of amyloid. (a) Tissue staining with antibodies specific for the protein forming the aggregates. (b) Electron microscopy shows typical straight, unbranched fibrils, which are 5–10 nm in diameter and 100 nm in length. (c) Structure of the typical amyloid-binding dyes Congo red and Thioflavine T. (d) Cong red staining visualized under polarized light shows green/yellow birefringency. X-ray ˚ and 9.7A ˚. fiber diffraction studies show a characteristic pattern known as cross-b, represented by signals at 4.7A (e) A diagrammatic picture for the cross-b structure showing the periodicity observed in X-ray diffraction studies. (f) Computer modeling of the putative tridimensional structure of a prototype amyloid fibril viewed from the side and from the top

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Role of Protein Misfolding and Aggregation in Disease

Neuropathologic and genetic studies, along with the development of transgenic animal models, have provided strong evidence for the involvement of protein misfolding in ND. Nearly 100 years ago, the German neuropathologist Alois Alzheimer described for the first time the typical aggregates observed in the

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brain parenchyma of demented people (Alzheimer et al., 1995). These aggregates have now been well characterized as formed by protein fibrils called amyloid plaques. The end point of protein misfolding is usually aberrant protein aggregation and accumulation as amyloid-like deposits in the brain (Glenner, 1980; Sipe, 1992; Soto, 2003; Dimakopoulos and Dimakopoulos, 2005; Ross and Poirier, 2005). The involvement of amyloid deposition in the pathogenesis of ND has been suggested by the colocalization of protein aggregates with sites of tissue degeneration and its correlation with the symptoms of the disease. Furthermore, protein deposits have become so frequent in affected individuals that their presence is used for definitive diagnosis (Westermark, 1995; Gillmore et al., 1997). On the other hand, absolute proof is still awaiting confirmation that aggregation itself is the culprit of the disease and not only an inseparable epiphenomenon (Carrell and Gooptu, 1998; Tran and Miller, 1999; Goldberg and Lansbury, 2000; Caughey and Lansbury, 2003). Indeed, today the most accepted view is that the process of misfolding and early stages of oligomerization, rather than the mature compacted aggregates deposited in the brain, are the real culprits in neurodegeneration (Glabe and Kayed, 2006; Lansbury and Lashuel, 2006; Haass and Selkoe, 2007). Genetic studies have provided strong support for the role of protein misfolding in ND (Hardy and Gwinn-Hardy, 1998; Selkoe et al., 2002). In addition to sporadic and infectious origin, ND can also be caused by inherited defects. Epidemiological analysis has shown the presence of several loci in the human genome responsible for diverse ND. Importantly, in most of the inherited cases, the mutated gene encodes the protein that undergoes misfolding and aggregation. Thus it is believed that specific mutations in critical amino acids may destabilize the normal protein folding, increasing its propensity to misfold and aggregate. Mutations in the respective fibrillar proteins have been associated with familial forms of all ND, and also with many other forms of systemic and brain disorders associated with protein misfolding and aggregation, including amyloid polyneuropathy, cardiac amyloidosis, visceral amyloidosis, cerebral hemorrhage with amyloidosis of the Dutch and Icelandic type, cerebral amyloidosis of the British and Danish type, thromboembolic disease, angioedema, emphysema, sickle cell anemia, and diabetes type 2 (Jacobson and Buxbaum, 1991; Kelly, 1996; Buxbaum and Tagoe, 2000). The familial forms usually have an earlier onset and greater severity than sporadic cases. As described later in this chapter, another strong evidence for the role of protein misfolding and aggregation in ND has been the successful production of animal models by solely introducing the mutated human gene encoding for the protein undergoing misfolding. In most of cases, this is enough to produce a disease in the animals with similar pathological, clinical, and biochemical features as the human disease.

3

Clinical, Neuropathological, and Molecular Features of Neurodegenerative Diseases

3.1 Alzheimer’s Disease AD is the most prevalent ND, known to affect at least 5 million people in the United States alone, with more than 300,000 new cases each year. About 10% of people over the age of 65 are affected by AD, and the proportion increases dramatically with age. The patients suffer from progressive memory impairments, disordered cognitive function, and decline in language function among other signs of dementia (Small and Cappai, 2006). The neuropathological features of AD include extensive neuronal loss and synaptic alterations mostly in areas implicated in memory and cognition, and massive deposition of amyloid plaques and neurofibrillary tangles (NFT) (Selkoe, 2004a; Small and Cappai, 2006). Amyloid plaques are composed of deposits of the amyloid-b peptide (Ab). Ab is derived from a larger precursor protein, termed amyloid precursor protein (APP), by sequential proteolytic cleavages (Selkoe, 1998). Ab deposits, in the form of insoluble fibrils, are mostly found in the brain parenchyma and on the walls of cerebral blood vessels (Selkoe, 2001). The familial forms of AD, although representing less than 5% of the patients affected, have provided strong evidence that Ab is involved in the disorder and have been used to develop in vitro and experimental animal models of AD. Several gene mutations have been identified to be involved in early-onset AD, including those encoding

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for APP and the presenilins (PS1 and PS2) (Selkoe, 2001, 2004b). The presence of the e4 allele of apolipoprotein E (ApoE) has been shown to be a major predisposition factor for the late-onset form of AD (Rebeck et al., 1993; Gearing et al., 1996; Lane and Farlow, 2005). APP is a transmembrane cell surface glycoprotein that can be found in several forms arising both from alternative splicing and posttranslational modifications (Walter et al., 1997; Selkoe, 2004b). The APP695 isoform is the most abundant in neurons; other forms are expressed in other cell types of the brain, as well as in other tissues. Although the function of APP is unclear, its soluble form has been described as an autocrine (Saitoh et al., 1989) and a neuroprotective (Mattson et al., 1993) factor. The presence of an ectodomain containing a KPI (serine protease inhibitor domain of Kunitz type) motif has raised the possibility that APP may function as a protease inhibitor. Indeed, APP has been shown to inhibit the serine protease factor XIa in the clotting cascade (Smith et al., 1990). Other suggested functions include participation in cell–cell and cell–substrate adhesions (Schubert et al., 1989; Qiu et al., 1995), and more recent findings have shown that APP may be involved in the brain regulation of neural progenitor cell proliferation (Caille et al., 2004; Conti and Cattaneo, 2005) as well as in axonal outgrowth after injury in a fly model (Leyssen et al., 2005). No evidence has been obtained to support the hypothesis that a loss of cellular function of APP may explain AD pathogenesis. Indeed, knockout mice with the APP gene are viable and fertile; however, they exhibit a decreased locomotor activity and forelimb grip strength, both events occurring later in adult life (Zheng et al., 1995). APP can be cleaved by three different complexes of proteases; a, b, and g secretases. The Ab fragment comprises the first 12–14 residues of the transmembrane domain plus 28 extracellular residues (Sisodia, 1992). Cleavage by a-secretase gives rise to a large soluble fragment (sAPPa) and a smaller peptide of 83 aa (c83), which is retained in the membrane. The c83 peptide can be further cleaved by g-secretase to generate a small peptide (p3) that encompasses two-thirds of Ab, but is not involved in amyloid formation (Chyung et al., 2005; Agiostratidou et al., 2006). Alternative cleavage of APP is carried out by b-secretase, also called b-amyloid cleaving enzyme 1 (BACE), an aspartyl protease. BACE cleavage gives rise to the sAPPb (which is soluble and secreted from the cell membrane) and the 99 c-terminal amino acids retained in the membrane. The c99 fragment is heterogeneously cleaved by g-secretase to produce Ab 40 and 42 peptides, ending at the amino acid 40 and 42, respectively (Selkoe, 2004a). Ab42 is the most prevalent form found in amyloid plaques. It is more hydrophobic than Ab40 and thus more prone to aggregation, despite the fact that it is normally less abundant than its counterpart in healthy individuals (Selkoe, 1998, 2004a). Even though Ab is embedded in the membrane at the time cleavage occurs, it is subsequently released to the extracellular space were it is able to initiate the formation of fibrils resulting in the genesis of amyloid plaques. Mutations observed in APP-, PS1-, and PS2-encoding genes result in an increased production of total Ab peptides, selective increase of the Ab42 variant, or in Ab forms more prone to misfold (Soto, 1999; Selkoe, 2001). Presenilin 1 missense mutations have been shown to be responsible of causing the earliest and most aggressive forms of AD. The genes of PS1 and PS2 were shown to encode for the two presenilins that form the catalytic core of the g-secretase complex (Wolfe et al., 1999). PS1 and PS2 are highly homologous and functionally redundant (Wolfe et al., 1999; Haass and Steiner, 2002; Brunkan and Goate, 2005). Mutations within the PS genes might modify the structure of the presenilin, affecting the precision of the g-secretase activity and usually resulting in an increase in production of the Ab42 peptide (Selkoe, 2001). Even though more than 100 mutations that cause AD have been found for the PS1 gene alone, genetically based AD represents a minority compared with sporadic cases. NFTs, the other specific characteristic lesion found in the brain of AD patients, are long bundles of fibers that form large vesicles within the cytoplasm. These fibers, structurally organized in pairs of approximately 10 nm in length, are composed of the microtubule-associated Tau proteins, whose main function is microtubule stabilization, but it has also been reported to be involved in membrane interaction or acting as an anchor for enzymes (Sontag et al., 1999). The majority of the Tau proteins found in NFTs are highly insoluble and hyperphosphorylated (Grundke-Iqbal et al., 1986; Kosik et al., 1986). NFTs are biochemically similar to amyloid plaques, although these two well-known lesions of AD have been observed independent of each other in affected patients. Unlike amyloid plaques, NFT formation has not been found to be because of known mutations within the genes associated with familial AD (Armstrong, 2006). Inherited mutations in the protein Tau lead to fronto-temporal dementia with parkinsonism (Goedert

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and Spillantini, 2000). In this disease, mutated Tau proteins accumulate in the cytoplasm as NFT without extracellular amyloid plaques. Even though this disease is far less common than AD, it leads to similar clinical manifestations (Hutton, 2001).

3.2 Parkinson Disease PD is less prominent than AD; in the elderly it is estimated to affect around 1% of individuals over 65 years old (Savitt et al., 2006). PD was first characterized by James Parkinson in 1817, but more than a century passed before the neuropathological features of PD were revealed. PD is a slowly progressive motor disorder, characterized by tremor at rest, rigidity, slowness, absence of voluntary movements, and freezing, as well as depression and dementia in older patients (Savitt et al., 2006). The disease is characterized by the progressive degeneration of dopaminergic cells and the presence of intracellular proteinaceous aggregates known as Lewy bodies (Forno, 1996). These two events occur in a region of the brain termed ‘‘substantia nigra.’’ Biochemical and neuropathological studies have identified a-synuclein as being the major constituent of the intracellular aggregates (Spillantini et al., 1997). a-Synuclein is a highly conserved 140 amino acids protein found in the central nervous system, particularly abundant in the presynaptic terminals in close association with synaptic vesicles (Clayton and George, 1998). The physiological role of a-synuclein is still unclear, but it may regulate synaptic vesicle function, or be involved in synaptic plasticity (Clayton and George, 1998). a-Synuclein has also been identified in several other neurologic disorders known as ‘‘a-synucleinopathies’’ (Spillantini et al., 1997; Spillantini and Goedert, 1998) and in the filamentous glial and neuronal inclusions of multiple-system atrophy (Goedert et al., 1998; Trojanowski and Lee, 2003). Genetic determinants have also been identified in PD, with familial cases representing less than 5% of the affected individuals. Three different genes have been identified to contain mutations correlated with the disease. In addition to a-synuclein, they encode parkin and the carboxy-terminal hydrolase L1 (UCHL1) (Bertoli-Avella et al., 2004; Gandhi and Wood, 2005). Several additional loci have been shown to be involved as risk factors for PD. Interestingly, as it is the case for AD, certain apolipoprotein E polymorphisms have been shown to represent a risk factor for sporadic PD (Huang et al., 2004, 2006). Parkin is a large protein of 456 amino acids, mainly expressed in the brain and muscles, and functions as an E3 ubiquitin ligase, a component of the ubiquitin–proteasome system that plays a role in targeting misfolded proteins to the proteasome for degradation (Sherman and Goldberg, 2001). UCHL1 is a thiol protease known to hydrolyze ubiquitin chains back to the ubiquitin monomer (Wilkinson, 2000).

3.3 Huntington Disease HD is an autosomal dominant inherited disorder characterized by uncontrolled movements, loss of intellectual faculties, and emotional disturbance (Vonsattel and DiFiglia, 1998). In the United States alone, about 30,000 people have HD; estimates of its prevalence are about 1 in every 10,000 persons. The rate of disease progression and the age at onset vary from person to person. Adult-onset HD, with its disabling, uncontrolled movements, most often begins in middle age. Some individuals develop symptoms of HD when they are very young—before age 20. The terms ‘‘early-onset’’ or ‘‘juvenile’’ HD are often used to describe this form of the disease (Gonzalez-Alegre and Afifi, 2006). Symptoms of juvenile HD include subtle changes in handwriting and slight problems with movement, such as slowness, rigidity, tremor, and myoclonus. Several of these symptoms are similar to those seen in PD, and they differ from the chorea seen in individuals who develop the disease as adults. People with juvenile HD may also have seizures and mental disabilities. The earlier the onset, the faster the disease seems to progress, and death often follows within 10 years. The neuropathologic abnormalities include massive neuronal death mostly in the basal ganglia, which is a brain region that participates in coordinating movement (Vonsattel and DiFiglia, 1998). Within the

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basal ganglia, HD especially targets neurons of the striatum, particularly those in the caudate nuclei and the pallidum. In severe cases, the brain cortex is also largely affected. Nuclear protein aggregates composed of the huntingtin protein are a typical feature of HD (DiFiglia et al., 1997). The large majority of HD cases are inherited in an autosomal dominant way, due to a triplet repeat expansion of CAG (coding for the amino acid glutamine) in exon 1 of the gene encoding for the protein huntingtin (Wanker, 2000). The HD gene comprises a region coding for a stretch of polyglutamine residues close to the N terminus of the protein. In the case of the triplet expansion observed in patients, the longer glutamine portion promotes protein aggregation in the brain (Wanker, 2000). These deposits are known to trigger oxidative and endoplasmic stress along with proteosomal and mitochondrial dysfunction (Meriin et al., 2002; Nishitoh et al., 2002). Individuals with 35 or fewer triplet repeats do not develop HD, in contrast, those with more than 40 repeats always develop the disease. Several biological activities have been attributed to huntingtin, including cytoskeletal anchoring, axonal transport, endocytosis, and intracellular trafficking (Cattaneo et al., 2001; Smith et al., 2005). Evidence also points to a role in brain development and neuroprotection. Ablation of huntingtin in transgenic mice results in embryonic death, and conditional deletion results in neurodegeneration, suggesting that huntingtin has a protective role (Reiner et al., 2003). Furthermore, huntingtin has been shown to function as a caspase substrate that is actively cleaved during apoptosis in cultured cells (Cattaneo et al., 2001). In vitro experiments have revealed the ability of huntingtin to protect CNS cells from a variety of apoptotic stimuli, including serum withdrawal, death receptors, and pro-apoptotic proteins activation (Cattaneo et al., 2001; Reiner et al., 2003).

3.4 Amyotrophic Lateral Sclerosis ALS, also called Lou Gehrig’s disease, is a rapidly progressive, fatal ND that attacks motor neurons in the brain and spinal cord. ALS is one of the most common neuromuscular diseases, which most commonly affects people between 40 and 60 years of age, but younger and older people also can develop the disease (Pasinelli and Brown, 2006). It is estimated that around 20,000 people in the United States have ALS, and around 5,000 new cases are diagnosed every year in this country alone. The disease appears mostly in a sporadic form, but around 5–10% of the cases are inherited. About 20% of all familial cases result from mutations in the gene encoding for the enzyme known as Cu/Zn superoxide dismutase 1 (SOD1) (Pasinelli and Brown, 2006). Recently, several other genes have been linked to familial ALS, including the vascular endothelial growth factor, Angiogenin, and the vesicle-associated membrane protein B (Lambrechts et al., 2006). The other genes responsible for familial ALS remain to be identified. ALS is characterized by progressive degeneration of both the upper and lower motor neurons. The neuronal degeneration produces a decreased muscle function, leading to the gradual weakening of the muscles, which finally waste away and twitch (Price et al., 1996; Pasinelli and Brown, 2006). Individuals with ALS lose their ability to start and control voluntary movements, and the ability to move their arms, legs, and body. When muscles in the diaphragm and chest wall fail, individuals lose the ability to breathe without external support. Patients in the later stages of the disease may become totally paralyzed, yet, for the vast majority of people, their sensorial and cognitive abilities remain unaffected. Most people with ALS die from respiratory failure, usually within 3–5 years from the onset of symptoms. Cytoplasmic protein aggregates in cell bodies and axons of motor neurons are observed in both sporadic and familial ALS cases (Bruijn et al., 1997). However, in ALS the role of protein misfolding and aggregation has been studied much lesser than that in other ND, and the term ‘‘aggregate’’ has been used to refer to a variety of different morphological structures. The major component of ALS aggregates appears to be SOD1, but they also contain substantial quantities of ubiquitin and other proteins. ALS pathology has been observed in mice bearing the human mutated SOD1 gene (Gurney et al., 1994). These mice develop motor neuron dysfunction and typical pathological alterations, including the presence of hyaline inclusion bodies in degenerating axons, muscle atrophy, astrocytic damage, and extensive loss of large myelinated axons of motor neurons (Gurney et al., 1994).

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3.5 Prion Diseases Prion diseases, also called TSE, are a group of fatal neurodegenerative disorders that affect humans and other mammals (Collinge, 2001). A unique feature of these diseases is that they can have three different origins: sporadic, inherited, and infectious. The clinical, epidemiological, and neuropathological features can be very different in each of the diseases, but they are classified together because the key molecular event appears to be the same, that is, the misfolding of the prion protein (Prusiner, 1998; Collinge, 2001). Creutzfeldt–Jakob disease (CJD) is the most common form of TSE in humans. CJD is a rare disease with an estimated incidence rate of about one new case per million people each year. There are four different forms of CJD: (1) Sporadic CJD (sCJD) refers to those cases in which there is no known infectious source and no evidence of the disease in the prior or subsequent generations of the patient’s family. This is the most common subtype corresponding to about 85% of cases. Patients are usually between 50 and 75 years old and typical clinical features include a rapidly progressive dementia and myoclonus followed by loss of the ability to move or speak (Johnson and Gibbs, 1998). The disease is 100% fatal and in most of the cases death occurs within a year since the appearance of first clinical symptoms (Brown et al., 1986; Johnson and Gibbs, 1998; Weber, 2000; Collinge, 2001). (2) Familial CJD (fCJD) is an inherited disease that represents approximately 10–15% of the total CJD cases. In most of these kindreds, point mutations, deletions, or insertions are found in the coding sequence of the prion protein gene (Prusiner and Scott, 1997; Kovacs et al., 2002). More than 30 mutations in this gene have been described that are associated with phenotypes mimicking typical CJD or that induce distinctive progressive diseases with spongiform changes in the nervous system (Prusiner and Scott, 1997; Kovacs et al., 2002). In general, fCJD has an earlier age of onset and a more prolonged course than sporadic disease. (3) Iatrogenic CJD (iCJD) represents less than 5% of the cases and results from transmission of the causative agent via medical or surgical interventions using accidentally contaminated materials (Brown et al., 2000; Will, 2003). Iatrogenic transmission of CJD has occurred in cases involving corneal transplants, implantation of electrodes in the brain, dura mater grafts, contaminated surgical instruments, and treatment with human growth hormone derived from cadaveric pituitaries (Brown et al., 2000; Will, 2003). (4) Variant CJD (vCJD) is a new disease, which was first described in March 1996 (Will et al., 1996). In contrast to typical cases of sCJD, this variant form affects young patients (average age 27 years old) and has a relatively long duration of illness (median 14 months vs. 4.5 months in traditional sCJD). vCJD patients usually experience psychiatric symptoms early in the illness, which most commonly take the form of depression or less often a schizophrenia-like psychosis (Will et al., 2000). Compelling evidence suggest that vCJD has an infectious origin and is acquired by consumption or exposition to cattle meat contaminated by the cow form of TSE, known as bovine spongiform encephalopathy (BSE) (Bruce et al., 1997; Smith et al., 2004). Insufficient information is available at present to make any well-founded prediction about the future number of vCJD cases (Hilton, 2000; Balter, 2001; Smith et al., 2004). In addition to CJD, three other clinically or pathologically similar prion diseases have been recognized in humans, including kuru, Gerstmann–Straussler–Scheinker (GSS) disease, and fatal insomnia (Collinge, 2001). TSEs are known to affect various animal species including sheep, goats, mink, deer, elk cows, cats and exotic felines, and ungulates (Collinge, 2001). The most common animal TSE is scrapie, a disorder of sheep and goats that was first recognized in 1730 and became an endemic problem in several countries (Detwiler, 1992). However, the most worrisome zoonotic TSEs from the point of view of public health are BSE, the cattle disease that is known to have infected humans, and chronic wasting disease (CWD), a disease of wildrange and captive cervids that has spread uncontrollably in North America. The typical neuropathological alterations in TSEs are vacuolation of the neuropil in the gray matter, prominent neuronal loss, exuberant reactive astrogliosis, and a variable degree of cerebral accumulation of prion protein aggregates (Wells, 1993; Budka et al., 1995; MacDonald et al., 1996). The most specific of these abnormalities is the vacuolation, giving the brain the appearance of a sponge and hence the name of spongiform encephalopathies. A characteristic feature of TSE neuropathology is the large degree of variation in the distribution and magnitude of the abnormalities (Budka et al., 1995; Jeffrey et al., 1995). These variations have been attributed to several factors including the kinetics of prion propagation, cell tropism, differential toxicity, host neuro-anatomy, and genetic makeup.

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The nature of the transmissible agent is perhaps the most extensively studied aspect of TSE research (Soto and Castilla, 2004). Compelling evidence suggest that the infectious agent is composed mainly (or exclusively) of a misfolded protein that can propagate in the absence of nucleic acid. The proteinaceous nature of the agent led to its denomination as prion, and extensive research has enabled to identify that a b-sheet-rich misfolded form of the prion protein (termed PrPSc) is the main component (Prusiner, 1998). Infection with PrPSc produces the autocatalytic conversion of the normal form of the prion protein (termed PrPC) into more of PrPSc in a process that results in the formation of a toxic structure responsible for brain damage and disease. The fact that administration of highly purified PrPSc (>99%) is sufficient to induce the disease in experimental animals provides a strong support not only for the prion hypothesis, but also for the key role of protein misfolding in neurodegeneration.

4

Animal Models of Neurodegenerative Diseases

Several animal models have provided tremendous amount of information in understanding the mechanisms underlying ND (Bilen and Bonini, 2005; Marsh and Thompson, 2006). Drosophila has been used for modeling most syndromes, because the availability of powerful genetic tools allows testing many possible genetic modifiers. In addition, the architecture of the fly nervous system presents several similarities with the mammalian CNS, like compartmentalized brain functions such as olfaction, vision, or memory. Fly models are engineered by cloning the genes responsible for the diseases observed in humans into transposable p-element vectors under the control of a yeast transcription system and injected into fly embryos to obtain transgenic animals (Bilen and Bonini, 2005). Neuronal degeneration is typically measured by observation of the structure of the photoreceptor neurons of the eyes and motor function. Like in humans, transgenic drosophila models for human genes involved in ND develop progressive neuropathology (Bilen and Bonini, 2005; Marsh and Thompson, 2006). For example, upon expressing polyQtract extended protein or mutant alpha synuclein, tau, or Ab the number of intact photoreceptors rapidly decreased after the day of eclosion (Jackson et al., 1998). Aggregates of misfolded proteins are also found in transgenic flies, expressing the mutant human proteins, supporting the fact that fly is a suitable model for neurodegeneration (Bilen and Bonini, 2005; Marsh and Thompson, 2006). However, major limitations exist, the most significant ones including differences in morphology and functionality of the brain, differences in metabolism, a lack of or reduced inflammatory response, or the absence of the blood–brain barrier. Another animal model used to study ND is the worm Caenorhabditis elegans (Link, 2006; Brignull et al., 2007). This organism has an invariant number of 959 cells, of which, remarkably around one-third (302) are neuronal. Microinjection of mutated proteins results in several models of human disease (Kraemer et al., 2003; Miyasaka et al., 2005; Wu et al., 2005). Transgenic C. elegans expressing Ab presents deposits of the peptide in muscle cells with formation of vacuolation and motor impairment (Link, 1995). The Huntingtin gene fused with expanded triplet repeats has been shown to aggregate in the cytoplasm of the worm and to induce progressive cell death. Interestingly, the toxicity of polyQ-expanded protein was shown to occur at a threshold of approximately 40 glutamine residues, similar to the human disease (Morley et al., 2002). RNA interference (RNAi) technology has recently demonstrated that up to 186 genes might be involved in the response to protein misfolding events in transgenic worms expressing yellow fluorescent protein fused with an expanded polyQ tract (Nollen et al., 2004). Considering the complexity of neurological diseases, it is no surprise that transgenic mice have been the most popular model to study the contribution of protein misfolding in the pathogenesis of these diseases (Price et al., 2000). Several pathological, biochemical, and clinical features of diverse ND have been reproduced in transgenic mice models expressing mutant forms of the human genes encoding the corresponding fibrillar protein (Tom Van Dooren et al., 5 A.D.; Araki et al., 1994; Janson et al., 1996; Price et al., 1998; Weissmann et al., 1998; Emilien et al., 2000; Gurney, 2000; Price et al., 2000; Grieb, 2004). Transgenic mice overexpressing the human APP containing diverse mutations responsible for AD progressively develop many of the pathological hallmarks observed in humans, including cerebral amyloid deposits, neuritic dystrophy, astrogliosis, and cognitive and behavioral alterations (Duff, 1998; Price

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et al., 1998; Emilien et al., 2000). Similarly, transgenic mice overexpressing the human-mutated SOD1 gene involved in ALS pathology have shown to develop signs corresponding to the disease found in ALS patients (Price et al., 1997, 1998). Some of these mice developed motor neuron dysfunction and typical pathological alterations, including the presence of hyaline inclusion bodies in degenerating axons, muscle atrophy, astrocyte damage, and extensive loss of large myelinated axons of motor neuronal cells (Grieb, 2004). Transgenic mice expressing the wild-type form of the human a-synuclein gene develop several of the clinicopathological features of PD (Hashimoto et al., 2003). Among them are Lewy bodies accumulated in neurons of the neocortex, hippocampus, and substantia nigra, loss of dopaminergic terminals in the basal ganglia, and the associated motor impairments (Klement et al., 1998; Masliah et al., 2000). The transgenic mice model of HD expressing exon 1 of the human huntingtin and carrying 115–156 CAG repeat expansions develop pronounced neuronal intranuclear inclusions (Davies et al., 1997). These typical cerebral lesions were found to contain huntingtin and ubiquitin protein aggregates that appeared prior to the development of the neurological phenotype and were strikingly similar to those observed in HD patients. Furthermore, these mice develop a progressive neurological dysfunction with a movement disorder and weight loss similar to HD (Sathasivam et al., 1999). One of the first animal models mimicking a human misfolding protein disorder was obtained when the mutated gene PrnP, coding for human prion protein (PrP), was overexpressed in a transgenic mice (Hsiao et al., 1990). Spontaneous neurologic disease with spongiform degeneration was observed in these mice. Moreover, these abnormalities were found to be transmissible to some transgenic mice by inoculation of the sick brain homogenate. Thus, the critical role for protein misfolding and aggregation as the culprit of neurodegeneration is strongly supported by transgenic animal models in which expression of the human proteins involved in abnormal folding resulted in typical clinical and pathological features. However, time course studies of the appearance of neurological signs of the disorder in some of the transgenic models have shown that visible protein aggregates appear after clinical symptoms and significant tissue damage (Janson et al., 1996; Moechars et al., 1999; Van Leuven, 2000). These observations suggest the presence of a misfolded soluble intermediate that does not form large detectable aggregates and is not accumulated in the brain, which could be the real culprit for the pathogenesis (Tran and Miller, 1999; Goldberg and Lansbury, 2000; Caughey and Lansbury, 2003). This hypothesis implies that the formation of the characteristic aggregates could be considered as a protective mechanism working by sequestering the possible toxic misfolded proteins (Caughey and Lansbury, 2003).

5

Structural and Mechanistic Basis of Protein Misfolding and Aggregation

No sequence or structural homology has been found among the native proteins implicated in ND. However, there is accumulating evidence that the aggregates formed by the different misfolded proteins have a similar molecular structure (Soto, 2003; Glabe and Kayed, 2006; Sawaya et al., 2007). The process of misfolding and aggregation of most of the proteins involved in ND has been modeled in vitro. Large secondary structural differences between the monomeric native protein and the aggregated material have been observed using low-resolution structural studies (Makin and Serpell, 2005). The native protein is generally folded into a ahelix structure while the misfolded protein is rich in b-sheet conformation. High-resolution studies have been difficult due to the insoluble and noncrystalline nature of the aggregated proteins. However, recent studies using X-ray fiber diffraction and solid-state nuclear magnetic resonance have confirmed the b-sheet structure of the protein aggregates implicated in ND (Makin and Serpell, 2005; Nelson and Eisenberg, 2006; Tycko, 2006). Structural studies performed on misfolded proteins reveal that amyloid-like fibrils are composed of several protofilaments that consist of hydrogen-bonded b-sheets with the b-strands running perpendicular to the long fiber axis, a structure known as a cross-b conformation (Makin and Serpell, 2005; Makin et al., 2005). These structural analyses demonstrate that a large conformational rearrangement of the polypeptide chain occurs during misfolding and aggregation. However, it is presently unclear whether the misfolding triggers protein aggregation or whether it is the protein oligomerization that induces the conformational changes. This issue is of the highest interest, especially for the design of effective therapeutic strategies.

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Although the detailed mechanism for the formation of fibrillar amyloid-like aggregates is not entirely clear, the initiating event is protein misfolding, which results in the formation of aggregation-prone structures that can eventually grow by an autocatalytic mechanism (Soto, 2003). Kinetic studies have suggested that the critical event is the formation of protein oligomers that act as seeds to further propagate protein misfolding (Soto et al., 2006). This is the basis for the currently accepted nucleation-dependent polymerization model of amyloid formation (Gajdusek, 1994; Harper and Lansbury, 1997; Soto et al., 2006). Diverse proteins have been shown to follow this crystallization-like process, including Ab, huntingtin, and a-synuclein among others. According to this model, aggregation starts after the protein concentration exceeds a point known as the critical concentration (Harper and Lansbury, 1997). Unfavorable interactions between monomers determine a slow phase (termed lag phase) in which oligomers are formed, providing an ordered nucleus to catalyze further growth of the polymers. The addition of preformed nuclei (seeds) serves as templates for the reaction and, as a result, the initial, slow phase of primary nucleation is eliminated (Harper and Lansbury, 1997; Soto et al., 2006). In vitro studies have been useful for understanding the structural requirements for protein misfolding and the fragments of the protein mostly implicated in the conformational changes. Using mutated peptides or shorter Ab fragments it has been shown that the internal hydrophobic region between amino acids 17 and 21 is essential in the early steps of Ab misfolding and aggregation (Soto, 1999). This finding demonstrates that Ab assembly is partially driven by hydrophobic interactions (Hilbich et al., 1992; Soto et al., 1995; Wood et al., 1995; Lazo et al., 2005), which is consistent with the higher ability of Ab peptides to aggregate with two or three extra hydrophobic amino acids at the carboxy terminus (Jarrett et al., 1993). Indeed, hydrophobic forces appear to be crucial in the aggregation process of most of the misfolded proteins. In PrPc misfolding, the hydrophobic fragment 106–126 of PrPc has been mapped to represent the most relevant sequence for protein oligomerization (Tagliavini et al., 1993; Ziegler et al., 2006). A similar observation has been reported concerning the fibrillogenesis of a-synuclein. Although less is known about this process, evidence indicates that the amino-terminal fragment 1–87 might be crucial (Serpell et al., 1995, 2000). However, the aggregation process involved in huntingtin and other polyglutamine-containing proteins seems to be different. In HD and other polyglutamine diseases, both disease and protein aggregation are associated with an inherited expansion of CAG (the codon for glutamine) repeats. In the case of HD, aggregation of huntingtin in vitro depends on the length of the polyglutamine repeat (Scherzinger et al., 1997; Burke et al., 2003), and the aggregation process is driven by the glutamine residue, which contains an amide group that provides a polar side chain and the potential to form a hydrogen bond with water in a model termed ‘‘polar zipper’’ (Perutz et al., 1994; Chan et al., 2005). In this model, b-sheets are formed and stabilized by the collective strength of cooperative hydrogen bonding involving the amide groups of the glutamine residues. Therefore, protein aggregation can arise from two different (and, in some respects, opposite) driving forces: hydrophobic interactions and polar hydrogen bonding among side-chain groups. In addition to mature fibrils, several other structures have been described as part of the protein misfolding and aggregation process, including soluble oligomers, pores, annular structures, spherical micelles, and protofibrils (Caughey and Lansbury, 2003; Glabe and Kayed, 2006; Haass and Selkoe, 2007). Interestingly, these diverse structures have been identified in the amyloidogenesis process of various disease-associated proteins, suggesting common misfolding pathways (Caughey and Lansbury, 2003; Glabe and Kayed, 2006; Haass and Selkoe, 2007). However, currently, the biological relevance of these intermediates is not clear, and it is even unknown whether or not they exist in the diseased brain. Furthermore, although it is likely that these metastable species assemble in a stepwise process, the relative implication of each of them is difficult to assess because they are too unstable to study (Glabe and Kayed, 2006; Teplow et al., 2006). Recently, a set of antibodies that recognize specifically different type of aggregated species, such as oligomers, annular assemblies, protofibrils, and fibrils, have been produced (Glabe and Kayed, 2006; Kayed and Glabe, 2006). Strikingly, these antibodies are not sequence specific, but conformation specific and thus they can recognize intermediate species formed by different proteins (Glabe and Kayed, 2006; Kayed and Glabe, 2006). In summary, the biophysical studies of the intermediates in the amyloid formation process indicate that diverse species with progressive degree of aggregation are present simultaneously and in a dynamic equilibrium between each other (Caughey and Lansbury, 2003; Glabe and Kayed, 2006;

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Teplow et al., 2006). This makes evaluation of the relative contribution of different protein structures to neurodegeneration very difficult.

6

Mechanism of Brain Degeneration

Besides of protein deposits, the typical pattern of brain damage in ND includes selective neuronal death, synaptic alterations, and astrogliosis (Martin, 1999). The different clinical picture associated with distinct ND is most likely determined by the diverse brain regions most affected in each case. Although it was widely thought that neuronal apoptosis was the most important problem in neurodegeneration, recent evidence from different diseases suggest that extensive neuronal death may not be the initial cause of the disease (Haass and Selkoe, 2007). Indeed, clinical symptoms can be observed before significant neuronal loss and a better temporal and topographic correlation is found with synaptic dysfunction (Haass and Selkoe, 2007). Although protein misfolding and aggregation is undoubtedly associated with neurodegeneration and disease, the mechanism by which this process leads to brain damage is unknown. The most widely accepted theory of brain degeneration in ND proposes that misfolding and aggregation results in the acquisition of a neurotoxic function by the misfolded protein (Soto, 2003). Although the possibility that neuronal damage may be produced by the loss of activity of the protein upon the conformational changes has been extensively studied, most of the evidence argues against this mechanism (Soto, 2003). The mechanism by which misfolded aggregates produce synaptic dysfunction and neuronal death is unknown. It is also unknown which of the different polymeric structures formed in the process of amyloidogenesis is the triggering factor of brain damage (Lansbury and Lashuel, 2006; Haass and Selkoe, 2007) (> Figure 12-1). For many years it was thought that big amyloid-like protein deposits were the species responsible for brain damage. However, the hypothesis that large aggregates accumulated in the brain are toxic has been challenged by histopathological, biochemical, and cell biology studies (Lansbury and Lashuel, 2006; Haass and Selkoe, 2007). The current view is that the process of misfolding and early stages of oligomerization, rather than the mature compacted aggregates deposited in the brain, are the real culprits in neurodegeneration (Glabe and Kayed, 2006; Lansbury and Lashuel, 2006; Haass and Selkoe, 2007). This hypothesis is supported by results showing that purified oligomeric species and protofibrils are toxic to cultured neurons, inhibit hippocampal longterm potentiation, impair synaptic functions, and disrupt cognition and learned behavior in rats (for list of references, see Glabe and Kayed (2006), Lansbury and Lashuel (2006), and Haass and Selkoe (2007)). Several mechanisms have been proposed for the neurotoxic activity of misfolded oligomers, and it is likely that different pathways operate, depending on whether the proteins accumulate intra- or extracellularly (Soto, 2003). Extracellular oligomers might activate a signal transduction pathway leading to apoptosis by interacting with specific cellular receptors. Intracellular polymers might damage cells by, for example, recruiting factors essential for cell viability. Another well-supported mechanism by which oligomeric and annular pore-like structures may damage cells is by membrane disruption and depolarization, resulting in alterations of ion homeostasis and disregulation of cellular signal transduction (Glabe and Kayed, 2006). Finally, misfolded proteins could induce cellular oxidative stress by producing free radical species, resulting in protein and lipid oxidation, elevation of intracellular calcium, and mitochondrial disfunction (Behl et al., 1994; Hsu et al., 2000).

7

Targets for Therapy

In spite of the extensive knowledge collected in recent years toward understanding the pathogenesis of ND, there are no effective therapies directed to prevent or reverse the disease progression. If protein misfolding and aggregation play a central role in the pathogenesis of ND, then the strategy to follow should be the prevention and/or the correction of the misfolding process. At least three different strategies have been proposed to intervene against protein misfolding and aggregation (Soto, 2003): (1) stabilization of the native protein conformation; (2) inhibition and reversion of protein conformational changes; and (3) increase the clearance of the misfolded protein.

Protein misfolding, a common mechanism in the pathogenesis of neurodegenerative diseases

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Stabilization of the native folding of the normal protein has been attempted in several protein misfolding disorders. In the case of AD, nicotine and cotinine have been shown to inhibit Ab amyloid formation by specifically binding to the peptide and stabilizing its alpha helix structure (Salomon et al., 1996; Ono et al., 2002). In prion-infected neuroblastoma cells, treatment with chemical chaperones (reagents known to stabilize native conformation of proteins) results in prevention of PrP misfolding (Tatzelt et al., 1996; Bennion et al., 2004). Protein engineering has been proposed as an approach to create sequence-modified proteins with higher stability, lower tendency to misfold, and ability to trans-suppress the aggregation of wild-type proteins (Villegas et al., 2000). Although the idea is attractive, its therapeutic application would require sophisticated gene therapy technologies. Several small molecules, peptide fragments, specific antibodies, and proteins capable of interacting with b-sheet aggregates have been shown to specifically interact and inhibit protein aggregation (Aguzzi et al., 2001; LeVine, 2002; Sacchettini and Kelly, 2002; Bose et al., 2005; Soto and Estrada, 2005). Several small chemical molecules have been reported to have this activity, including Congo red, 40 -iodo-40 -deoxydoxorubicin, curcumin, rosmarinic acid, rifampicine, polyphenol, tetracycline, minocyclin, and ferulic acid among others (Forloni et al., 2001, 2002; Cardoso et al., 2003; Ono et al., 2005; Familian et al., 2006). The majority of the active compounds produce their effect by competitively blocking protein–protein interactions. These compounds can be classified in two classes depending on their ability to either interact with the complexes in between monomers or to bind at the edge of b-sheet oligomers, preventing their growth. One of the major problems with this approach is to determine the mechanism of action of these competitive inhibitors. Depending on their activity, it is likely that some inhibitors may lead to accumulation of toxic intermediates and thus may aggravate the disease. Another class of inhibitors compounds act by destabilization of the pathological b-sheet conformation of misfolded proteins. One example of this strategy are b-sheet breaker peptides that have been shown to inhibit and reverse protein misfolding in vitro and in vivo (Soto and Estrada, 2005). b-Sheet breakers are short synthetic peptides that can specifically interact with the protein fragment that is undergoing misfolding but has been designed to be unable to get incorporated into the b-sheet structure. b-Sheet breaker peptides have been designed to block the conformational changes and aggregation of both Ab and PrP in vitro and in various animal models (Soto et al., 1998, 2000; Permanne et al., 2002). Another strategy is to increase the clearance of misfolded and aggregated proteins. This alternative represents an interesting approach based on findings showing that accumulation of protein aggregates is dependent on a balance between deposition and clearance. In a conditional transgenic model of HD, preventing the production of mutant fragments caused nuclear inclusions to disappear, indicating that cells can metabolize the aggregated material (Yamamoto et al., 2000). Perhaps the most promising strategy to increase the clearance of misfolded proteins is the immunization approach first described for AD (Schenk et al., 1999). Aggregates of synthetic Ab protein were used as antigens to induce the immune system to produce antibodies to clear them. Immunization can reduce amyloid load, cerebral damage, and behavioral impairments in transgenic animal models of AD (Schenk et al., 1999; Janus et al., 2000; Morgan et al., 2000; Boche et al., 2005; Gandy and Heppner, 2005; Goni and Sigurdsson, 2005; McGeer et al., 2005; McGavern, 2006). A similar approach has been used for the treatment of TSE (Sigurdsson et al., 2002; Cashman and Caughey, 2004; Boche et al., 2005; Goni and Sigurdsson, 2005; Magri et al., 2005). However, a clinical trial to evaluate the efficacy of the immunization strategy in humans affected by AD was stopped because of several cases of meningoencephalitis (Schenk, 2002). Little is known about the reason for this side effect, but future research should bring both more knowledge about this problem and also should result in new strategies to minimize brain inflammation after vaccination. Immunization with fragments of Ab that preferentially stimulate the B cells (and not the T cells) or passive immunization with antibodies against the misfolded proteins might be safer strategies (Schenk, 2002). Enhancement of the clearance of amyloid-like deposits has been also attempted by removing some accessory constituents found in plaques. The underlying idea is that other factors that tightly bind to the aggregates might increase their insolubility and resistance to proteolytic degradation. Strategies for removing specifically the amyloid-P component, proteoglycans, and metal ions have shown the best results in models of AD and systemic amyloidosis (Kisilevsky et al., 1995; Cherny et al., 2001; Pepys et al., 2002; Lee et al., 2004; Maynard et al., 2005).

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Conclusions and Perspectives

Over the last 10 years considerable progress has been made in our understanding of the molecular mechanisms responsible for ND. The observation that several syndromes are histologically characterized by accumulation in the brain of normal proteins has led to the hypothesis that most ND are caused by a very similar cascade of events. Misfolding of distinct proteins appears to be the initiating step that eventually leads to neuronal dysfunction and clinical symptoms specific for each disease. Formation of amyloid structures in the brain is the visible consequence of abnormalities in protein folding, but recent data suggest that the plaques themselves may not be the triggering factor in neurodegeneration. The similarities observed in the different ND provide hope for a common therapeutic strategy to treat these devastating illnesses. The development of a therapy based on the misfolding concept may also enable a better understanding of the mechanisms that eventually lead to amyloid formation. The concept that protein misfolding leads to severe illness is relatively new; thus, one important research for the future is to find out whether other diseases are associated with similar alterations. For example, a report demonstrated that atherosclerosis might indeed be a consequence of ApoB misfolding induced by high cholesterol level (Ursini et al., 2002). This raises the possibility that other common diseases might directly or indirectly be a consequence of the misfolding of proteins. During many years it was thought that only certain proteins could undergo misfolding and eventually form amyloid-like structures, but this concept has been reconsidered since recent studies have shown that many, if not all, proteins can form amyloid-like structures under appropriate conditions (Fandrich et al., 2001; Stefani and Dobson, 2003). Even though most of the data were obtained for proteins incubated in nonphysiological pH or salt concentrations, it cannot be excluded that in certain cellular compartments, these conditions could be reached transitorily, leading to a seeding event that would trigger the aggregation process. One important open question is whether protein misfolding always leads to disease or rather a similar phenomenon may play a normal biological function. The recent demonstration that several proteins can misfold and aggregate into amyloid-like structures (Lindquist, 1997; Wickner et al., 1999; Iconomidou et al., 2000; Wosten and de Vocht, 2000; Chapman et al., 2002; Kenney et al., 2002; Berson et al., 2003; Bieler et al., 2005), changing the biological activity of the protein suggest that the acquisition of alternative protein folding is indeed not only a disease-associated process. The possibility that many proteins may adopt multiple conformations to exert different functions might revolutionize our understanding of biology.

Acknowledgments This project was supported in part by NIH grant R01 AG028821 and an award from CART Foundation.

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Anti‐Aging Strategies

J. A. Joseph . J. R. Perez‐Polo

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306 2 Muscarinic Receptors and Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 3 Muscarinic Receptors and Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308 4 Consequences of Aging on Cholinergic Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 5 The Effects of Fruit and Vegetable Supplementation on Behavioral and Neuronal Deficits in Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

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Abstract: There are age-associated motor and cognitive deficits, even in the absence of neurodegenerative disease, that result from alterations in the striatal dopamine or cholinergic systems, respectively. In both instances, oxidative stress and inflammation are contributing factors to the observed behavioral impairments. Muscarinic acetycholine receptors (mAChR) play important roles in neuronal and vascular function. There is a loss of sensitivity in classic receptor systems that include the dopaminergic and muscarinic systems. It appears that a major factor that may be important in the loss of sensitivity in mAChR with aging may be increased sensitivity to oxidative stress that is at least partially determined by changes in membranes. Also possibly contributing to the loss of cholinergic integrity and ultimately behavioral function in aging are perturbations in ChAT function. There are fewer cholinergic neurons in the aged basal forebrain that express lower levels of cholinergic activity. For example, decreased ACh synthesis in cholinergic neurons of the aged rat basal forebrain may result in part from altered binding of NF-kB to a cognate DNA-binding repressor sites within the ChAT promoter. Given that NF-kB DNA-binding activity is elevated in the aged rat basal forebrain and hippocampus, the result is age-associated decreases in ChAT expression that have consequences on cognitive function in the aged. Given the role of oxidative stress in aging, Anti-oxidant based fruit and vegetable supplementation could forestall the consequences of oxidative stress on behavioral and neuronal deficits associated with aging. List of Abbreviations: BC, black currant; BY, boysenberry; CB, cranberry; CHL, cholesterol; DA, dopamine; DP, dried plums; ERK, extracellular signal‐regulated kinase; GR, grape; MPT, mitochondrial pore transition; ORAC, oxygen‐radical absorbance capacity assay; PKC, protein kinase Ca; RIP, receptor‐ interacting protein; SAM, S‐adenosylmethionine; SB, strawberry; TNFR, tumor necrosis factor receptor; TRAF2, TNFR‐associated factor 2; VaD, vascular disease

1 Introduction There is a plethora of research indicating the occurrence of numerous behavioral deficits during aging, even in the absence of neurodegenerative disease. These decrements can be expressed, ultimately, as alterations in both motor (Joseph et al., 1983; Kluger et al., 1997) and cognitive behaviors (Bartus, 1990). The alterations in motor function may include decreases in balance, muscle strength, and coordination (Joseph et al., 1983), while memory deficits are seen on cognitive tasks that require the use of spatial learning and memory (Ingram et al., 1994; Shukitt‐Hale et al., 1998). Indeed, these characterizations have been supported by a great deal of research both in animals (Bartus, 1990; Ingram et al., 1994; Shukitt‐Hale et al., 1998) and humans (West, 1996; Muir, 1997). Age‐related deficits in motor performance are thought to be the result of alterations in the striatal dopamine (DA) system as the striatum shows marked neurodegenerative changes with age (Joseph, 1992) or in the cerebellum which also shows age‐related alterations (Bickford et al., 1992; Bickford, 1993). Memory alterations appear to occur primarily in secondary memory systems and are reflected in the storage of newly acquired information (Bartus et al., 1982; Joseph, 1992). It is thought that the hippocampus mediates allocentric spatial navigation (i.e., place learning), and that the prefrontal cortex is critical for acquiring the rules that govern performance in particular tasks (i.e., procedural knowledge), while the dorsomedial striatum mediates egocentric spatial orientation (i.e., response and cue learning) (McDonald and White, 1994; Zyzak et al., 1995; Devan et al., 1996). It appears that oxidative stress (OS) (Shukitt‐Hale, 1999) and inflammation (Hauss‐Wegrzyniak et al., 1999, 2000) are contributing factors to the behavioral decrements seen in aging. There are numerous factors involved in mediating these behavioral deficits in aging that include decrements in calcium buffering (Landfield and Eldridge, 1994), as well as declines in the sensitivity of several receptor systems including the (1) adrenergic (Gould and Bickford, 1997) and (2) dopaminergic (Joseph et al., 1978; Levine and Cepeda, 1998) systems. However, one of the most notable changes are those involving the muscarinic cholinergic system. There is a long association between cognitive behavior and cholinergic functioning in the literature (Wo¨rtwein et al., 1998), and there are many reports of significant declines in cholinergic functioning in aging. While we realize that there is an involvement of the nicotinic

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cholinergic system in learning and memory that mediates cognition, those changes that have been observed in the muscarinic system in aging and the possible mechanisms involved in these changes have not been well defined.

2 Muscarinic Receptors and Aging Research indicates that muscarinic acetylcholine receptors (mAChRs) are intimately involved in various aspects of both neuronal (e.g., amyloid precursor protein (APP) processing) (Rossner et al., 1998) and vascular functioning (Elhusseiny et al., 1999). The finding concerned with mAChR/vascular interaction is important, since it also appears that vascular disease and Alzheimer’s disease (AD) can occur in concert in aging and that vascular disease may exacerbate cognitive dysfunction in AD (Morris et al., 2000; Deschamps et al., 2001). In fact, data suggest that there may be complex interactions between vascular dementia (VaD) and AD (Morris, 2000; Deschamps et al., 2001). There is a long history of research showing a loss of sensitivity in classic receptor systems including the dopaminergic and muscarinic (see Joseph et al., 1992). Numerous studies have shown that the PI‐linked muscarinic receptors (mAChRs) show deficits in signal transduction, which contributes to their loss in sensitivity (Joseph and Roth, 1991). Joseph et al. (1991) have shown that mAChR agonist enhancement of IP3 formations is reduced in striatal tissue obtained from senescent Wistar rats. Additional studies have suggested that the site of the deficit in agonist responsiveness in mAChR in aging may be at the mAChR/G‐ protein interface (Joseph et al., 1988), which may involve deficits in mAChR/G‐protein coupling/uncoupling (Yamagami et al., 1992). This is an important consideration since research also indicates that decrements in mAChR/G‐protein coupling/uncoupling, as assessed via carbachol‐stimulated GTPase activity, may also be greater in (Cutler et al., 1994) than that seen in aging. Additionally, there also appear to be reductions in anterior, cortical PI levels, as well as a 70% loss of IP3 binding in the temporal cortex and hippocampus (Young et al., 1988) and a decrease in the number of mAChRs that are coupled to G proteins in the high‐affinity states (Flynn et al., 1991). The mechanisms involved in these deficits have yet to be determined, but such factors as decreases in membrane fluidity may also be involved. Findings showed that when striatal tissue was incubated with S‐adenosylmethionine (SAM), a potent membrane‐fluidizing agent, it increased the sensitivity of the mAChR to agonist stimulation in striatal slices, while exposure of the slices to cholesterol (CHL), which increases membrane viscosity, decreased this sensitivity (Joseph et al., 1991). There are decreases in membrane fluidity in aging that involves the translocation of CHL to the outer leaflet of the membrane where it is more readily oxidized. It also appears that sphingomyelin metabolites such as sphingosine‐1‐ phosphate may increase sensitivity to OS in aging. Recent studies have also suggested an involvement of lipid rafts with OS sensitivity (Shen et al., 2004). The findings of Shen and coworkers (2004) indicate that receptor‐interacting protein (RIP) and tumor necrosis factor receptor (TNFR)‐associated factor 2 (TRAF2), which are two primary molecules involved TNF signaling, may be necessary for ROS‐induced cell death of TNFR1. The colocalization of RIP with a membrane lipid raft marker (GM‐1) suggested that these rafts might participate in the formation of a complex between RIP and TRAF‐2 during OS. The study suggests that this complex may be part of a novel signaling pathway that mediates oxidant‐induced cell death. Thus, it appears that a major factor that there may be important in the loss of sensitivity in mAChR with aging may be increased sensitivity to OS, which is at least partially determined by changes in membranes. Another component of the cholinergic signaling system that is affected in AD pathophysiology is regulation of the deposition of b‐amyloid (Ab) peptides due to increased APP processing, resulting in increased levels of Ab. For example, AChE inhibitor treatments increase the secretion of nonamyloidogenic‐ processing products and decreases Ab formation. (Mori et al., 1995; Pakaski et al., 2001). There is also in vivo evidence that APP processing underlies cholinergic regulation in the rat brain (Rossner et al., 1998). When M1‐MAChRs are selectively stimulated in tissue slices, the nonamyloidogenic processing of APP increases, suggesting that activation of the M2‐MAChR subtype results in a suppression of the

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nonamyloidogenic processing pathway (Farber et al., 1995). Furthermore, BACE‐1 expression is regulated in different ways through MAChR. Since increased M1‐MAChR increases BACE‐1 expression but increased M2‐MAChR decreases BACE‐1 expression (Zuchner et al., 2004), cholinergic function is important for APP processing and AD pathophysiology. This is also consistent with the observation that there is demonstrable downregulation of M2‐MAChR in those brain regions of AD patients which contain senile plaques (Boulay et al., 1996; Lai et al., 2001; Apelt et al., 2003).

3 Muscarinic Receptors and Oxidative Stress Several experiments conducted over the last several years have indicated that there are considerable parallels between the loss of sensitivity seen in aging in muscarinic receptors and those seen following heavy‐particle irradiation. Thus, studies have shown that, just as was seen in aging, striatal tissue obtained from young rats exposed to various doses of 56Fe irradiation (0.1–1 Gy) showed reductions in oxotremorine enhancement of Kþ‐evoked dopamine release (Kþ‐EDAR) (Joseph et al., 1992), as well as decreases in carbachol‐stimulated IP3 activity (Joseph et al., 1993a) and carbachol‐stimulated GTPase activity, an indicator of receptor/ G‐protein coupling/uncoupling. Further studies showed that the deficit appeared to be at the muscarinic receptor/G‐protein interface (Joseph et al., 1993a). As early as 1991, Stark postulated that certain free radicals were able to gain access into the interior of cellular membranes and interact with the lipid matrix or with membrane‐bound proteins, altering their integrity. Importantly, in this regard, subsequent research indicated that as was seen previously in aging, increasing membrane fluidity via exposure of striatal tissue obtained from the irradiated animals to SAM reversed the irradiated‐induced deficits in Kþ‐ERDA ( Joseph et al., 1999b). Even more important are recent findings that suggest that there may be differences among MAChR subtypes in response to OS in aging. Clearly, if this is the case, it might provide some explanation for findings such as those showing that there is differential aging among various brain regions. Areas such as the hippocampus (Nyakas et al., 1997; Kaufmann et al., 2001), cerebellum (Hartmann et al., 1996; Kaufmann et al., 2001), and striatum (Joseph et al., 1996; Kaasinen et al., 2000) show profound alterations in aging in such factors as morphology, electrophysiology, and receptor sensitivity. Interestingly, studies that have examined regional differences in mAChR subtypes have shown that the mAChRs that show increased vulnerability to OS appear to be localized in areas that show profound alterations in aging; these include the dentate, striatum, and forebrain (e.g., see Levey, 1996 for review). Thus, we assessed differential vulnerability to OS among the various mAChR subtypes by transfecting one of five MAChR subtypes (M1–M5) in COS‐7 cells. The results showed that when the cells were exposed to DA (Joseph et al., 2002) or Ab (Joseph and Fisher, 2003), differences in OS sensitivity, expressed as a function of Ca2þ buffering as assessed by examining the ability of the cell to extrude or sequester Ca2þ following oxotremorine‐induced depolarization, were observed. The COS‐7 cells transfected with M1, M2, or M4‐AChRs showed greater sensitivity to DA‐ or Ab‐induced disruptions in calcium buffering than those transfected with M3‐ or M5‐MAChRs. An additional study ( Joseph et al., 2004) has suggested that the locus of the differential sensitivity to OS among the various receptor subtypes may involve the i3 loop, and studies are being carried out to determine possible differences among the i3 loops of these mAChR subtypes that could contribute to these differences. Importantly, the findings with respect to COS‐7 cells suggest that, as mentioned above, brain areas showing increased effects of aging contain high concentrations of M1, M2, or M4 receptors (Flynn and Mash, 1993; Levey, 1994; Hersch et al., 1999). Additionally, it should be noted that the loss in calcium buffering could lead to further decrements in cell functioning and OS, since the intracellular compartmentalization of high amounts of calcium (Brini, 2003) and mitochondrial pore transition (MPT) (Green and Kroemer, 2004) can mediate both cell necrosis and apoptosis, with MPT possibly releasing cytochrome c. Thus, in the case of aging, OS appears to impinge on systems that are already compromised in their ability to regulate Ca2þ flux. Additionally, it also appears that the membrane alterations and decrements in calcium buffering may reduce the effectiveness of endogenous antioxidants in the aged organism (Joseph et al., 1998a, 2001).

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4 Consequences of Aging on Cholinergic Function Also possibly contributing to the loss of cholinergic integrity and ultimately behavioral function in aging are perturbations in choline acetyltransferase (ChAT) function. There are fewer cholinergic neurons in the aged basal forebrain, and they express lower levels of ChAT and ACh uptake than younger rats. These differences are consistent with age‐associated increases in pro‐apoptotic markers and decreases in ChAT activity (Drachman and Leavitt, 1974; Davies and Maloney, 1976; Drachman and Sahakian, 1980; Bartus et al., 1982; Whitehouse et al., 1982; Davies, 1985; Fischer et al., 1987; Williams et al., 1993; De Lacalle et al., 1996; Clemens et al., 1997). For example, cholinergic deafferentations carried out by transection of the fimbria fornix or selective killing of basal forebrain cholinergic neurons by immunolesion fail to increase neurotrophin protein levels in aged rat brains (Whitehouse et al., 1982; Fischer et al., 1987; Wo¨rtwein et al., 1998). Taken together, it would appear that there is an aging‐associated impairment of stress response mechanisms. Determining the different contributions of cholinergic cell losses versus changes in the cholinergic phenotype in the aged brain remains a disputed area. For example, it could be that the activities of transcription factors that regulate gene expression responsible for cholinergic function and apoptotic responses to OS and trauma are altered in the aged brain (Tong et al., 2002). Although the activation of the transcription factor NF‐kB has been determined in the aged rat and AD brains, the assessments have been limited to binding to oligonucleotide preparations displaying the IgG‐kB promoter consensus sequence and have not differentiated among the specific sequences present in individual promoters. Choline acetyltransferase is rate limiting for acetylcholine synthesis (Nachmansohn and Machado, 1943). ChAT is expressed in basal forebrain cholinergic neurons, important in memory and cognition, whose deterioration is associated with cognitive deficits. Decreases in ChAT and acetylcholine release are pathological markers in AD and aging‐associated dementias (Armstrong et al., 1983; Davies, 1985; Williams et al., 1993). The cDNAs and regulatory regions for mouse, rat (Ishii et al., 1990), human (Toussaint et al., 1992), and porcine (Berrard et al., 1987) ChATs have been cloned. They are highly homologous, and while the 50 ‐flanking sequences have greater interspecies variation, they share common regulatory elements, such as a silencer element restricting expression to cholinergic neurons (Lonnerberg et al., 1996). ChAT promoters also have a general enhancer and an NGF‐responsive enhancer (Bejanin et al., 1992; Pu et al., 1993). The mouse ChAT promoter 50 ‐flanking sequence is alternatively spliced to yield seven different mRNAs (R1–4, N1–2, and the most predominant M form) that differ in their 50 ‐noncoding regions (Misawa et al., 1992). Misawa and coworkers (1992) cloned a 4,014 base pair (bp) region of the promoter, including the 1,470 nucleotides reported by Pu and coworkers (1993). Here, promoter positions are numerically referenced as described by Misawa and coworkers (1993). The 50 ‐flanking region lacks a consensus TATA box, but contains TATA‐like elements and multiple transcription initiation start sites. There is a strong enhancer element that is not modulated by NGF (–3021 to –2706) and an NGF‐inducible enhancer (–3416 to –3021). Region –3516 to –3148 confers cell‐type specificity, acting as a repressor element in noncholinergic cells (Pu et al., 1993). Although there are AP‐1 (–2934 to –2928 and –2858 to –2852), serum response element (–3159 to –3145), cAMP response element (–3083 to –3076), NGFI‐A (–3190 to –3182), and NF‐kB (–3174 to –3165) consensus sites within the NGF‐responsive element, it is not known if the cognate transcription factors regulate ChAT. Transcription of R‐type mRNAs is controlled by regulatory elements upstream of the R promoter, and transcription of N‐ and M‐type mRNAs by elements surrounding the N and M promoters. The most common form of ChAT mRNA, which is present in the brain and relevant to this project, is the M‐form. Misawa and coworkers (1992) cloned a 4014‐bp flanking region, which includes the N‐ and M‐type exons and the 1470 nucleotides reported by Pu and coworkers (1993); numbering of mouse ChAT promoter nucleotide positions will be as in Misawa et al. (1992). The transcription factor NF‐kB belongs to a family of homo‐ and heterodimeric proteins that are related by a conserved
300 residue NH2 terminal Rel/homology domain and includes p50, p65 (or RelA), p52 (or p49), c‐Rel, and RelB proteins (Bours et al., 1993; Nolan et al., 1993; Ruben et al., 1991; Schmid et al., 1991; Bours et al., 1993). Heterodimerization of NF‐kB proteins produces species with different intrinsic DNA‐binding specificities (p52, p50) and transactivation properties (p65, c‐Rel), an important

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functional distinction (Baeuerle, 1991; Siebenlist et al., 1994; Chen et al., 1998). The most commonly described active subunit combination (Flohe et al., 1997) is the p50/p65 heterodimer, present in the cytoplasm, bound to an inhibitory IkB subunit. There are several inhibitory IkB proteins (IkBa, IkBb, IkBe, IkBg, and Bcl‐3). Upon stimulation, IkBa is phosphorylated, ubiquitinated, and degraded (Ghosh and Baltimore, 1990; Liu et al., 1993), exposing nuclear localization signals on NF‐kB proteins, which allow translocation to the nucleus for DNA binding. NF‐kB is stimulated by OS or receptor ligands via increased IkBa degradation and NF‐kB nuclear translocation, but also via an independent pathway involving Bcl‐3 (Gozal et al., 1998; Zhang et al., 1998; Qiu et al., 2001). Bcl‐3, of the IkB family, functions in the nucleus by drawing NF‐kB p50 or p52 homodimers away from NF‐kB‐binding sites in promoters (Bours et al., 1993; Franzoso et al., 1993; Fujita et al., 1993; Nolan et al., 1993; Bundy and McKeithan, 1997). Thus, Bcl‐3 can inhibit p50 or p52 homodimer binding to promoter sites (Franzoso et al., 1993; Nolan et al., 1993; Siebenlist et al., 1994; Zhang et al., 1998), allowing cRel/p50 or p65/p50 binding and activation (Heissmeyer et al., 1999; Qiu et al., 2001). We and others have shown that NF‐kB DNA‐binding activity is elevated in the aged rat basal forebrain (Toliver‐Kinsky et al., 1997; 2000). Our hypothesis is that age‐associated alterations in these two regulatory factors contribute to the age‐associated decreases in ChAT expression, which has consequences on cognitive function in the aged. We have shown that a 20‐bp ChAT (NF‐kB) oligonucleotide probe containing the ChAT (NF‐kB) site binds to NF‐kB p49, somewhat less to p65, but not at all to p50, cRel, or RelB. This observation supports the idea that different consensus sequences are regulated by different subunits in a gene‐specific and tissue‐specific manner. When PC12 cells are transfected with two ChAT promoter reporter constructs containing the wild‐type ChAT promoter and a mutant construct where the NF‐kB binding site is replaced with a Bgl2 restriction site, there was a threefold increase in promoter activity in the cultures transfected with the mutant reporter plasmid as compared with those transfected with the wild‐type plasmid, consistent with the assignment of a repressor function to the (ChAT) NF‐kB site. Furthermore, the NF‐kB proteins p49, p65, and p50 were overexpressed in NGF‐treated PC12 cells, and ChAT promoter and enzymatic activities were measured. The overexpression of p49 and of p49 and p65 significantly decreased total ChAT activity as compared with controls. The assignment of a repressor role to NF‐kB on the ChAT promoter agrees with reports of increased NF‐kB binding and decreased ChAT in aged and AD brains (Coyle et al., 1983; Vile et al., 1995; Helenius et al., 1996; Taglialatela et al., 1996; Terai et al., 1996; Boissiere et al., 1997a, b; Kitamura et al., 1997). We measured NF‐kB activity by EMSA and Western blots on nuclear fractions from brains of 3‐ and 30‐month‐old male F344  BN hybrid rats. p49 appeared to be higher in the basal forebrain nuclear extracts prepared from 30‐month‐old rats. Thus, NF‐kB DNA‐binding activity increases in the aged basal forebrain where it may contribute to decreased ChAT expression. Interestingly, this increase supports the hypothesis that changes in NF‐kB are gradual and proportional to the aging process.

5 The Effects of Fruit and Vegetable Supplementation on Behavioral and Neuronal Deficits in Aging We have attempted to outline the possible role of OS in mediating decrements in cholinergic functioning in aging. It is evident that methods to reduce stress may forestall or even reduce these deficits in the aged organism. While there are numerous studies suggesting that various antioxidant supplements (see Casadesus et al., 2002 for review) may be effective in this regard, our research suggests that the combinations of antioxidant/antiinflammatory polyphenolics found in fruits and vegetables may show efficacy in aging. All plants, including fruit or vegetable bearing plants, synthesize a vast array of chemical compounds that are not necessarily involved in the plant’s metabolism. These ‘‘secondary compounds’’ instead serve a variety of functions that serve to enhance the plant’s survivability. These compounds may be responsible for the putative multitude of beneficial effects of fruits and vegetables on health‐related issues, two of the most important of which may be their antioxidant and antiinflammatory properties. The anthocyanins are among the plant polyphenols that have potent antioxidant and antiinflammatory activities. These are natural pigments responsible for the orange, red, and blue colors of fruits, flowers,

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vegetables, and other storage tissues in plants (Wang et al., 1999; Seeram et al., 2001a, b). Anthocyanins have been reported to affect many of the parameters discussed above by inhibiting lipid peroxidation and the activity of cycloxygenase‐1 and ‐2 (COX) enzymes (Seeram et al., 2001b, 2003). The chemistry of the anthocyanins can be reduced to six major anthocyanidins: delphinidin, cyanidin, pelargonidin, petunidin, peonidin, and malvidin. Among berry fruits, blueberries contain high levels of a wide variety of anthocyanins including glycosides of four of the six major anthocyanidins: malvidin, petunidin, peonidin, and cyanidin (Kalt et al., 1999). Therefore, blueberries may have very potent effects on the ‘‘fires of aging.’’ Anthocyanins are a subset of a larger class of polyphenols known as flavonoids. Over 4000 flavonoids have been identified in plants. They are also abundant in seeds, fruits, and plant‐derived oils such as olive oils, as well as tea and red wine. Thus, they are part of the human diet, and plants and spices containing them have been used for many years in Eastern medicine. As might be expected from the above discussion of the anthocyanins, flavonoids have been reported to inhibit lipid peroxidation in several biological systems including mitochondria and microsomes (Bindoli et al., 1977; Cavallini et al., 1978) as well as erythrocytes (Sorata et al., 1984; Maridonneau‐Parini et al., 1986) and liver (Kimura et al., 1984). They appear to be potent inhibitors of both NADPH and CCl4‐induced lipid peroxidation (Afanas’ev et al., 1989). It appears that the iron‐chelating ability of the flavonoids may be very important in mediating their potent inhibitory effects on 5‐LOX (Hoult et al., 1994), while CO inhibition appears to involve other mechanisms. The antioxidant effects of flavonoids may be derived in part from their ability to upregulate antioxidant enzymes (e.g., glutathione) or enzymes related to glutathione synthesis. One mechanism that may be operational in these beneficial effects may be the direct enhancement of transcription factors that enhance antioxidant enzymes or their signaling cascades. It is known, for example, that the enzymes for glutathione (reviewed in Zipper and Mulcahy, 2000; Schroeter et al., 2002) or heme oxygenase (Chen and Maines, 2000) synthesis exhibit extracellular signal‐regulated kinase 1/2 (ERK1/2) dependency in the regulation of their expression, while Cu/ZnSOD is regulated by ELK‐1 (Chang et al., 1999), and MnSOD expression contains binding sites for Sp1, AP‐1, and CREB (Das et al., 1995; Chang et al., 1999), which are ERK1/2 (Sgambato et al., 1998; Chang and Karin, 2001) regulated. Finally, it also appears that flavonoids regulating ERK1/2 may influence iNOS activity. Thus, there is a great deal of evidence to suggest that a possible link exists between the antioxidant activity of flavonoids and their putative MAP‐kinases altering activity. Since MAPKs are involved in numerous biological activities, the findings that flavonoids may influence such signaling suggests that their potential benefits may involve properties other than those involving antioxidant or antiinflammatory effects. As examples, delphinidin inhibits endothelial cell proliferation and cell‐cycle progression by ERK1/2 activation (Martin et al., 2003), while grape seed proanthocyanidin can reduce ischemia/reperfusion‐induced activation of JNK‐1 and c‐Jun and reduce cardiomyocyte apoptosis (Sato et al., 2001). Additional research indicates that phytochemicals can regulate MAP kinase and other signaling pathways at the level of transcription (Frigo et al., 2002). These findings, coupled with a plethora of studies showing the involvement of ERK in diverse forms of memory, such as contextual fear conditioning (English and Sweatt, 1996), long‐term potentiation (English and Sweatt, 1997), striatal‐dependent learning and memory (Mazzucchelli and Brambilla, 2000), hippocampal‐dependent spatial memory (Selcher et al., 1999), and inhibitory avoidance (Schafe et al., 1999), suggest that interventions that influence MAPK signaling may have beneficial effects on cognition. Given the findings reviewed above showing alterations in signaling as a function of age, the putative signaling modifying properties of flavonoids may prove to be invaluable in altering the neuronal and behavioral effects of aging. Thus, we believed that given multiple properties of fruits and vegetables, they might show considerable efficacy in reducing the deleterious effects of aging on neuronal function and behavior. In the mAChR‐transfected COS‐7 cells, research showed that M1‐MAChR transfected cells that had been pretreated with various high antioxidants (boysenberry (BY); cranberry (CB); black currant (BC); strawberry (SB); dried plums (DP); and grape (GR)) and exposed to Ab25–35 or DA. Calcium buffering was examined following oxotremorine‐induced depolarization in M1‐MAChR‐transfected COS‐7 cells, and on cell viability following DA (4 h) exposure. The results suggested that, while there were differential levels of

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protection among the various fruit extracts, all of them decreased to varying degrees, the decrements in calcium buffering induced by Ab or DA (Joseph et al., 2004). Extending these findings, additional research showed that feeding high antioxidants (via the oxygen‐ radical absorbance capacity assay (ORAC)) (Cao et al., 1997; Wang et al., 1996; Prior et al., 1998) could both prevent the onset of age‐related deficits in several indices (e.g., cognitive behavior, Morris water maze performance) (Joseph et al., 1998) or reverse them (Joseph et al., 1999). Interestingly, examinations of various brain regions from the supplemented animals showed only small levels of antioxidant activity, which was insufficient to account for the observed significant beneficial effects of blueberry (BB) supplementation on motor and cognitive function. Findings from this (Joseph et al., 1999) and a subsequent study (Youdim et al., 2000) suggested that there are beneficial properties, in addition to those involving antioxidants, of BBs on both motor and cognitive behavior; these may involve alterations in neuronal signaling and communication. This was observed recently in a study (Joseph et al., 2003) carried out in APP/PS1 transgenic mice, which serve as a model for AD since these mutations promote the production of Ab, and subsequently Alzheimer‐like plaques in several brain regions, which are accompanied in middle age by cognitive deficits. A group of these mice was given BB supplementation beginning at four months of age (as in Joseph et al., 1999) and continued until they were 12 months of age, when their performance was tested in a Y‐maze. The results indicated that mice supplemented with BB exhibited Y‐maze performance that was similar to those seen in nontransgenic mice and significantly greater than that seen in the nonsupplemented transgenic animals. Interestingly, there was a dichotomy between the plaque burden and behavior in the BB‐supplemented transgenic mice. No differences between the supplemented and nonsupplemented APP/ PS1 mice in the number of plaques were observed, even though behavioral declines were prevented in the BB‐supplemented animals. One possible reason that the behavior did not reflect the morphology may be that there was enhanced signaling present in the BB‐supplemented transgenic mice, which acted to prevent or circumvent any putative deleterious effects of the amyloid plaques on behavior. The evidence for this possibility is provided by data showing that the BB‐supplemented APP/PS1 mice exhibited greater levels of hippocampal ERK as well as striatal and hippocampal protein kinase Ca (PKC) than that seen in the transgenic mice maintained on the control diet. As pointed out above, ERK and PKC have been shown to be important in mediating cognitive function, especially conversion of short‐term to long‐term memory (Micheau and Riedel, 1999). Enhancement was also seen in the BB‐supplemented group in the sensitivity of muscarinic receptors (i.e., increasing striatal, carbachol‐stimulated GTPase activity), which has been found to be associated with learning and memory in numerous studies. These findings, combined with additional preliminary research showing that BB supplementation in addition to altering ERK activity may also increase hippocampal neurogenesis (Casadesus et al., 2004), suggests that at least part of the efficacy of the BB supplementation may be to enhance neuronal function in areas of the brain affected by aging or disease. This would allow more effective intra‐ and interarea communication and ultimately facilitate both cognitive and motor function.

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Green DR, Kroemer G. 2004. The pathophysiology of mitochondrial cell death. Science 305(5684): 626-629. Hartmann H, Velbinger K, Eckert A, Muller WE. 1996. Region‐specific downregulation of free intracellular calcium in the aged rat brain. Neurobiol Aging 17: 557-563. Hauss‐Wegrzyniak B, Vannucchi MG, Wenk GL. 2000. Behavioral and ultrastructural changes induced by chronic neuroinflammation in young rats. Brain Res 859: 157-166. Hauss‐Wegrzyniak B, Vraniak P, Wenk GL. 1999. The effects of a novel NSAID on chronic neuroinflammation are age dependent. Neurobiol Aging 20: 305-313. Heissmeyer V, Krappmann D, Wulczyn FG, Scheidereit C. 1999. NF‐kB p105 is a target of IkB kinases and controls signal induction of Bcl‐3‐p50 complexes. EMBO J 18: 47664778. Helenius M, Hanninen M, Lehtinen SK, Salminen A. 1996. Changes associated with aging and replicative senescence in the regulation of transcription factor nuclear factor‐kB. Biochem J 318: 603-608. Hersch SM, Gutekunst CA, Rees HD, Heilman CJ, Levey AI. 1994. Distribution of M1‐M4 muscarinic receptor proteins in the rat striatum: Light and electron microscopic immunocytochemistry using subtype‐specific antibodies. J Neurosci 14: 3351-3363. Hoult JR, Moroney MA, Paya M. 1994. Actions of flavonoids and coumarins on lipoxygenase and cyclooxygenase. Methods Enzymol 234: 443-454. Ingram DK, Jucker M, Spangler E. 1994. Behavioral manifestations of aging. Pathobiology of the Aging Rat. Vol. 2. Mohr U, Cungworth DL, Capen CC, editors. Washington: ILSI Press; pp. 149-170. Ishii K, Oda Y, Ichikawa T, Deguchi T. 1990. Complementary DNAs for choline acetyltransferase from spinal cords of rat and mouse: Nucleotide sequences, expression in mammalian cells, and in situ hybridization. Mol Brain Res 7: 151-159. Joseph JA. 1992. The putative role of free radicals in the loss of neuronal functioning in senescence. Integr Physiol Behav Sci 27: 216-227. Joseph JA, Fisher DR. 2003. Muscarinic receptor subtype determines vulnerability to amyloid b toxicity in transfected COS‐7 cells. J Alzheimers Dis 5: 197-208. Joseph JA, Roth GS. 1992. Loss of muscarinic regulation of striatal dopamine function in senescence. Neurochem Int 20 Suppl: 237S-240S. Joseph JA, Roth GS. 1994. Oxidative stress and reduced receptor responsiveness senescence. Trophic Regulation of the Basal Ganglia: Focus on Dopamine Neurons. Fuxe K, Agnati LF, Leon A, Ottoson D, editors. Pergamon Press Oxford. Joseph JA, Arendash G, Gordon M, Diamond D, Shukitt‐Hale B, et al. 2003. Blueberry supplementation enhances

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