Apoptosis in motor neuron degenerative diseases

APOPTOSIS IN M O T O R N E U R O N D E G E N E R A T I V E DISEASES

W A R D A. PEDERSEN, INNA K R U M A N and M A R K P. M A T r S O N

Table of Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pro-/Anti-Apoptotic Proteins in the Brain and Spinal Cord . . . . . . . . . . . . . . . . . . . . . . . . . . Anti-Apoptotic Proteins in Neuroprotective Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evidence for Apoptosis in Motor Neuron Degenerative Diseases . . . . . . . . . . . . . . . . . . . . . . Studies in Human Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cell Culture Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ALS Overview: Integration of Proposed Cell Death Cascades . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction One unresolved issue in elucidating the pathogenesis of neurodegenerative diseases is whether cells die by apoptosis, necrosis, or both. Necrosis, a relatively "fast" type of cell death compared to apoptosis, has been a prevalent area of research concerning the mechanisms of neuronal degeneration in conditions such as ischemia and stroke. The progression of neurodegenerative diseases usually occurs in a time frame of years to decades, and one could therefore argue that apoptosis plays a particularly important role in the pathogenesis of these disorders. In Alzheimer's disease (AD), for example, the onset of clinical symptoms can precede death by more than 20 years. It is estimated that a 50% neuronal loss over this 20-year period corresponds to an overall apoptotic rate of 1 in 10,000 cells per day (Perry et al., 1998). Such an estimate is based on the assumption that apoptotic death follows an exponential pattern. This raises one of many intriguing questions concerning the study of apoptosis in neurodegenerative diseases. Does apoptosis proceed in a more or less linear fashion, or is there an exponential rise in apoptotic activity at a certain stage in the disease? In the most common motor neuron degenerative disease, Amyotrophic Lateral Sclerosis (ALS), the time from onset of symptoms to death of the patient in inherited forms alone can vary from one to twenty years (Abe et al., 1996; Cudkowicz et al., 1997; Juneja et al., 1997; Radunovic and Leigh, 1996). Epidemiological studies have not been able to identify significant environmental risk factors for ALS (Chancellor and Warlow, 1992), which suggests that genetic factors have a particularly important influence on the neurodegenerative process and hence the clinical phenotype. For instance, as in AD, the duration of ALS may be related 225 Role in Disease, Pathogenesis and Prevention Ed. by M.P. Mattson, S. Estus and V.M. Rangnekar. © 2 0 0 1 Elsevier Science. Printed in the Netherlands.

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to apolipoprotein E genotype (Moulard et al., 1996). Does apolipoprotein E genotype impact upon the apoptotic process in ALS? Clearly, identifying the factors that influence the expression of a particular neurodegenerative disease and how they relate to one another is a complex issue. Even more complex and uncertain at this point is the relationship between factors which influence the clinical phenotype and the induction and rate of apoptosis during the course of neurodegenerative diseases. In studying motor neuron degenerative diseases, particularly ALS, several parallels can be drawn from investigations of the pathogenesis of AD. A number of studies carried out in cell culture and with animal models of AD have correlated pathological determinants with apoptosis. The ~4 kDa amyloid-fl peptide (At), derived from the 110-130 kDa transmembrane glycoprotein known as the amyloid precursor protein (APP), deposits in the brains of AD patients in the form of the neuritic plaque (see Selkoe, 1994 for review). The neuritic plaque is an invariant feature of AD brain, and AI~ has been implicated as having a causative role in the neurodegenerative process in this disease. For instance, AI$ has been shown to induce apoptosis in neurons cultured from brain regions vulnerable in AD, i.e. the hippocampus (cf. Kruman et al., 1997). Evidence suggests that the mechanism by which AI3 exerts its neurotoxic effect involves increased production of reactive oxygen species, membrane lipid peroxidation, and disruption of calcium ion homeostasis (see Mattson et al., 1996; Mattson, 1998 for review). The mechanism of motor neuron degeneration in ALS is also proposed to involve increased production of reactive oxygen species, membrane lipid peroxidation, and disruption of calcium ion homeostasis based on data from cell culture experiments, transgenic mouse models, and from studies with patient material (Gurney et al., 1994, 1996; Hall et al., 1998; Liu et al., 1998; Pedersen et al., 1998; Siklos et al., 1996; Smith et al., 1996, 1998; Wiedau-Pazos et al., 1996; Yim et al., 1996). What remains as unclear in AD research as it does in ALS research is why only certain neuronal populations are vulnerable to degeneration. Among the populations vulnerable in AD are basal forebrain cholinergic neurons (Bartus et al., 1982; Harkany et al., 1995a,b; Whitehouse et al., 1982), whose cell bodies reside within the medial septum, the diagonal band of Broca and the nucleus basalis magnocellularis, and which project to the neocortex and hippocampus (Dutar et al., 1995; Mesulam et al., 1983; Woolf, 1991). In contrast, ~,-aminobutyric acid-synthesizing neurons of the medial septum are relatively resistant to the toxic effects of AI3 (Harkany et al., 1995a; Hof et al., 1991). This appears to be due, at least in part, to expression of the calcium-buffering protein parvalbumin in the latter population of neurons and the lack thereof in medial septal cholinergic neurons (Freund, 1989). Similarly, it has been shown that ALS-resistant motor neurons contain calbindin-D28 K and/or parvalbumin whereas ALS-vulnerable motor neurons do not (Alexianu et al., 1994; Elliott and Snider, 1995). Elevations in intracellular calcium concentrations can lead to apoptosis, e.g. by up-regulating the expression of pro-apoptotic genes, and the lack of calcium-buffering proteins in spinal cord motor neurons may render the cells vulnerable to apoptotic insults. It is fascinating to consider that there is the potential for extensive overlap of the components of the cell death mechanism in two neurodegenerative diseases that differ as greatly as AD and ALS. If current findings are any indication, further elucidation of the pathogenesis of either of these disorders may lead to new insights for the other.

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One distinct advantage of AD researchers not enjoyed by those of us attempting to understand the pathogenesis of motor neuron degenerative diseases is the existence of a common causative agent. Unlike Af5 for AD, there is not a single pathological determinant which unifies all variants of motor neuron degenerative diseases. In ALS, mutations in copper/zinc-superoxide dismutase (Cu/Zn-SOD) occur in approximately one-fifth of inherited forms (Cudkowicz et al., 1997; Deng et al., 1993; Rosen et al., 1993), which account for about 10% of all cases. Studies with purified Cu/Zn-SOD revealed that familial ALS mutations confer upon this anti-oxidant enzyme increased ability to generate reactive oxygen species (Wiedau-Pazos et al., 1996; Yim et al., 1996). Increased levels of markers of oxidative stress have been observed in the spinal cords of both familial and sporadic ALS patients relative to control spinal cords (cf. Ferrante et al., 1997a), yet the underlying cause of this stress in most ALS cases is unknown. A major challenge in ALS research, then, is to determine the unifying pathogenetic features leading to the convergence of all cases upon the same clinical phenotype. Are mechanisms consistent with apoptosis common features of motor neuron degenerative diseases, despite different etiologies? If so, to what extent does apoptosis occur relative to other forms of cell death in vulnerable motor neuron populations? In this chapter, we review evidence from studies in cell culture, with transgenic mouse models, and with patient material that apoptosis plays a role in motor neuron degenerative diseases. We will discuss the relationship between proposed mechanisms of the pathogenesis of motor neuron degenerative diseases, with emphasis on ALS, and apoptosis. We will also briefly compare proposed apoptotic mechanisms in AD and ALS.

Pro-/Anti-Apoptotic Proteins in the Brain and Spinal Cord Apoptosis is a form of cell death that has classically been defined by distinct morphological criteria. It is an active process that requires an up-regulation in the expression of certain genes and/or the function of their protein products to promote these morphological changes, and perhaps a down-regulation in the expression of certain genes and/or the function of their protein products which promote survival. Thus, apoptosis can more precisely be characterized by the involvement of a set of pro-death proteins leading to specific changes in cellular and nuclear morphologies. The term "programmed cell death" is used to describe an apoptotic form of cell loss that occurs under normal or physiological conditions, i.e. during development and in adult tissues where there is continual turnover of cells throughout the life of the organism. Mature, fully differentiated neurons have lost the ability to enter the cell cycle and loss of cells is not accompanied by regeneration. If neurons do in fact die by apoptosis in the adult brain and spinal cord, then it would seem more appropriate to refer to this as "dys-programmed cell death" in order to distinguish between physiological and pathological neuronal apoptosis. Nonetheless, apoptosis is a useful generic term and the morphological criteria used to characterize this form of cell death in neuronal systems are the same as those used for non-neuronal tissues, i.e. cell shrinkage, plasma membrane blebbing, chromatin condensation, DNA and nuclear fragmentation, and ultimately formation of small membrane-bound bodies

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(Lo et al., 1995; Wyllie, 1987). However, although most genes encoding either pro- or anti-apoptotic proteins are widely expressed, there also appears to be apoptotic genes that are differentially expressed among neuronal and non-neuronal tissues. It remains to be determined if there are apoptotic genes that are exclusively expressed in neurons. As a preface to the discussion of the potential role of apoptotic proteins in the pathogenesis of motor neuron degenerative diseases, we will briefly review what is known about the expression of apoptotic genes in the brain and spinal cord. Programmed cell death is a mechanism for the determination of cell number and structural organization in all tissues during vertebrate development, but this process is exemplified by the mammalian nervous system where establishing its intricate circuitry requires the tightly regulated death of as much as 50% of some types of neurons to ensure precise and appropriate target innervation (see Oppenheim, 1991; Raft et al., 1993; Burek and Oppenheim, 1999 for review). Ironically, the study of apoptosis in the mammalian nervous system has been catapulted by discoveries borne out of research into understanding the pathogenesis of cancer. For instance, the most intensely studied anti-apoptotic protein in the nervous system under both physiological and pathological conditions is that encoded by the oncogene bcl-2, which was originally discovered by cloning the t(14; 18) (q32;q21 ) chromosomal breakpoint characteristic of non-Hodgkin's follicular lymphoma (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto et al., 1984). The initial evidence for a role for Bcl-2 in programmed cell death came from studies where over-expression of the gene in certain hematopoietic cell lines was found to promote cell survival following growth factor withdrawal (Hockenbery et al., 1990; Nunez et al., 1990; Vaux et al., 1988). There are two particularly interesting features to note about the Bcl-2 protein. First, Bcl-2 promotes cell survival without affecting cell proliferation (see Korsmeyer, 1992 for review). Thus, the protein appears to function without the cell cycle machinery, supporting an anti-death role in proliferating and non-proliferating cells. Second, by analysis of subcellular fractions of the hematopoietic RL-7 cell line, the 25-kDa Bcl-2 protein was found to be predominantly localized to the inner mitochondrial membrane (Hockenbery et al., 1990). Subsequent studies revealed that Bcl-2 is also localized to the nuclear envelope, the endoplasmic reticulum, and the outer mitochondrial membrane (Krajewski et al., 1993). A C-terminal region of 19 amino acids in the Bcl-2 protein serves as an integral membrane anchor, at least for mitochondrial membranes (Nguyen et al., 1993). The functional significance of the subcellular localization of Bcl-2, however, remains unclear. There is increasing evidence that mitochondrial dysfunction is an early event and has a causative role in the cell death process in neurodegenerative diseases such as ALS (cf. Carri et al., 1997). Thus, understanding how Bcl-2 impacts upon mitochondrial function may be critical for elucidating the pathogenesis of neurodegenerative diseases. The topographical distribution of Bcl-2 in adult tissues is restricted to cells that are associated with an apoptotic form of cell death (Hockenbery et al., 1991). In particular, Bcl-2 is present in the germinal center of the lymphoid follicle where plasma and memory B cells are generated, in all hematopoietic lineages derived from a renewing stem cell, in the epithelium of intestine and skin characterized by long-lived stem cell populations, in the epithelium of breast, thyroid, prostate, and pancreatic glands, and in neurons (Hockenbery et al., 1991). The differential expression of bcl-2 among

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neuronal populations is viewed as a mechanism for eliminating unwanted cells in remodeling the nervous system during development, and maintaining Bcl-2 in adult neurons implies a standing protective role for these exceptionally sensitive cells. It has been more than 10 years since the first report of bcl-2 mRNA in the newborn and adult nervous systems (Negrini et al., 1987), and several detailed analyses have demonstrated that Bcl-2 is widely present in the adult brain and spinal cord. The levels of Bcl-2 are substantially higher in the developing versus adult central nervous system, but several sub-structures of the adult brain and spinal cord retain a low level of expression of the gene (Abe-Dohmae et al., 1993; Castren et al., 1994; Ferrer et al., 1994; Merry et al., 1994; Yachnis et al., 1998). In the adult mammalian brain, bcl-2 expression is detected in the olfactory bulb, cerebral cortex, striatum, hippocampus, basal forebrain, midbrain, thalamus, hypothalamus, pons and medulla, and cerebellum, but not in white matter (Castren et al., 1994; Merry et al., 1994). Specifically, the highest levels of bcl-2 mRNA in the adult rat brain have been reported in the mitral cells of the olfactory bulb, the piriform, temporal and cingulate cortices, the subfornical organ, the ependyma, the supraoptic nucleus of the hypothalamus, the anteroventral nucleus of the thalamus, the dentate gyrus granule cell layer, CA1-CA3 regions, and subiculum of the hippocampus, the pontine nuclei, the principal sensory trigeminal nucleus, and the granule cell layer of the cerebellum (Castren et al., 1994). Notably, however, most of the Bcl-2 in adult brain appears to be present in microglial cells (Merry et al., 1994), which proliferate in response to neuronal injury. Between embryonic days 13 and 18 of the developing mouse spinal cord, alpha motor neurons in the lateral motor column undergo cell death and are observed to have higher bcl-2 expression than surrounding cells in the lumbar and cervical spinal cords (Merry et al., 1994). Only large ventral horn neurons which have regenerative capacity appear to retain a relatively high level of bcl-2 expression in the adult spinal cord. In the human spinal cord, Bcl-2 immunostaining was observed in cells lining the central canal, neurons of the dorsal horn (particularly laminae I and II), and in anterior horn cells between 10 and 14 weeks gestation, but could only be detected in ependymal cells by 30 weeks gestation (Yachnis et al., 1998). A number of proteins homologous to Bcl-2 have been identified, and a family of bcl-2-related genes encoding pro- and anti-apoptotic proteins has emerged (see Merry and Korsemeyer, 1997 for review). The members of the Bcl-2 family share two highly conserved regions designated BH1 and BH2 (for Bcl-2 homology 1 and 2 domains). The BH1- and BH2-containing proteins include Bcl-xL, Bax, Bad, Bak, and Mcl-1 in humans, A1 in the mouse, Ced-9 in C. elegans, and LMW5-HL and BHRF1 in viruses. A third domain, BH3, with lesser homology among family members than BH1 and BH2, is shared between Bcl-2, Bcl-xL, Bax, Bak, Mcl-1, and Bik, the latter of which does not contain BH1 or BH2 domains. Of the human proteins, Bcl-2, Bcl-xL, and Mcl-1 protect against cell death, whereas Bax, Bad, Bak, and Bik promote cell death. A complex set of interactions between the Bcl-2 family members has been realized from yeast two-hybrid studies. Bax interacts with Bcl-2, Bcl-xL, and Mcl-1 (Sedlak et al., 1995). Along with homodimer formation, Bcl-2 and Bcl-xL also interact with Bad, Bak, Bik, and Bcl-xS (a splicing product of bcl-x mRNA lacking the BH1 and BH2 domains) (see Merry and Korsemeyer, 1997 for review). The BH1 and BH2

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domains have been shown to be necessary for heterodimerization and anti-apoptotic function, but not for homodimerization. For instance, substitution of Gly 145 in BH1 or Trp 188 in BH2 of Bcl-2 eliminates its interaction with Bax and its ability to block apoptosis caused by interleukin-3 withdrawal, T-irradiation, or glucocorticoid treatment in hematopoietic cell lines, yet homodimerization of the mutant Bcl-2 protein is not affected (Yin et al., 1994). The BH3 domain appears to be required for the interactions of Bax, Bak, and Bik with Bcl-2 and Bcl-xL (Boyd et al., 1995; Chittenden et al., 1995). Why Bad lacks the BH3 domain and how this this impacts on its pro-apoptotic action relative to other members remains unclear. Further complicating the understanding of apoptotic pathways is the fact that there are also interactions of the Bcl-2-related proteins with non-family members, such as BAG-1 (Takayama et al., 1995). The protein with the highest homology to Bcl-2 is Bcl-x, and Bcl-xL can inhibit cell death as well as Bcl-2 (Boise et al., 1993). However, there are great differences in the levels and distribution patterns of the two proteins in the nervous system. In contrast to Bcl-2, which is at its highest level during development of the nervous system and is down-regulated considerably after birth, Bcl-x increases after birth and peaks in the adult brain and spinal cord (Frankowski et al., 1995; Gonzalez-Garcia et al., 1994; Yachnis et al., 1998). The major form of Bcl-x in mammalian brain and spinal cord is Bcl-xL (Gonzalez-Garcia et al., 1994). There is widespread expression of the bcl-x gene in the adult mammalian brain, with the highest levels of its RNA and protein in the olfactory bulb, cerebral cortex, paraventricular and supraoptic nuclei of the hypothalamus, the CA1-CA3 regions and dentate gyrus of the hippocampus, pontine and facial nuclei, Purkinje cells of the cerebellum, and in the ependyma (Frankowski et al., 1995; Gonzalez-Garcia et al., 1994). Like Bcl-2, Bcl-x is not found in white matter; unlike Bcl-2, however, it appears to be restricted to neurons (Frankowski et al., 1995; Gonzalez-Garcia et al., 1994; Krajewski et al., 1994b). Bcl-2 and Bcl-x both display a punctate staining pattern in the cytosol of neurons consistent with their localization to intracellular organelles, and neither protein appears to be present in axons (Hockenbery et al., 1991; Krajewski et al., 1994b). In the adult human spinal cord, strong Bcl-x immunostaining is observed in neurons of the intermediolateral cell column, Clarke's column, and in anterior horn cells (Yachnis et al., 1998). The only other anti-apoptotic Bcl-2 family member identified in humans to date, Mcl-1, is either not present or is present at low levels in neurons of the adult brain and spinal cord (Krajewski et al., 1995). Several tissues in the adult human show an inverse relationship between the levels of Bcl-2 and Mcl-1, indicating that the two proteins have differential roles in programmed cell death. Neuroendocrine ceils, for example, including adrenal cortical cells, do not contain Bcl-2 but have high levels of Mcl-1 (Krajewski et al., 1995). In contrast, thyroid epithelial cells are strongly immunoreactive for Bcl-2 but are negative for Mcl-1. The same inverse expression pattern is also observed between Mcl-1 and both Bcl-2 and Bcl-x in many neurons of the central nervous system. Neurons of the adult peripheral nervous system, however, which have regenerative capabilites, contain high levels of Mcl-1, Bcl-2, and Bcl-x (Krajewski et al., 1995; Merry et al., 1994). Curiously, most of the pro-apoptotic members of the Bcl-2 family are found in the adult mammalian central and peripheral nervous systems (Boyd et al., 1995; Kiefer et al., 1995; Kitada et al., 1998; Krawjewski et al., 1994a;

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Krawjewski et al., 1996; Oltvai et al., 1993). Bax has been shown to be present at high levels in Purkinje cells of the cerebellum, in large neurons of the cortex and brainstem, and in neurons of sympathetic ganglia of the mouse (Krajewski et al., 1994a). In the mouse spinal cord, weak or absent Bax immunostaining was observed in healthy ventral hom motor neurons, whereas intense staining for Bax was typically found only in ventral horn motor neurons that showed signs of degeneration (Krajewski et al., 1994a). The axonal fibers in the gray matter of mouse spinal cord showed weak to moderate levels of Bax immunostaining, yet Bcl-2 levels were generally higher in the fibers. In the adult human brain, Bad was reported to be detected only in neurons of the gray matter of cortex and basal ganglia, in activated but not resting microglia, and in the ependyma, leptomeninges, and choroid plexus (Kitada et al., 1998). The axons and neuropil of the cortex and basal ganglia displayed absent to weak Bad immunostaining, and only astrocytes in the cerebellum appear to contain the protein. In the adult human spinal cord, only absent to weak immunostaining for Bad was observed (Kitada et al., 1998). Specifically, Bad was found in axons of white matter and in ventral horn motor neurons, dorsal horn sensory neurons, and the neuropil of gray matter. A very different situation arises for Bak, which is virtually absent in neurons and microglia of the brain and spinal cord (Krajewski et al., 1996). It has been detected, however, in axons and degenerating neurons of the brain and spinal cord, and in the ependyma, leptomeninges, and choroid plexus. Overall, an apparently consistent finding in the studies of the distribution of Bcl-2 family members in normal mammalian tissues is that the pro-apoptotic proteins are up-regulated in neurons undergoing degeneration. Thus, understanding the relative role of these proteins in the neuronal death process may be critical to understanding the pathogenesis of neurodegenerative diseases. Several pro-/anti-apoptotic proteins not belonging to the Bcl-2 family have now been detected in the adult brain and spinal cord. The proteolytic breakdown of a number of proteins is a general apoptotic event. This is largely due to a family of at least 11 cysteine proteases which cleave proteins at aspartate residues, and are accordingly named "caspases" (Alnemri et al., 1996). One of the substrates for the caspase-3 family member is Bcl-2, which results in a C-terminal cleavage product with pro-apoptotic activity that is dependent on the BH3 and transmembrane domains (Cheng et al., 1997). The interleukin-lB converting enzyme was the first family member to be identified in mammals (Yuan et al., 1993). There is little or no detectable caspase expression in adult mammalian brain, but increased expression of interleukin-ll3 converting enzyme and other caspases has been reported following ischemia (see Gorman et al., 1998 for review). An anti-apoptotic protein that has been studied most extensively in motor neuron degenerative diseases is the neuronal apoptosis inhibitory protein (NAIP; Roy et al., 1995). The distribution of NAIP is widespread in the adult rat brain (Xu et al., 1997). The highest levels of NAIP in adult rat spinal cord were observed primarily in cells of the ventral hom (laminae VIII and IX) and of the intermediate zone (lamina VII); in ventral hom regions, NAIP immunoreactivity was found to be greatest in what appeared to be alpha motor neurons (Xu et al., 1997). Our laboratory has recently focussed on the role of a protein known as prostate apoptosis response 4 (Par-4) in neurodegenerative diseases. As the name implies, Par-4 was identified by differential screening for genes that are up-regulated after an apoptotic insult in

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a rat prostatic cancer cell line (see Rangnekar, 1998 for review). The par-4 gene is ubiquitously expressed, and particularly high levels of the protein are found in brain (Boghaert et al., 1997). Intense Par-4 immunostaining has been observed, for example, in neurons of the rat cortex. We will discuss the evidence supporting a role for Par-4 in the cell death process of neurodegenerative diseases in a subsequent section.

Anti-Apoptotic Proteins in Neuroprotective Roles The identification of apoptotic genes has progressed so rapidly that researchers are now faced with a collection of pro-/anti-apoptotic proteins for which the specific functions are poorly understood. A challenging task also lies in determining at what level(s) of the apoptotic cascade do the proteins act to result in the morphological changes characteristic of this form of cell death. There is now a wealth of evidence supporting a protective role for anti-apoptotic proteins during development of the nervous system and in experimentally-induced neuronal death, and we will discuss only select examples here. There is an important consideration when interpreting the results of studies involving an examination of the effects and/or levels of apoptotic proteins in neuronal systems. That is, at least some proteins with pro-/anti-apoptotic activity can also regulate other cell death pathways. For instance, Kane et al. (1995) showed that overexpression of bcl-2 in GT1-7 neural cells inhibited necrotic death caused by glutathione depletion. Their results demonstrate that Bcl-2 can modulate both apoptotic and necrotic processes, but suggest it does not directly inhibit the cell death program. There are neurodegenerative conditions where apoptosis, necrosis, or both, can be envisioned. However, it is possible that there are other forms of cell death that contribute to the pathogenesis of neurodegenerative diseases, and these may also be modulated by proteins involved in apoptotic pathways. Thus, a broad neuroprotective role may apply to proteins with anti-apoptotic activity, and their effects can be described as anti-apoptotic in only defined experimental situations. A neuroprotective role for Bcl-2 family members has been demonstrated in a variety of experimental models, and there is evidence that they are more effective in certain neuronal populations, such as motor neurons. Sensory neurons from dorsal root ganglia and sympathetic neurons from superior cervical ganglia of newborn rodents are dependent on nerve growth factor for survival in culture. However, in the absence of nerve growth factor, it has been shown that microinjection of a cDNA construct containing human bcl-2 into cultures of sympathetic neurons from newborn rats significantly increases survival relative to control cultures (Garcia et al., 1992). Farlie et al. (1995) generated transgenic mice that express the human bcl-2 gene under the control of the neuron-specific enolase promoter, which limited expression to neurons. In nerve growth factor-deprived cultures of sensory neurons from dorsal root ganglia of newborn transgenic mice, they similarly observed a significant increase in survival relative to control cultures. These transgenic mice showed a 30% increase in the number of cells in certain neuronal populations of the central and peripheral nervous systems, supporting a neuroprotective role for Bcl-2 during development. Furthermore, neuron-specific expression of human bcl-2 in transgenic mice was reported

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to completely prevent the death of spinal cord motor neurons following transection of the sciatic nerve and facial motor neurons after axotomy of the facial nerve in newborns (Dubois-Dauphin et al., 1994; Farlie et al., 1995). The surviving facial motor neurons of axotomized transgenic mice retained electrophysiological responses to AMPA, NMDA, and vasopression (Alberi et al., 1996), indicating that bcl-2 expression not only keeps the cells alive but preserves their function as well. These results suggest that Bcl-2 may have a particularly important protective role in motor neurons. Interestingly, mice with targeted disruption of the bcl-2 gene appear grossly normal at birth and do not have neuronal developmental abnormalities (Veis et al., 1993). Within one week after birth, however, bcl-2 -/- mice were observed to be smaller than their littermates and to have immature facial features. The mice develop polycystic kidney disease, have an abnormally high rate of apoptosis in the thymus and spleen, and most die between 10 days to 10 weeks of age (Veis et al., 1993). Subsequently, it was reported that bcl-2 -/- mice have a normal number of sensory, sympathetic, and facial motor neurons at birth, and, furthermore, that axotomy-induced degeneration of facial motor neurons is prevented by either brain-derived neurotrophic factor or ciliary neurotrophic factor (Michaelidis et al., 1996). Yet, in early postnatal development, there is a progressive loss of sensory, sympathetic, and facial motor neurons in these mice. Upon deprivation of nerve growth factor, sympathetic neurons from embryonic bcl-2 -/- mice die more rapidly in culture than those from wild-type littermates (Greenlund et al., 1995b), and it is likely that other neurotrophic factors can substitute for nerve growth factor in preventing the death of these neurons during development in bcl-2 -/- mice. Thus, it appears that Bcl-2 does not mediate growth factor neuroprotection during the physiologic cell death period, but that it is required for the survival of certain populations in the postnatal period. These results also suggest that a decrease in Bcl-2 levels during apoptosis may have particularly important consequences for the survival of certain neuronal populations, such as motor neurons, in disease states. In contrast to the effects of bcl-2 knock-out, targeted disruption of the bcl-x gene is embryonic lethal in mice (Motoyama et al., 1995). As evidenced by the presence of fragmented DNA, extensive apoptotic cell death was observed in regions of differentiating neurons in the developing brain, spinal cord, and dorsal root ganglia of bcl-x -/- mice. Although this knock-out model cannot tell us anything about the neuroprotective role of Bcl-x postnatally, we can infer from this study that Bcl-x is a more potent regulator of neuronal survival than Bcl-2. In support of this, over-expression of bcl-x in cultures of rat embryonic sympathetic neurons afforded a greater degree of protection than was observed for over-expression of bcl-2 in the mouse models (Frankowski et al., 1995). Limiting amounts of target-derived trophic factors has long been viewed as a mechanism by which cell number is regulated in the majority, if not all, neuronal populations during development (see Henderson, 1996; Johnson and Deckwerth, 1993; Oppenheim, 1991 for review). In the developing chick embryo, treatment with inhibitors of RNA or protein synthesis in the early absence of limbs markedly reduced the loss of spinal cord motor and sympathetic neurons (Oppenheim et al., 1990), suggesting that in these neuronal cells, target-derived neurotrophic factors play a particularly important role in suppressing an active gene expression program required for death

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(Oppenheim et al., 1990). In culture, pure populations of motor neurons from embryonic chick spinal cord die within 48 h if muscle extract is not present (Milligan et al., 1994). Some of the cells were found to die by apoptosis as evidenced by DNA fragmentation and nuclear condensation, which was blocked by inhibiting RNA synthesis with actinomycin D at the time when the cells became committed to die. Interestingly, in the presence of the protein synthesis inhibitor cycloheximide and in the absence of trophic support, Comella et al. (1994) found that muscle extracts could still prevent apoptosis of chick spinal cord motor neurons when added back at a time when transcription of cell death genes would have been initiated. This suggests that there is a window of opportunity for rescuing motor neurons after activation of the apoptotic program. These studies did not, however, identify the trophic factors responsible for the motor neuron survival effects. In cultures of rat motor neurons, brain-derived neurotrophic factor or neurotrophin-4 alone could not substitute for muscle extracts in reducing the numbers of apoptotic cells (Kaal et al., 1997). Because numerous growth factors have been shown to promote the survival of motor neurons in vitro and in vivo (see Elliott and Snider, 1996; Oppenheim, 1996 for review), any one type of motor neuron is likely to depend on a combination of these growth factors to survive. The neuroprotective function of Bcl-2 may be selective for certain types of neurons depending on their growth factor dependence (Allsopp et al., 1993). Trophic factor withdrawal is only one of many paradigms where Bcl-2 has been shown to prevent neuronal death. In cultures of rat pheochromocytoma PC12 cells, which do not express bcl-2, transfection with a construct containing human bcl-2 cDNA increased cell survival following serum or nerve growth factor withdrawal and in the presence of calcium ionophore relative to untransfected cells (Mah et al., 1993). Cell death was characterized as apoptotic by internucleosomal fragmentation of DNA. A more broad study revealed that over-expression of bcl-2 in conditionally immortalized neural cells reduced apoptotic death caused by serum or growth factor withdrawal, glucose deprivation, an inducer of free radical formation, menadione, the membrane peroxidizing agent t-BOOH, and calcium ionophore (Zhong et al., 1993b). The toxicity caused by glutamate application can also be blocked by over-expression of bcl-2 in neuronal cell lines (Behl et al., 1993; Zhong et al., 1993a). Although A8 has been shown to induce apoptosis in primary cultures of mouse cortical neurons (Loo et al., 1993), no protective effect of Bcl-2 against AB toxicity was reported in neuronal cell lines (Behl et al., 1993). This discrepancy may reflect differences in cell death pathways activated by AB in primary cultures versus cell lines, i.e. differences in neuronal cell type activation of death pathways by AI3. The protective effects of Bcl-2 in cell lines have also been observed in primary neuronal cultures. In hippocampal neurons cultured from rat, over-expression of bcl-2 using herpes simplex virus vectors increased survival relative to untransfected cultures in the presence of glutamate and adriamycin, a potent inducer of oxyradical formation, and in hypoglycemic conditions (Lawrence et al., 1996). Furthermore, delivery of Bcl-2 into rat brain via herpes simplex virus vectors was found to protect against damage to striatum following focal ischemia (Lawrence et al., 1996). Transgenic mice which express human bcl-2 under the control of the neuron-specific enolase promoter were shown to be more resistant to ischemic damage caused by permanent occlusion of the middle cerebral artery

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(Martinou et al., 1994). Indeed, increased expression of bcl-2 and bcl-xL has been observed in cells of rat hippocampus after global ischemia (Chen et al., 1997). The levels of bcl-2 and bcl-xL mRNA were increased in surviving and dying hippocampal neurons, whereas the proteins were detected only in surviving cells. Following intraperitoneal injections of kainic acid in mice, there is a down-regulation in bcl-2 expression and an up-regulation in bax expression in hippocampal and cortical cells coincident with DNA fragmentation (Gillardon et al., 1995). Of particular interest to neuroscientists investigating the pathogenesis of motor neuron degenerative diseases is the evidence that Bcl-2 alters oxyradical production and lipid peroxidation. Oxidative stress is at least a common correlate of different forms of ALS if not a common cause of motor neuron degeneration in this disorder (the evidence for a protective role for Bcl-2 in motor neuron degeneration diseases will be discussed in the subsequent section). Although Bcl-2 has been localized to the inner mitochondrial membrane (Hockenbery et al., 1990), over-expression of bcl-2 in hematopoietic cell lines did not alter the cyanide-resistant oxidative burst caused by treatment with menadione (Hockenbery et al., 1993), supporting the results of other studies showing that Bcl-2 does not affect respiratory chain activity and that its anti-apoptotic function is not dependent on respiration (Jacobson et al., 1993). Instead, Bcl-2 was found to completely suppress lipid peroxidation caused by treatment with H202 or menadione. By thiobarbituric acid reactivity and using electron paramagnetic resonance analysis of signals generated by nitroxyl stearate spin labels, attenuated lipid peroxidation in isolated AB- or H202-treated plasma and mitochondrial membranes from PC12 cells over-expressing bcl-2 was demonstrated (Bruce-Keller et al., 1998). This may be due to suppressive effects of Bcl-2 on the production of reactive oxygen species and on accumulation of peroxides (Kane et al., 1993), thereby blocking free radical attack on membrane lipids. In support of this hypothesis, a higher content of oxidized proteins has been detected in the brains of Bcl-2 -/- mice versus controls (Hochman et al., 1998). Apoptosis can be induced in PC12 cells cultured in an atmosphere of 50% oxygen, and this can be blocked by either treatment with vitamin E or over-expression of bcl-2 (Kubo et al., 1996). In contrast, Bcl-2 and Bcl-xL were found to inhibit apoptosis in cells cultured in near anaerobic conditions (Jacobson and Raft, 1995; Shimizu et al., 1995), indicating that Bcl-2 and Bcl-xL can modulate other activating events in the apoptotic process. For instance, Bcl-xL may directly regulate the permeability of membranes to which it is associated by forming ion-conducting channels (Minn et al., 1997). Bcl-2 has been shown to block calcium efflux from the endoplasmic reticulum and to reduce the viability of lymphoma cells treated with thapsigargin, a CaZ+-ATPase inhibitor (Lam et al., 1994). This appears to be dependent on capacitative entry and elevation of cytosolic calcium concentration (He et al., 1997). In neural cells, Bcl-2 promotes mitochondrial sequestration of large amounts of calcium without altering respiratory activity (Murphy et al., 1996). Another potential regulatory effect of Bcl-2 is activation of the transcription factor NF-nB (Moissac et al., 1998), which can protect neurons from oxidative stress-induced apoptosis (Mattson et al., 1997). Thus, Bcl-2 may exert a flexible control on the neuronal death process, depending on the neurotoxic insult involved.

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A small number of proteins not homologous to the Bcl-2 family members have been shown to inhibit the death of neuronal cells in culture, some of which are viral in origin but have provided potentially important information regarding mammalian neuronal apoptosis. After Bcl-2, the next protein demonstrated to be protective in mammalian neural cells was that encoded by the apoptosis suppressor gene p35, derived from the baculovirus Autographa californica nuclear polyhedrosis virus. As with Bcl-2, expression of p35 in conditionally immortalized neural cells increases survival relative to untransfected cells following glucose or serum withdrawal, and treatment with ionophore (Rabizadeh et al., 1993). Because there is no homology between p35 and Bcl-2, and presumably no interaction, the potential for modulating the apoptotic process at multiple points was realized. Along these lines, the protein encoded by crmA, a cytokine response modifier gene from cowpox virus, blocks apoptosis by specifically inhibiting the activity of interleukin-ll3 converting enzyme (Gagliardini et al., 1994). The expression of crmA in chicken dorsal root ganglion neurons increased cell survival relative to controls in the absence of nerve growth factor, and this was the first demonstration that caspases participate in vertebrate neuronal death and revealed a new level by which apoptosis may be regulated. In mammalian cells, the protein BAG-1 binds to Bcl-2, but they share no homology. In a human lymphoid cell line, it was shown that expression of both genes afforded a greater protection against apoptotic insults than expression of either gene alone (Takayama et al., 1995). Thus, along with its interaction and suppression of the death-promoting activity of family members such as Bax, Bcl-2 also appears to interact with non-family members to cooperatively inhibit apoptotic pathways. NAIP has been shown to suppress apoptosis in non-neuronal cells (Liston et al., 1996), and it will be discussed in the next section as it is particularly implicated in the pathogenesis of a certain form of motor neuron degenerative disease.

Evidence for Apoptosis in Motor Neuron Degenerative Diseases Studies in Human Tissue There is a progressive loss of lower and upper motor neurons during the course of ALS, ultimately resulting in paralysis and death of the patients by respiratory failure (see Brooks, 1996; Mitsumoto, 1997 for review). The familial forms of ALS are clinically indistinguishable from sporadic forms, yet progression of the disease in patients with certain Cu/Zn-SOD mutations is much more rapid (Cudkowicz et al., 1997). The causative agent(s) in most cases of ALS is unknown, but the clinical data suggest that there may be a common final pathway leading to motor neuron degeneration in different forms of ALS. There is evidence that this pathway can trigger apoptosis in lower and upper motor neurons in ALS. Some researchers have observed markers of apoptosis in post-mortem tissue samples from affected regions of ALS patients (summarized in Table 1), but others have not. Yoshiyama et al. (1994) used two markers to detect apoptotic ceils in spinal cord sections from ALS patients. First, based on the correlation between changes in specific glycosylation patterns and apoptosis in human tissues (Hiraishi et al., 1993), they carried out immunohistochemical analysis with

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a mouse monoclonal antibody that recognizes the antigen Le v, a difucosylated type 2 chain determinant. Second, they used the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) method to detect DNA strand breaks (Gavrieli et al., 1992). In 7 out of 10 ALS spinal cord sections, a small number of motor neurons were positive for Le v. Of these 7 cases, double labeling revealed that 4 also had nick-end labeled motor neurons. All of the ALS cases were sporadic. In contrast, neither apoptotic marker was observed in spinal cord sections from patients with progressive supranuclear palsy, lacunar stroke, polyarteritis nodosa, or non-neurological diseases. Although less than 20% of surviving neurons had apoptotic markers in the ALS spinal cord sections, this does not necessarily reflect the percentage of neurons that underwent apoptosis during the course of the disease. Also using in situ nick-end labeling to detect DNA strand breaks, Migheli et al. (1994) were unable to demonstrate apoptotic neurons in ALS spinal cord sections. They did observe, however, diffuse labeling of neuronal and glial nuclei in spinal cord and motor cortex sections from the ALS patients, but this did not correspond to the presence of isolated pyknotic nuclei and they concluded that the DNA fragmentation was a post-mortem artifact. Again, a small number of motor neurons with apoptotic markers or the lack thereof in post-mortem tissue is not necessarily an indication of the extent to which apoptosis occurred in affected ALS regions prior to death of the patients. Studies attempting to provide evidence for apoptosis in ALS based on changes in the levels of Bcl-2 family members or expression of their genes in post-mortem spinal cord samples have yielded conflicting results. In a study of 12 sporadic ALS cases selected by the presence of neuronophagia in the cerebral cortex, nick-end labeling of DNA and/or increased Bcl-2 immunoreactivity was observed in one or all paraffin-embedded sections of spinal cord, brainstem, and cerebral cortex in each patient (Troost et al., 1995). However, only nick-end labeling corresponded with the morphological features of apoptosis, such as cell shrinkage. The presence of DNA strand breaks in only the cerebral cortex or brain stem in some of the ALS cases is unlikely to be due to region-specific apoptosis, but may be the result of an early loss of apoptotic motor neurons in the spinal cord as the disease progresses to upper motor neurons, i.e. in no case was apoptosis observed in only the spinal cord. Nick-end labeled DNA was observed in the spinal cord and/or cerebral cortex of 4 out of 5 non-neurological control sections, but in fewer neurons than in any of the ALS sections. No inverse relationship between apoptosis and extent of Bcl-2 immunoreactivity was found, and the levels of Bcl-2 in spinal cord motor neurons were, in fact, the same or even higher than in the ALS sections. Moreover, in some of the ALS sections, Bcl-2 levels were found to be higher in normal cells of the border zone between affected motor cortex and the adjacent post-central gyrus. One explanation for these results is the involvement of Bcl-2 in a protective pathway that is activated in surviving cells, possibly, particularly in the latter case, in response to dying neighboring cells. Interestingly, DNA fragmentation and increased levels of Bcl-2, Bcl-x, Bax, and interleukin-lB converting enzyme have been observed in denervated muscle fibers of some ALS patients (Tews et al., 1997). Contradictory results were obtained by Mu et al. (1996) in their study of post-mortem spinal cord samples from sporadic ALS patients. Using in situ hybridization, they found that the levels of bcl-2 mRNA were significantly reduced in lamina IX alpha motor

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neurons of ALS spinal cord relative to those of non-neurological control spinal cord. This reduction was selective, in that bcl-2 mRNA levels in neurons of the proper sensory nucleus of laminae III and IV, which do not degenerate in ALS, were similar in disease and control spinal cord sections. It was further reported by Mu et al. (1996) that the levels of bax mRNA were significantly and selectively increased in lamina IX alpha motor neurons of ALS spinal cord relative to those of control spinal cord. Although Bcl-2 and Bax protein levels were not determined in this study, the results suggest a reduction in the levels of Bcl-2/Bax heterodimers and perhaps an increase in the levels of Bax homodimers in ALS-vulnerable motor neurons, thereby promoting apoptosis. Overall, not a single apoptotic marker has been consistently detected in post-mortem tissue from ALS patients, and a major contributing factor for this is likely to be the variation in the clinical and pathological features of the ALS cases from study to study. What is needed is an analysis of the correlation between morphological apoptotic markers, DNA fragmentation, and both mRNA and protein levels of Bcl-2 family members in a broad variety of ALS patients. It has also not been reported if there is a difference in the extent to which apoptotic markers occur in sporadic versus familial ALS spinal cord. Nonetheless, current evidence from studies with human tissue does support an apoptotic form of cell death in at least a subset of motor neurons that degenerate in ALS. The most common motor neuron degenerative disease in children is proximal spinal muscular atrophy (SMA), but unlike ALS, it is characterized by the loss of only lower motor neurons. There are four types of SMA classified by the extent of motor development and age at onset (see Zerres and Rudnik-Schoneborn, 1995 for review). Type I SMA, or Werdnig-Hoffmann disease, is the most severe form with an age at onset of <2 months and death of most afflicted infants by 1-2 years. The type I SMA infants never sit alone. Type II SMA has an age of onset of <1 year, and infants can sit alone but never walk. There are two sub-classifications of the third type of SMA where patients walk without support: IIIa, with an age at onset of <3 years and IIIb, with an age at onset of 3-30 years. The fourth type of SMA is an adult-onset form, where symptoms begin after the age of 30. SMA is one of the most common autosomal recessive disorders, and all childhood forms are linked to the chromosome 5q 11.3-13.1 region (Gilliam et al., 1990; Melki et al., 1990). The gene encoding NAIP is located within this region (Roy et al., 1995), as is the gene encoding what is known as the survival motor neuron (SMN) protein (Lefebvre et al., 1995). The 5ql 1.3-13.1 region is polymorphic and contains several tandomly repeated genes, including N A I P and SMN, with some variability in orientation and gene copy number between individuals. The S M N gene is present as two highly homologous copies, one telomeric and one centromeric (Lefebvre et al., 1995). The N A I P gene was named based on its homology with two baculovirus genes that encode proteins functioning to inhibit apoptosis upon viral infection. Indeed, infection of mammalian cells with adenoviral vectors containing the human N A I P gene results in increased survival upon serum withdrawal, menadione treatment, and upon exposure to tumor necrosis factor ct (Liston et al, 1996). Evidence for an involvement of NAIP in the pathogenesis of SMA was first provided by Roy et al. (1995). These authors reported that a deletion in the first two coding exons of the NAIP gene occurs in approximately

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Table 1.

Evidence for apoptosis in motor neuron degenerative diseases

Summary of published data

Disease

Reference

ALS

Yoshiyama et al. (1994)

ALS

Troost et al. (1995)

ALS

Tews et al. (1997)

ALS

Mu et al. (1996)

SMA SMA

Roy et al. (1995) Lefebvre et al. (1995)

ALS

Kostic et al. (1997)

ALS

Friedlander et al. (1997)

ALS

Pasinelli et al. (1998)

SMA

Schrank et al. (1997)

ALS

Rabizadeh et al. (1995)

ALS

Wiedau-Pazos et al. (1996)

ALS

Kaal et al. (1998)

ALS

Pedersen et al. (1999); Smirnova et al., 1998a Ellerby et al. (1999)

Studies in human tissue Spinal cord motor neurons labeled by TUNEL and/or an antibody for the LeVantigen Nick-end labeling of DNA and/or increased Bcl-2 immunoreactivity in motor neurons of spinal cord, brainstem, and cerebral cortex DNA fragmentation, increased levels of Bcl-2, Bcl-x, Bax, and interleukin-113 converting enzyme in denervated muscle fibers Reduced bcl-2 and increased bax mRNA levels specifically in spinal cord alpha motor neurons Deletion in coding exons of NAIP gene Deletion of telomeric SMN gene Studies in animal models Delayed onset of phenotype and increased survival in transgenic mice over-expressing mutant human Cu/Zn-SOD gene and human bcl-2 gene Increased survival in transgenic mice over-expressing mutant human Cu/Zn-SOD gene and a dominant negative mutant of interleukin-113 converting enzyme Proteolytic processing characteristic of caspase-1 activation in mutant Cu/Zn-SOD expressing mice Homozygous deletion of the SMN gene in mice causes massive cell death in early embryonic development with characteristics of apoptosis Cell culture studies Familial mutations convert the effect of Cu/Zn-SOD from anti-apoptotic to pro-apoptotic in neural cells Expression of mutant Cu/Zn-SOD in nigral neural cells enhances serum withdrawal-induced apoptosis Oxidative stress induces apoptosis in primary spinal cord motor neuron cultures Oxidative and lipid peroxidative stress induces apoptosis in a spinal cord motor neuron cell line Caspase-3 cleavage of the androgen receptor containing a polyglutamine stretch results in apoptosis in a kidney cell line

KD

ALS, Amyotrophic Lateral Sclerosis; KD, Kennedy's Disease; NAIP, neuronal apoptosis inhibitory protein; SMA, Spinal Muscular Atrophy; SMN, survival motor neuron.

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67% of type I SMA chromosomes, 42% of type II and III SMA chromosomes, and only 2% of non-SMA chromosomes. Thus, it is proposed that an apoptotic cell death program is continually activated or reactivated in SMA contributing to motor neuron degeneration in this disorder. Deletions in the telomeric copy of the SMN gene occur in greater than 90% of type I SMA patients, with lower frequencies in types II and III, and the lowest frequency in adult-onset forms (Campbell et al., 1997; Chang et al., 1997; Lefebvre et al., 1995; Simard et al., 1997; Sommerville et al., 1997). A small number of SMA patients also have an interrupted telomeric SMN gene, resulting in a chimeric centromeric-telomeric gene. The mechanisms underlying these genetic rearrangements vary from case to case, and may involve unequal recombination, double recombination, gene conversion, and intrachromosomal deletion (Wirth et al., 1997). There is no direct correlation between the severity of the clinical phenotype and the genetic rearrangements that occur in SMA, but the most severe cases appear to have deletions in both the NAIP and telomeric SMN genes. The clinical phenotype of SMA is, therefore, likely to be determined by an interaction between different alleles of the 5q11.3-13.1 region. The SMN protein appears to have a role in mRNA processing (Liu and Dreyfuss, 1996), and mice with a homozygous deletion in the SMN gene exhibit massive cell death in early embryonic development that is characteristic of apoptosis (Schrank et al., 1997). Mice have only one copy of the SMN gene, and the variable clinical phenotype in humans may be attributable, in part, to the extent to which expression of the centromeric copy of the SMN gene compensates for the lack of telomeric SMN. The specific functions of SMN and NAIP in inhibiting apoptosis are unclear, and it is not yet known if other proteins encoded by SMA-determining genes participate in apoptotic pathways. Notably, one out of 152 apparently sporadic ALS patients was found to have a homozygous deletion in exon 5 of the NAIP gene, but the SMN gene was unaffected (Jackson et al., 1996). It is possible that this case was adult-onset SMA rather than ALS, as suggested by the authors, and defects in the SMN and NAIP genes appear to be predominantly, if not exclusively, associated with SMA. Animal Models Several transgenic and naturally occurring mouse models are available to study the pathogenesis of motor neuron degenerative diseases (see Cleveland et al., 1996; Ludolph, 1996; Rabin and Borchelt, 1999 for review). Transgenic mice expressing the human Cu/Zn-SOD gene containing familial ALS mutations develop a clinical phenotype and have pathological features similar to that seen in humans (Gurney et al., 1994). Studies with these transgenic mice have demonstrated that proteins with pro- or anti-apoptotic activity can alter the clinical course of the disease. Kostic et al. (1997) generated mice who over-express both the human Cu/Zn-SOD gene with the G93A mutation and the human bcl-2 gene. There was a statistically significant delay in the onset of symptoms from 170 days in the G93A mice to 203 days in the G93A/Bcl-2 mice. Furthermore, there was a statistically significant increase in the survival time for G93A/Bcl-2 mice relative to that for G93A mice i.e. the time from symptom onset to complete paralysis. By counts of Nissl-stained and choline

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acetyltransferase-positive motor neurons in spinal cord sections, the delay in onset of symptoms in G93A/Bcl-2 mice corresponded to a greater number of surviving cells. Notably, the presence of apoptotic markers was not determined in this study, and it cannot be concluded that the effects of Bcl-2 on the clinical course were due to modulation of apoptotic pathways. As discussed in the previous section, there is evidence that Bcl-2 can reduce cellular levels of reactive oxygen species. Familial ALS mutations in Cu/Zn-SOD confer upon the enzyme the ability to efficiently catalyze the oxidation of substrates by H202 and lead to increased oxyradical production (Bogdanov et al., 1998; Liu et al., 1998; Wiedau-Pazos et al., 1996; Yim et al., 1996). Yet, because oxidative stress can manifest as either apoptosis or necrosis (cf. Tan et al., 1998), and because Bcl-2 may be able to protect against both of these forms of cell death (see previous section), a mechanism that involves an anti-oxidant effect of Bcl-2 in the transgenic mice of Kostic et al. (1997) does not necessarily indicate apoptotic cell death in ALS. If we assume that the modulation of the clinical course is due to anti-apoptotic effects, then possible explanations are that the protective response of Bcl-2 is overwhelmed, or, that other cell death mechanisms play a prominent role in the pathogenesis of ALS. In mice expressing a dominant negative mutant of interleukin-lB converting enzyme and the G93R mutation in human Cu/Zn-SOD, Friedlander et al. (1997) reported a statistically significant increase in survival time and thus delay in mortality relative to mice expressing only mutant Cu/Zn-SOD. There was, however, no alteration in the time from birth to onset of symptoms in this transgenic model. More recent studies have shown increased proteolytic processing characteristic of caspase-1 activation in the spinal cords of mice expressing mutant Cu/Zn-SOD and in differentiated N2a neuroblastoma cells (Pasinelli et al., 1998). Activation of caspase-1 was enhanced by treatment with pro-oxidants, resulting in cleavage of pro-interleukin-1B and induction of apoptosis. Unlike with other neurodegenerative diseases, there are mice with spontaneous mutations inherited in an autosomal-recessive manner that serve as useful models to investigate the pathogenesis of human motor neuron degenerative diseases. One of these models is the wobbler mouse, where a mutation of an unidentified gene on chromosome 11 is inherited as an autosomal recessive trait leading to progressive degeneration of spinal cord and brainstem motor neurons (see Ludolph, 1996 for review). Studies with these mice aimed at elucidating the motor neuron protective role of Bcl-2 have yielded very different results than those obtained from studies with transgenic ALS mice or with axotomy-induced cell death paradigms. Ikeda et al. (1995) reported that intraperitoneal injection of phosphatidylcholine-bound human Cu/Zn-SOD partially protects against the effects of the wobbler mutation, suggesting a role for oxidative stress in motor neuron degeneration in this model. However, in contrast to what was observed in transgenic ALS mice, neuron-specific over-expression of human bcl-2 did not delay the onset or slow the progression of disease in the wobbler mouse (Ait-Ikhlef et al., 1995; Coulpier et al., 1996). Moreover, wobbler mice with and without the human bcl-2 gene had identical pathological features, i.e. vacuolar degeneration of motor neurons in the ventral root, degeneration of large myelinated axons in the cervical ventral roots, and astrogliosis. The expression of human bcl-2 in these mice did protect motor neurons in the spinal cord and brainstem from developmental death, and protected

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motor neurons of the facial nucleus from axotomy-induced cell death in postnatal mice (Coulpier et al., 1996). Thus, the results of these studies indicate that there are cell death pathways insensitive to Bcl-2 that result in selective motor neuron degeneration as seen in human disease, and are consistent with the partial protective effects of bcl-2 expression in ALS transgenic mice. Whether or not these Bcl-2-insensitive cell death pathways are apoptotic, perhaps regulated by Bcl-xL or non-family members such as BAG-l, remains to be determined. A consistent finding in mice over-expressing the human bcl-2 gene is that motor neurons are protected from death caused by axotomy. In postnatal day 1 rats, facial nerve transection results in morphological changes characteristic of apoptosis within the facial motor nucleus, which begins about 3 days after axotomy and is maximal at 4 days after axotomy (see Elliott and Snider, 1999 for review). Indeed, when the same experiment is carried out in mice with a homozygous deletion of the bax gene, virtually all facial motor neurons survive up to 1 week post-axotomy. Facial motor neurons in bax-/- mice can, in fact, survive for several weeks after axotomy of the facial nerve at postnatal day 1. These results, and the remarkable protection of Bcl-2 against motor neuron degeneration upon transection of the sciatic nerve or axotomy of the facial nerve (see previous section), argues that apoptosis is the predominant form of cell death in this experimental paradigm. The axotomy model relates to the "dying-back" process proposed as a way in which cell loss occurs in motor neuron degenerative diseases. This process is believed to follow a sequence beginning with synaptic loss, which initiates a cell death signal resulting in axonal degeneration and ultimately the loss of cell bodies. It has been shown that mice over-expressing the human neurofilament heavy-subunit gene, variant alleles of which are found in ALS patients, have dramatic defects in axonal transport (Collard et al., 1995), which may render synapses abnormally vulnerable to insult. Another model for the dying-back hypothesis is the progressive motor neuropathy (pmn) mouse, which is caused by autosomal-recessive inheritance of a spontaneous mutation on chromosome 13 (see Ludolph, 1996 for review). In the pnm mouse, disease begins with weakness in the hindlimbs at 3 weeks of age and rapidly progresses to complete paralysis and death by 6 weeks of age due to respiratory failure. This is caused by distal axonal degeneration with relative sparing of proximal axons and cell bodies. Sagot et al. (1995) reported that over-expression of human bcl-2 in pmn mice prevents motor neuron cell body loss but not axonal degeneration. Collectively, the results obtained with axotomy paradigms versus the results obtained with ALS transgenic mice and other mouse models of motor neuron degenerative diseases demonstrate that Bcl-2 is less protective against cell loss by a dying-back process in pathological conditions than in experimentally-induced conditions. Similar experiments with other pro-/anti-apoptotic proteins will therefore be required to gain a complete understanding of the extent to which apoptotic cell death occurs in motor neuron degenerative diseases. For instance, mutations in a gene encoding a putative transcriptional activator and ATPase/DNA helicase were recently identified in the neuromuscular degeneration (nmd) mouse (Cox et al., 1998). Some members of the ATPase/DNA helicase superfamily, such as TFIIH, have a role in general transcription, DNA repair, and apoptosis (see Svejstrup et al., 1996; Warbrick, 1996 for review).

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Cell Culture Studies The in vitro studies showing increased oxidative stress resulting from mutations in Cu/Zn-SOD in familial ALS and the evidence that oxidative stress can lead to an apoptotic form of death in neurons provides, at present, the best conceptual model for apoptosis in a motor neuron degenerative disease. An anti-apoptotic role for Cu/Zn-SOD has been demonstrated in cell culture studies. In PC 12 cells, Troy et al. (1994) showed that down-regulation of Cu/Zn-SOD with antisense oligonucleotides decreased survival, an effect that was more pronounced in untreated cells than in cells differentiated with nerve growth factor. Cell death was characterized as apoptotic based on the DNA degradation pattern. Interestingly, pre-treatment with vitamin E inhibited cell death caused by the antisense oligonucleotides, suggesting that Cu/Zn-SOD blocks apoptosis by suppressing the levels of free radicals. In cultures of rat sympathetic neurons deprived of nerve growth factor, apoptosis can be delayed by injection of purified bovine Cu/Zn-SOD or of an expression vector containing human Cu/Zn-SOD cDNA (Greenlund et al., 1995a). The delay in apoptosis occurs to a similar extent as obtained by over-expression of human bcl-2 (see previous section). Greenlund et al. (1995a) further reported that an increase in production of reactive oxygen species peaked at 3 h following trophic factor withdrawal in the sympathetic neuronal cultures. After the peak period, apoptosis could be prevented if nerve growth factor was added back but not by injection of SOD. These results further support an anti-apoptotic role for Cu/Zn-SOD through suppression of free radical production. The results of this study also reveal that the suppressive effect of Cu/Zn-SOD on the levels of reactive oxygen species occurs at an early step in the apoptotic cascade. It was initially believed that familial ALS mutations in Cu/Zn-SOD result in a loss of the ability of the enzyme to catalyze the dismutation of superoxide and to therefore protect motor neurons from oxidative stress-induced death. However, the majority of familial ALS mutations do not occur in the active site of Cu/Zn-SOD and mutant enzymes possess a significant level of dismutase activity (Borchelt et al., 1994). In transgenic mice expressing Cu/Zn-SOD with familial ALS mutations, increases in enzyme activity correspond to the level of transgene expression, yet there is no correlation between enzyme activity and disease severity (Gurney et al., 1994; Ripps et al., 1995; Wong et al., 1995). Motor neurons in knockout mice lacking Cu/Zn-SOD develop normally, the mice live well beyond one year of age, and the mice fail to develop motor neuron disease (Reaume et al., 1996). Thus, the pathogenic effect of mutant Cu/Zn-SOD is not due to loss of enzyme activity. Instead, as mentioned previously, familial ALS mutations confer upon Cu/Zn-SOD a peroxidase activity. This gain-of-function has been shown in cell culture to shift the anti-apoptotic effect of Cu/Zn-SOD to a pro-apoptotic effect. In yeast that are deficient in Cu/Zn-SOD, Rabizadeh et al. (1995) were able to restore the wild-type phenotype by expressing the human enzyme with familial ALS mutations, an effect that was due to the mutant enzyme retaining functional dismutase activity. These investigators further showed that expression of the human wild-type Cu/Zn-SOD gene was able to protect rat nigral neural cells from apoptotic insults, but that expression of the mutant enzyme promoted apoptosis in a dominant fashion. Additionally, Wiedau-Pazos et al. (1996) reported

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that nigral neural cells expressing mutant human Cu/Zn-SOD are more vulnerable to apoptosis induced by serum withdrawal. These results support an apoptotic form of motor neuron death in the subset of patients with Cu/Zn-SOD mutations. Oxidative damage occurs in both familial and sporadic ALS, and because oxidative stress can induce apoptosis in primary cultures of purified spinal cord motor neurons (Kaal et al., 1998) and in the spinal cord motor neuron cell line, NSC-19 (Pedersen et al., 1999; Smirnova et al., 1998a), motor neuron degeneration by oxidative stress-induced apoptosis may occur in all forms of ALS. A number of other studies have been carried out in vitro demonstrating apoptotic events leading to the death of motor neurons, but in most cases their significance for the pathogenesis of motor neuron degenerative diseases has not been substantiated. An approach to studying apoptosis in neurodegenerative diseases is to determine if compounds that play a role in programmed cell death during development of the nervous system inappropriately activate apoptotic pathways in the adult CNS and thus contribute to disease. One candidate is thrombin, a serine protease that acts through a cell-surface receptor known as the protease-activated receptor 1. This receptor is differentially expressed in neurons and glial cells of the adult CNS (see Festoff et al., 1996; Turgeon and Houenou, 1997 for review). In cultures of embryonic chick spinal cord motor neurons, thrombin was reported to decrease mean neurite length, to prevent neurite branching, and to induce cell death (Turgeon et al., 1998). Thrombin caused morphological changes consistent with apoptosis in these cultures, and cell death could be prevented by caspase inhibitors. The same group further reported that in NSC-19 cells, thrombin induced cell body rounding, neurite retraction, cell aggregation, chromatin condensation, DNA fragmentation, and caspase cleavage of nonerythroid spectrin (Smirnova et al., 1998b). Thrombin activity can be regulated by a group of serine protease inhibitors known as serpins. One member of this group is protease nexin-1, which has been shown to protect cultured hippocampal neurons from glucose-induced injury by a mechanism that involves stabilization of calcium homeostasis (Smith-Swintosky et al., 1995), and to protect motor neurons from naturally occurring and axotomy-induced cell death (Houenou et al., 1995). Although certain isoforms of APP contain a Kunitz-type protease inhibitor domain (see Selkoe, 1994 for review), this does not appear to be necessary for the neuroprotective function of the secreted products. Rather, secreted APP activates rB-dependent transcription (Barger and Mattson, 1996), regulates calcium levels (Mattson et al., 1993), and increases cGMP production (Barger et al., 1995), the latter of which promotes the survival of spinal cord motor neurons in vitro (Estevez et al., 1998). An increase in APP mRNA and protein levels has been observed in dying spinal cord motor neurons in culture, but the cell prevents this potential protective response by cleaving APP with caspase-3 (Barnes et al., 1998). While the relevance of these findings for the involvement of apoptosis in motor neuron degenerative diseases is unclear, the results of in vitro studies carried out by Ellerby et al. (1999) provide a direct link between apoptosis and the pathogenesis of Kennedy's disease, also known as spinobulbar muscular atrophy, an X-linked disorder characterized by degeneration of lower but not upper motor neurons (see Parboosingh et al., 1997 for review). There is an increase in the number of CAG repeats encoding a polyglutamine stretch in the androgen receptor

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in Kennedy's disease, and Ellerby et al. (1999) showed that expression of the androgen receptor with the polyglutamine stretch in a kidney cell line induces apoptosis. Induction of apoptosis required that the receptor be cleaved by caspase-3. These results, along with the effects of mutant Cu/Zn-SOD, provide mechanistic links between genetic aberrancies and apoptosis in motor neuron degenerative diseases.

ALS Overview: Integration of Proposed Cell Death Cascades There is now considerable evidence that oxidative damage to proteins and nucleic acids in motor neurons is a common correlate of all forms of ALS. The fact that transgenic mice expressing the human Cu/Zn-SOD gene with familial ALS mutations develop a clinical phenotype and neuropathological changes seen in humans (Gurney et al., 1994; Dal Canto et al., 1995; Tu et al., 1996; Wong et al., 1995) supports a causative role for oxidative stress in motor neuron degeneration in this disorder. Further support for this hypothesis comes from studies showing that greater oxyradical production and lipid peroxidation in the spinal cords of transgenic ALS mice precede onset of motor neuron degeneration (Bogdanov et al., 1998; Liu et al., 1998). Enhanced free radical production by mutant Cu/Zn-SOD is consistent with the observations of increased levels of free and protein-bound 3-nitrotyrosine (Bruijn et al., 1997; Ferrante et al., 1997b) and protein carbonyl groups (Andrus et al., 1998) in the spinal cords of transgenic ALS mice relative to non-transgenic littermates. From studies with patient material, the evidence for an involvement of oxidative stress in the pathogenesis of ALS includes: fibroblasts from ALS patients, particularly those harboring Cu/Zn-SOD mutations, are more sensitive to oxidative stress caused by treatment with H202 (Aguirre et al., 1998), increased levels of nuclear DNA 8-hydroxy-2'-deoxyguanosine, 3-nitro-4-hydroxy-phenylacetic acid, free and protein-bound 3-nitrotyrosine, protein carbonylation, malondialdehyde-modified proteins, and 4-hydroxynonenal (HNE)-modified proteins in the spinal cords of familial and/or sporadic ALS patients (Beal et al., 1997; Ferrante et al., 1997a; Pedersen et al., 1998), and a higher concentration of free HNE in the cerebrospinal fluid of ALS patients relative to controls (Smith et al., 1998). The underlying cause(s) of oxidative damage to motor neurons in the majority of ALS cases is unknown. However, as discussed in the previous section, evidence linking oxidative stress and apoptosis in motor neurons suggests that activation of apoptotic cascades may be responsible for cell death, at least to some extent, in all ALS cases. Membrane lipid peroxidation appears to play a particularly important role in the cell death process in many different neurodegenerative disorders. Investigations of postmortem brain tissue and cerebrospinal fluid from patients with Alzheimer's disease (Lovell et al., 1995, 1997; Sayre et al., 1997) and Parkinson's disease (Yoritaka et al., 1996) have documented increased levels of free and protein-bound HNE. Peroxidation of polyunsaturated fatty acids leads to formation of aldehydic by-products with varying carbon chain length, such as malondialdehyde and HNE (see Esterbauer et al., 1991 for review). Among such aldehydes, HNE appears to play a central role in the impairment of protein function and in causing neuronal degeneration

246

W.A. Pedersen, 1. Kruman and M.P. Mattson

(see Mattson, 1998 for review). Detailed studies of the impact of lipid peroxidation on cortical and hippocampal neurons in culture suggest a scenario in which oxidative stress leads to HNE production and subsequent impairment of membrane ion-motive ATPases (Na+/K+-ATPase and Ca2+-ATPase) and glucose transporters (Mark et al., 1995, 1997a,b). In addition, HNE results in impaired glutamate transport in both astrocytes and neurons (Keller et al., 1997; Blanc et al., 1998). These effects of HNE would result in membrane depolarization, energy depletion, and disruption of cellular calcium homeostasis; each of these changes renders neurons vulnerable to excitotoxicity and apoptosis (Figure I).

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Figure 1. Working model for the involvement of oxidative stress, lipid peroxidation, and excitotoxicity in ALS. Elevation of intracellular calcium levels and increased production of reactive oxygen species can damage proteins, lipids, and DNA, ultimately resulting in death. Increased oxidative stress, as caused by familial ALS mutations in Cu/Zn-SOD, results in lipid peroxidation (LP) and production of 4-hydroxynonenal (HNE). The latter can impair the function of membrane ion-motive ATPases, and glucose and glutamate transporters, which would reduce ATP levels and promote excitotoxicity. In motor neurons in ALS, overactivation of AMPA ionotropic receptors by glutamate causes excessive influx of calcium. The calcium buffering capacity of mitochondrial would be overwhelmed, disrupting the transmembrane potential (MPT) and increasing production of free radicals (superoxide) and hydrogen peroxide (H202). By the Fenton reaction, iron converts H202 to hydroxyl radical. The cellular anti-oxidant systems include catalase and glutathione peroxidase (GSHPx), which convert H202 to water. Elevations in calcium concentrations can further promote oxidative stress by: (1) activating nitric oxide synthase by ealmodulin, resulting in peroxynitrite formation through the reaction of nitric oxide and superoxide, and (2) activation of phospholipase A 2 (PLA2), resulting in arachidonic acid formation and generation of reactive oxygen species from the subsequent reactions catalyzed by cyclooxygenase (COX) and lipoxygenase (LOX). Nitration of proteins by peroxynitrite contributes to impaired function. See text for details.

Apoptosis in Motor Neuron Degenerative Diseases

247

The above scenario evolved from investigations carded out that are relevant to the neurodegenerative process in AD. In particular, immunoprecipitation-western blot analyses using a monoclonal antibody that recognizes HNE-modified proteins, in combination with antibodies against specific transport proteins, demonstrated direct covalent modification of glucose (GLUT3; Mark et al., 1997b) and glutamate (GLT1; Keller et al., 1997; Blanc et al., 1998) proteins by HNE upon exposure of cultured neurons and synaptosomes to insults that induce lipid peroxidation, such as AlL Indeed, treatment of primary hippocampal cultures from rat causes an increase in both free and protein-bound HNE (Mark et al., 1997a). Exposure of primary hippocampal cultures to HNE results in nuclear changes characteristic of apoptosis (Kruman et al., 1997), and HNE may therefore be a mediator of apoptosis induced by AlL Of direct relevance to ALS, we observed an increase in modification of a number of proteins by HNE in the lumbar spinal cords of ALS patients compared to the levels in the lumbar spinal cords of non-neurological controls (Pedersen et al., 1998). One protein with increased HNE modification in the spinal cords of these patients was GLT-1/EAAT2. We carded out a functional analysis of the protein modification by HNE in the motor neuron cell line, NSC-19. Treatment of these cells with either Fe 2÷ or HNE dramatically impaired glutamate and glucose uptake (Pedersen et al., 1999). Importantly, the Fea÷-and HNE-induced impairment of glutamate and glucose transport in NSC-19 cells preceded induction of apoptosis. This study suggests that lipid peroxidation is sufficient to impair membrane transporters that are critical for preventing excitotoxic degeneration of motor neurons, which may manifest as apoptosis (see below). Thus, the scenario proposed for neuronal degeneration in AD may also apply to the pathogenesis of ALS (Figure 1). There is overwhelming evidence that the pathogenesis of motor neuron death in ALS involves an excitotoxic component (see Rothstein, 1996 for review). The initial evidence in support of this came from a study where elevated concentrations of glutamate were found in the cerebrospinal fluid of ALS patients relative to those of normal patients (Rothstein et al., 1990). Subsequently, impaired glutamate transport was observed in synaptosomes prepared from the spinal cords of ALS patients (Rothstein et al., 1992) and from transgenic ALS mice (Canton et al., 1998). The cause of impaired glutamate transport in ALS spinal cord was shown to be due to a selective loss of the astroglial glutamate transporter protein EAAT2 (Rothstein et al., 1995; Bristol and Rothstein, 1996), which appears to result from defects in EAAT2 mRNA processing (Lin et al., 1998). Pharmacological inhibition of glutamate transport in rat spinal cord organotypic slice cultures leads to a slow degeneration of motor neurons over several weeks (Rothstein et al., 1993). This toxic effect could be prevented by non-NMDA receptor antagonists but not by NMDA receptor antagonists, consistent with the evidence that motor neurons are selectively vulnerable to AMPA/kainate receptor-mediated injury (Carriedo et al., 1996). The use of antisense oligonucletides specific for EAAT2 mRNA in spinal cord organotypic slice cultures from rat revealed that astroglial transporters play the major role in protecting motor neurons from glutamate toxicity (Rothstein et al., 1996). As with oxidative stress, glutamate toxicity can manifest as either apoptosis or necrosis. Mild glutamate toxicity has been shown to cause apoptosis in primary neuronal cultures (Ankarcrona et al., 1995; Bonfoco et al., 1995), and glutamate exposed to

248

W.A. Pedersen, 1. Kruman and M.P. Mattson

primary neuronal cultures or when injected into the hippocampi of rats causes D N A fragmentation associated with cell death (Kure et al., 1991). In the latter study, DNA fragmentation and cell death could be prevented by inhibitors of endonucleases and m_RNA synthesis. Furthermore, we have demonstrated that glutamate induces nuclear condensation and fragmentation in primary motor neuron cultures (Figure 2), and that the caspase inhibitor zVAD-fmk protects cultured motor neurons expressing mutant Cu/Zn-SOD from glutamte toxicity (I. Kruman and M.P. Mattson, unpublished data).

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There is a clear interrelationship between the proposed mechanisms of oxidative stress and excitotoxicity for motor neuron degeneration in ALS. The loss of ability of astrocytes, in particular, to transport glutamate would result in over-activation of the A M P A subtype of ionotropic glutamate receptors causing excessive influx of calcium into motor neurons. Abnormal elevations in the intracellular concentration o f calcium would promote oxidative stress as outlined in Figure 1. The calcium-buffering capacity of mitochondria would be quickly overwhelmed, thus disrupting mitochondrial function and increasing superoxide production. The latter may result in increased Cu/Zn-SOD activity, driving the reaction which converts superoxide to H202, followed by ironcatalyzed conversion of H202 to hydroxyl radical via the Fenton reaction, and lipid peroxidation. By neutron activation analysis, increased iron content has been detected in the lumbar spinal cords of ALS patients relative to controls (Markesbery et al., 1995), an observation that emphasizes the importance of lipid peroxidation in the pathogenesis

Apoptosis in Motor Neuron Degenerative Diseases

249

of this disorder. As shown in Figure 1, two reactions that protect cells from ironmediated production of hydroxyl radical involve glutathione and catalase, which convert HzO2 to water. However, catalase activity was reported by Przedborski et al. (1996) to not be different in affected brain regions in ALS patients versus controls, and although these authors observed a reduction in glutathione activity in the precentral gyrus of ALS patients relative to controls, others have found no difference in the activity of the latter enzyme in ALS spinal cord (Fujita et al., 1996). Other detrimental effects of increased cytosolic calcium levels include activation of neuronal nitric oxide synthase via calmodulin and activation of phospholipase A 2. Nitric oxide reacts with superoxide to form peroxynitrite, resulting in nitration of proteins on tyrosine residues. Activation of phospholipase A 2 increases arachidonic adid production, leading to enhanced levels of reactive oxygen species by cyclooxygenase and lipoxygenase. As disucssed above, HNE impairment of glutamate transport may provide a further link between oxidative stress and excitotoxicity. Mitochondrial dysfunction appears to be an early and critical event in motor neuron degeneration in ALS. Ultrastructural analysis of spinal cord tissue reveals mitochondrial swelling and perturbation of membrane structure in motor neurons of ALS patients (Sasaki and Iwata, 1996). Similar mitochondrial alterations have been documented in the early stages of motor neuron degeneration in mice expressing human Cu/Zn-SOD with familial ALS mutations (Wong et al., 1995; Kong and Xu, 1998), and following administration of excitotoxins to mice (Ikonomidou et al., 1996). Mitochondrial transmembrane potential, a key measure of mitochondrial function, was reported to be decreased in parallel with a rise in cytosolic calcium levels in neuroblastoma cells expressing mutant Cu/Zn-SOD (Carri et al., 1997). We have shown a reduction in mitochondrial transmembrane potential under basal conditions in motor neurons from mice expressing the G93A Cu/Zn-SOD mutation relative to wild-type motor neurons (I. Kruman and M.P. Mattson, unpublished data). This occurred concomitant with increases in levels of superoxide, protein tyrosine nitration, lipid peroxidation, and mitochondrial reactive oxygen species (Figure 3). Moreover, motor neurons from mutant Cu/Zn-SOD mice show a greater increase in mitochondrial reactive oxygen species than wild-type motor neurons upon treatment with glutamate (Figure 3). Previous studies have associated such mitochondrial alterations with both apoptosis and excitotoxicity (White and Reynolds, 1996; Susin et al., 1998). Indeed, motor neurons from wild-type mice are less vulnerable to glutamate-induced apoptosis than motor neurons from mice expressing mutant Cu/Zn-SOD (Figure 2). It has been shown in neuronal cells that mitochondrial function is a critical factor in determining whether glutamate-induced death occurs by apoptosis or necrosis (Ankarcrona et al., 1995). Opening of the mitochondrial permeability transition pore, resulting in loss of membrane potential, appears to be a central coordinating event in apoptosis (Marchetti et al., 1996). In mice expressing mutant Cu/Zn-SOD, administration of creatine, which inhibits calcium-induced opening of the mitochondrial transition pore, is the most effective intervention for prolonging survival reported to date (Klivenyi et al., 1999). Collectively, these results suggest that oxidative stress and excitotoxicity cause mitochondrial alterations that orchestrate the series of events leading to apoptotic motor neuron death in ALS.

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Cu/Zn-SOD. (A) Levels of hydroethidine fluorescence (HE, a measure of superoxide levels), protein tyrosine nitration, TBARS (a measure of lipid peroxidation), and dihydrorhodamine fluorescence (DHR, a measure of mitochondiral reactive oxygen species) were quantified under basal culture conditions in motor neurons from wild-type mice and Cu/Zn-SOD transgenice mice (SODMut). Values are the means and standard deviations of determinations made in at least 4 separate cultures (15-25 motor neurons assessed/culture). *p < 0.05, **p < 0.01 compared to corresponding value for wild-type mice (paired t-test). (B) Cultures were pretreated for 1 h with 200 ~M CNQX or vehicle, and were then exposed to 100 ~tM glutamate or vehicle (Control) for 6 h. The levels of DHR fluorescence in motor neurons were quantified and values are the means and standard deviations of determinations made in 4 separate cultures. *p < 0.01 compared to the Control value and the value for cultures exposed to CNQX+Glutamate; **p < 0.02 compared to corresponding value for glutamate-treated motor neurons from wild-type mice (ANOVA with Scheffe's post-hoc test).

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Apoptosis in Motor Neuron Degenerative Diseases

251

against neuronal voltage-dependent calcium channel subunits in both the plasma and cerebrospinal fluid of ALS patients (Smith et al., 1992). Subsequent cell culture studies revealed that exposure of cerebellar purkinje cells and a motor neuron cell line to ALS autoantibodies can induce calcium influx and cell death (Llinas et al., 1993; Smith et al., 1994), suggesting that autoimmune attack on motor neurons may be an important event in cell death in ALS. However, it seems likely that production of antibodies against the calcium channel subunits is a consequence of initial degeneration of some motor neurons, thereby exposing immune cells to the antigens, rather than an initiating event. This may serve, however, to propagate the motor neuron degeneration in ALS. This same group has provided ultrastructural evidence for increased calcium content of motor nerve terminals in muscle biopsy specimens from ALS patients (Siklos et al., 1996). These results further support a role for excessive calcium influx into motor neurons in the pathogenesis of ALS. In considering the contribution of disrupted calcium homeostasis to the degeneration of motor neurons in ALS, it is of interest that the most vulnerable populations of motor neurons in this disorder (lumbar cord) have the lowest levels of calcium-binding proteins such as calbindin-D28 k and parvalbumin, while resistant populations (e.g. cranial motor nuclei) have high levels of such calcium-binding proteins (Alexianu et al., 1994; Elliott and Snider, 1995). It has been shown that neurons which express calbindin D-28 k are relatively resistant to excitotoxicity (Mattson et al., 1991) and apoptosis (Wernyj et al., 1999). We recently reported that mitochondrial calcium uptake plays a pivotal role in both apoptosis and necrosis in neural cells (Kruman and Mattson, 1999). Thus, the selective vulnerability of certain populations of motor neurons in ALS may be due, in part, to their lack of expression of calcium- buffering proteins, rendering them vulnerable to apoptosis and necrosis. Another alteration that may contribute to increased oxidative stress in motor neurons in ALS is the formation of protein aggregates. Analyses of spinal cord motor neurons from familial ALS transgenic mice indicate that mutant Cu/Zn-SOD forms aggregates in the cells prior to degeneration (Bruijn et al., 1998). Moreover, over-expression of mutant Cu/Zn-SOD in cultured primary motor neurons results in formation of cytoplasmic Cu/Zn-SOD protein aggregates which precedes apoptotic death of the cells (Durham et al., 1997). The latter findings suggest a role for aberrant protein degradation in the pathogenesis of ALS. The results of more recent studies suggest that mutations in Cu/Zn-SOD perturb the molecular chaperoning system involving heat-shock proteins, and that relatively high levels of expression of HSP-70 can prevent aggregation of mutant Cu/Zn-SOD in motor neurons to prevent them from dying (Bruening et al., 1999). However, the latter study did not determine whether the mode of cell death was apoptosis. These findings are of broad interest in that studies of the pathogenic mechanisms underlying neuronal degeneration in Alzheimer's disease (Lee et al., 1999), Parkinson's disease (Duan et al., 1999a) and stroke (Lee et al., 1999; Yu et al., 1999; Yu and Mattson, 1999) also suggest an important neuroprotective role for the protein chaperone system. In particular, the latter studies showed that induction of HSP-70 and GRP-78 (an endoplasmic reticulum stress protein) is correlated with neuronal resistance to excitotoxic, metabolic and oxidative insults. It was further demonstrated that GRP-78 expression is necessary for increased resistance to such insults, and, specifically, is able to protect against apoptosis (Yu et al., 1999).

252

W.A. Pedersen, 1. Kruman and M.P. Mattson

A consequence of increased oxidative stress and disrupted calcium homeostasis potentially involved in the pathogenesis of neurodegenerative disorders is induction of prostate apoptosis response-4 (Par-4) transcription and/or translation. The par-4 gene was isolated from a rat prostatic cancer cell line by differential hybridization on a cDNA library prepared from the cells after treatment with ionomycin to induce apoptosis (see Rangnekar, 1988 for review). The 38 kDa Par-4 protein belongs to the family of immediate-early gene products, which include c-Myc, c-Fos, c-Jun, Nur77, and EGR-1. Unlike these genes, which are induced by apoptosis, growth arrest, or growth stimulation, par-4 expression is induced exclusively by apoptosis. The C-terminal portion of the Par-4 protein contains a death domain homologous to that of Fas and TRADD, and may therefore initiate a cascade of events analogous to that of other death domain-containing proteins. Within the death domain of Par-4 is a leucine zipper domain that may mediate protein-protein interactions (Diaz-Meco et al., 1996). Recently, we reported increased Par-4 protein and mRNA levels in the hippocampus of Alzheimer's disease patients relative to controls (Guo et al., 1998). We showed that over-expression of par-4 in PC12 cells increases their vulnerability to apoptosis induced by amyloid 8-peptide or staurosporine (Guo et al., 1998), both treatments of which increase oxidative stress. In contrast, over-expression of Par-4 lacking the leucine zipper domain did not enhance the induction of apoptosis by either treatment, suggesting a necessary role for protein-protein interactions in the pro-apoptotic effect of Par-4. These results implicate Par-4 in the neurodegenerative process in Alzheimer's disease. Interestingly, we have found that the levels of Par-4 protein can be regulated by translational mechanisms in synaptic compartments, and can act therein to induce caspase activation and mitochondrial dysfunction (Duan et al., 1999b). The ability of agents which increase oxidative stress and cause calcium influx to up-regulated Par-4 suggests that this protein may be a mediator of motor neuron degeneration in ALS. Indeed, we observed higher Par-4 levels in the lumbar spinal cords of ALS patients and of mice expressing mutant Cu/Zn-SOD relative to controls (W.A. Pedersen and M.P. Mattson, unpublished data). In NSC-19 cells, pre-treatment with a Par-4 antisense oligodeoxynucleotide prevents mitochondrial dysfunction caused by exposure to staurosporine, FeSO 4, or HNE (Figure 4), which indicates that Par-4 acts upstream of mitochondrial alterations. Moreover, Par-4 antisense pre-treatment can prevent the induction of apoptosis in NSC-19 cells by these agents and trophic factor withdrawal (Figure 4). These results implicate Par-4 in the pathogenesis of motor neuron degeneration in ALS as outlined in Figure 5. The interrelationships between cell death pathways involving oxidative stress and excitotoxicity may be generally applicable to neurodegenerative diseases (see Coyle and Puttfarcken, 1993; Mattson, 1998 for review). This may form the basis for an efficient therapeutic strategy for the treatment of neurodegenerative diseases, because an intervention that targets one disorder may also be useful against another. Although oxidative stress and excitotoxicity can result in apoptosis, necrosis, or both, they are unlikely to be the only forms of cell death that occur in neurons (see Clarke, 1999 for review). Determining the extent to which apoptosis contributes to the overall cell death in a neurodegenerative disease is extremely challenging. In particular, it is difficult to determine the influence of environmental and genetic modifying factors on the mode

253

Apoptosis in Motor Neuron Degenerative Diseases

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(A) Cells were pre-treated for 2 h with 20 ,uM Par-4 antisense DNA (AS) or 20 ,uM Par-4 nonsense DNA (NS) and were then exposed to 0.5% dimethylsulfoxide (Control), 1 uM staurosporine (STS), 1 mM FeSO4, or 10 uM HNE. The extent of MTT reduction, a measure of mitochondrial viability, was determined 8 h later. Values are the means and standarrd errors of determinations made in 4 cultures; *p < 0.01 versus corresponding value in cells pre-treated with Par-4 antisense DNA (ANOVA with Scheffe's post-hoc test). (B) Cells were pre-treated for 2 h with 20 ,uM Par-4 antisense DNA (AS) or with 20 ,uM Par-4 nonsense DNA (NS) and were then exposed to 0.5% dimethylsulfoxide (Control), 1 uM staurosporine (STS), 1 mM FeSO4, or 10/tM HNE, or were subjected to trophic factor withdrawal. The percentage of cells with apoptotic nuclei, as determined by Hoechst staining, were quantified 24 h later. Values are the means and standard errors of determinations made in 4 cultures; *p < 0.01 versus corresponding value in cells pre-treated with Par-4 antisense DNA (ANOVA with Scheffe's post-hoc test).

of cell death in a given neurodegenerative disease. No significant environmental risk factors for ALS have been identified in Western nations (Chancellor and Warlow, 1992). The possibility that environmental toxins play a role in ALS is suggested by the studies

254

W.A. Pedersen, 1. Kruman and M.P. Mattson

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of natives of a small group of islands in Guam, where ingestion of excitotoxins present in the cycad seed results in the so-called "ALS-Parkinson-dementia complex" (see Garruto, 1991 for review). There is also little known regarding genetic modifying factors in ALS. One possibility is that apolipoprotein E impacts on the course of disease. There are three alleles of apoE (e2, e3, e4), and the risk of developing AD is increased with each dose of E4 (see Roses, 1996 for review). Although less well established than in AD, evidence has been provided that apoE genotype influences the duration of ALS; patients carrying one e4 allele are reported to have earlier onset of disease versus patients carrying no e4 alleles (Moulard et al., 1996). The underlying mechanism for this observation may involve the differential anti-oxidant effects of apoE isoforms, which follows the order apoE2 > apoE3 > apoE4 (Miyata and Smith, 1996). The differences between the amino acid sequences of apoE isoforms are that apoE2 has cysteine residues at positions 112 and 158, apoE3 has a cysteine residue at position 112 and an arginine residue at position 158, and apoE4 has arginine residues at both of these positions. Given that HNE primarily modifies proteins at cysteine residues, we hypothesized that there would be differential protective effects of apoE isoforms against cell death caused by HNE. In primary motor neuron cultures, apoE2 prevented HNE-induced apoptosis, apoE3 was only partially effective, and apoE4 was completely

255

Apoptosis in Motor Neuron Degenerative Diseases

ineffective (Figure 6). These results suggest that genetic modifying factors influence the apoptotic process in ALS. However, to what extent this occurs in ALS, if at all, remains to be determined. In summary, there is sufficient evidence to suggest that interrupting apoptotic pathways may be an effective approach for the treatment of ALS and other motor neuron degenerative diseases.

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Mixed cultures of spinal cord motor neurons were established from day 12-14 embryos of B6/SJL mice. Cultures were treated in serum-free medium with HNE (1 IxM), apoE2 (20 nM), apoE3 (20 nM), apoE4 (20 nM), or combinations of HNE and each isoform. The cells were stained with Hoechst dye 24 h later, and the numbers of apoptotic nuclei determined. Values are means and standard deviations from determinations made in three cultures per treatment group. By one-way ANOVA and Scheffe's post-hoc test, there were statistically significant differences between: control and HNE, control and apoE3+HNE, control and apoE4+HNE, apoE2+HNE and apoE3+HNE, apoE2+HNE and apoE4+HNE. The differences between control and each isoform alone, control and apoE2+HNE, and HNE and apoE4+HNE were not statistically significant.

References Abe, K., Aoki, M., Ikeda, M., Watanabe, M., Hirai, S. & Itoyama, Y. (1996). Clinical characteristics of familial amyotrophic lateral sclerosis with Cu/Zn superoxide dismutase gene mutations. J. Neurol. Sci. 136, 108-116. Abe-Dohmae, S., Harada, N., Yamada, K. & Tanaka, R. (1993). bcl-2 gene is highly expressed during neurogenesis in the central nervous system. Biochem. Biophys. Res. Comm. 191, 915 -921. Aguirre, T., Van Den Bosch, L., Goetschalckx, K., Tilkin, P., Mathijs, G., Cassiman, J.J. 8¢. Robberecht, W. (1998). Increased sensitivity of fibroblasts from amyotrophic lateral sclerosis patients to oxidative stress. Ann. Neurol. 43,452-457. Ait-lkhlef, A., Murawsky, M., Blondet, B., Hantaz-Ambroise, D., Martinou, J.C. & Rieger, F. (1995).

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