Programmed cell death: Implications for neuropsychiatric disorders

Programmed Cell Death: Implications for Neuropsychiatric Disorders Russell L. Margolis, De-Maw Chuang, and Robert M. Post n

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Programmed cell death, sometimes referred to as apoptosis, occurs through an active process requiring new gone transcription, in contrast to the passive cell death produced by metabolic toxins. Programmed cell death is an essential part of normal development, particularly in the nervous system, Spatial, temporal, or quantitative errors in the stimuli that initiate programmed cell death, or errors within the programmed cell death pathway itse~ can result in an abnormal number of neurons and pathological neural development. Excesses and deficits in neuronal numbers have now been observed not only in typical neurodegenerative disorders such as Aizheimer's and Huntington's diseases, but also in several neurodevelopmental disorders, including schizophrenia and autism. Recent investigations into the mechanisms of ceU death during C. elegans neurodevelopment thymocyte negative selection, and wittulrawal of sympathetic ganglion cells trophic support provides intriguing clues to the etiology and pathophysioiogy of these neuropsychiatric disorders.

Key Words: Apoptosis, neuropsychiatry, cell death, neurodevelopment, neurodegeneration, molecular biology

Introduction Neuropsychiatric diseases can be classified as either neurodegenerative or neurodevelopmental. In the neurodegenerative category, which includes disorders such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and human immunodeficiency virus (HIV) dementia, neuronal death is a defining pathological feature. In the neurodevelopmental group of neuropsychiatric disorders,

From the Biological Psychiatry Branch, National Imiitete of Mental Health, Beth. esda, MD, Address reprint n~que.ststo Dr, Russell L. Mergolis,618 Ross Resea~h Building, ? 20 Rudand Avenue, Baltimo~ MD 2 i 205-2196, Dr. Margolis is now with the Delxmment of Ps~hiau'y and Behavioral Sciences. The Johns Hopkins UniveTsitySchool of Medicine,Baltimore MD. S ~ by a PBAT fellowship from the National Instilute of General Medical Sciences. Received April 5,1993: revised December 7, 1993. © 1994 Society of Biological Psychiatry

including autism, fragile X syndrome, various chromosomal abnormalities, schizophrenia, and potentially some forms of bipolar affective disorder, abnormal neuronal loss is now increasingly recognized as an important pathological feature. For instance, in the brains of schizophrenic patients, neuronal deficits have been observed in multiple areas of the cerebral cortex (Benes et al 1986, 1991; Falkai et al 1988; Jacob and Beckmann 1986), several regions of the hippocampus (Falkai and Bogerts 1986; Jeste and Lohr 1989), and in the mediodorsal thalamic nuclei and the nucleus accumbens (Pakkenberg 1990). Abnormal neuronal distribution, recently found in the dorsolateral prefrontal cortex and lateral and medial temporal lobes, may also stem from abnormal neuronal death (Akbarian et al 1993a, 1993b; Bloom 1993). Abnormal numbers of neurons have also been found in selected limbic regions and the cerebella (Bauman 1991) of autistic individuals. Even in bipolar affective disorder, preliminary data has demonstrated marked neuronal disorganization in the superficial layers of the entorhinal 0(X)6-3223/941507.00

ProgrammedCell Death

region and a deficit in neuronal number in a small part of the rostro-ventral insula (Beckmann and Jakob 1991). The evidence that abnormalities of neuronal death are associated with neuropsychiatric disease is not surprising, because neuronal death plays a fundamental role in the normal development of the nervous system. As many as 50% of all neurons that form in the developing nervous system die before reaching full maturity (Oppenheim 1991); the rate of neuronal loss in the primate dorsal lateral geniculate nucleus alone reaches 800 per hr during certain stages of embryonic development (Williams and Rakic 1988). Programmed cell death, the cellular death consequent to the activation of pathways intrinsic to the cell, accounts for much of this extraordinary neural pruning. With the rapidly accumulating evidence that cell death is a normal, indeed essential, process in the life of an organism, it is also increasingly apparent that abnormal programmed cell death---cell death at an inappropriate place or time, or in excess or insufficient quantity--is a potential etiologic or mediating event in central nervous system (CNS) pathology and pathophysiology. To develop this theme, we first define and describe programmed cell death, contrasting it with other forms of cell death. Next, we describe in some detail programmed cell death in three different systems, each of which provides a different sense of the initiation, biochemistry, and functional role of programmed cell death. Finally, we review several approaches useful to further understanding the role of programmed cell death in neuropsychiatric disorders.

Programmed Cell Death: Defining Characteristics The term "programmed cell death" (Lockshin and Zakeri 1991) originally defined an ontogenetic process--the controlled, regulated, and physiological cell death occurring as a normal part of development. The term "programmed" therefore did not imply either a specific morphologic pattern of death or a mechanism of action, though a genetic basis for the program was usually assumed. More recently, the term has been used to describe any cell death dependent on the synthesis of new gene products, and it is in this sense that we use the term. One form of programmed cell death, "apoptosis," was defined in 1972 (Kerr et al 1972). Derived from classical Greek, the term means "falling off," as in leaves from a tree or petals from a flower. Though often used synonymously with programmed cell death, apoptosis is best considered a subtype of programmed cell death (Schwartz et ~ 1993), distinguished from other forms of cell death on a morphological basis (Arends and Wyllie 1991) and typically, but not invariably (Tomei et al 1993), accompanied by a particular set of molecular events (Arends et ai 1990; Arends and Wyllie 1991; Duke et al 1983; Kerr and Harmon 1991;

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Table I. A Comparisonof Apoptosisand Necrosis Features Morphology

Apoptosis

Necrosis

Macromolecule synthesis DNA cleavage

Decreasedvolume, Increasedvolume. chromatincondensation, earlymembrane organellesintact rapture,damaged organeiles Typicallyessential; Unnecessary;rapidly increased early shutdown Intemucleosomal pattern Nodetectablepattern

Calcium

Moderate influx

Massive influx

Mechanism

Cascade of genetically controlled events Normal development, hormones, loss of trophic factors

Loss of water and electrolyte balance Hypoxia, hypothermia, complement attack, toxins

Phagocytosis

Inflammatory response

Typical causes

Immune

response

Waring 1990; Wyllie 1980). Necrosis, another morphological term, has been used to signify a type of cell death opposite to apoptosis (Fawthrop et a11991). Whereas apoptosis is the outcome of a cascade of modulated events, necrosis occurs when a toxin directly blocks cellular functions necessary for survival (Table l). Cell death, however, does not fall into the neat dichotomy of apoptosis versus necrosis, hence our broader use of the term "programmed" cell death. Clarke (1990) suggests that three different morphological forms of cell death occur in the developing nervous system. Type I is similar to apoptosis as morphologically defined in Table I, but type II (autophagic cell death) is characterized by numerous autophagic vacuoles, and type Ill (cytoplasmic cell death) features dilation of the rough endoplasmic reticulum, nuclear membrane, and golgi apparatus. Server and Mobley (1991) reviewed the literature using these criteria, and concluded that (1) controlled (what we refer to as programmed) neuronal death may take several morphological forms; (2) although these different morphologies may result from different genetic programs, the number of such programs is limited; (3) the mechanisms of cell death appear to be conserved among widely divergent species; and (4) genetic death programs normally operant during development may also function during aging or in disease states. This functional approach to programmed cell death allows us to compare ontogenetic, plastic, and pathologic processes by examing how each of these phenomena may depend on differential activation of related pathways. M o d e l s of P r o g r a m m e d Cell D e a t h

Intrinsic Initiation of Programmed Cell Death In the nematode C. elegans, cell division and cell death can be observed within the living animal, and even within larvae

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Figure I. Oenetic control ofcell death in C. elegans.ModifiedfromEIliset al(1991b),ces=cell death specification;egl = egg-laying abnormal, cad = cell death abnormal, nuc = nuclease.

still residing in the hermaphroditic mother. The hermaphrodite is composed of 1090 somatic cells, 131 of which die; some 20% of all generated neural cells eventually die (Ellis and Horvitz 1986). Figure i, modified from Ellis et al (1991b), diagramatically displays the sequence of events in programmed cell death in C ele&ans. The first part of the process, the specification of which cells die, in most cases appears to be genetically prepro. 8raramed, and independent of external cues from the envi. ronment or neighboring cells (Horvitz 1988; Robertson and Thomson 1982; $ulston and Horvitz 1977a, 1977b). Several genes have been identified that control the fate of specific cells. A mutation of the egg-laying abnormal- 1 re&i-l) gene leads to the death of two motor neurons involved in egg-laying in the hermaphrodite but does not affect other aspects of sexual differentiation (Ellis and Horvitz 1986; Trent et al 1983). Other mutations, such as of the hermaphrodite.l (her.l) gene, result in the death of these neurons but also have a variety ofother effects on sexual development (Desal et al 1988; Trent et al 1983). Alternatively, a dominant (gain-of-function) mutation of the cell death specification-i (ces-l) gene prevents the death of four specific neurons in the pharynx; ces.2 controls the fate of two of these neurons by inhibiting ces.l (Ellis and Horvitz 1991). These findings demonstrate that a single gene can selectively control the survival of a specific cell or a group of cells. The next pan of the sequence of genetically-regulated cell death in C. elegans is the actual killing of cells. Muta-

tions in either the cell death abnormal-3 (cad.3) or cad.4 gene block the death of nearly every cell that would otherwise die (Ellis and Horvitz 1986; 1991), and evidence from mosaic mutants indicates that these genes code for proteins that act within cells fated to die, rather than in neighboring cells (Driscoll 1992; Yuan and Horvitz 1990), A dominant (gain-of-function) mutation of the cad-9 gene, which normally inhibits cad-3 and cad.4, blocks all cell death (Hengartner et al 1992), whereas an inactivating mutation appears to kill all cells, hence killing the animal (Ellis et al 1991b). Mutations of either cad-3 and cad.4 prevent this lethal outcome, further documenting the modulatory role of cad-9. Both the ced-$ and cad.4 genes have been cloned (Driscoll 1992; Ellis et el 1991b). Analysis of primary structure suggests that cad.4 encodes a novel protein with two regions that may relate to calcium binding. The cad.3 protein, which is similar to mammalian interleukinlb-13converting enzyme, may act as a cysteine pretense (Miura et el 1993; Yuan et ill 1993). The specificity of these genes for cell death is suggested by the observation that cells that ordinarily undergo programmed cell death but are saved by engineered defects in death-inducing genes develop and function similarly to their normally surviving sister cells. For instance, mutations of the cad-3 or cad.4 genes allow certain cells that ordinarily die to survive; these saved or "undead" cells each differentiate to adult neurons (White et al 1991) that apparently remain functional (Avery and Horvitz 1987). The extra cells

Programmed Cell Death

cause subtle defects in growth and chemotaxis without grossly disturbing behavior (Driscoll 1992; Ellis et al 1991b). The final two stages of genetically regulated cell death in C. elegans involve phagocytosis and deoxyribonucleic acid (DNA) degradation. Engulfment appears to be mediated by two groups of genes that act in distinct, parallel, yet partially redundant processes to dictate either the recognition of dead cells or the actual mechanics of phagocytosis of cell corpses (Ellis et al 1991a; Hedgecock et al 1983). A final gene, nuclease- 1 (nuc-l), controls a calcium-independent endonuclease that degrades the DNA of dead cells after they have been engulfed (Hedgecock et al 1983; Hevelone and Hartman 1988). A markedly different form of cell death, which does not require ced-3 and ced.4, has also been identified in C ele. &ans (Chalfie and Wolinsky 1990; Driscoll and Chalfie 1991). The morphology of this type of cell death resembles necrosis: cells with the dominant mutations degeneration (deg-l) or mechanosensory abnormality (mec.4) swell, deteriorate, and form large vacuoles. The DNA sequence of these genes suggests that they each form some type of transmembrane receptor. Strikingly, the deg-I mutant causes death only after cells have begun to function at a mature stage of development. An even more divergent type of death, again involving dominant mutations, may stem from an abnormal activation of phagocytosing cells (Ellis et al 1991b). The genetic analysis of C elegans reveals a number of critical features about programmed cell death. First, genetic mutations may lead to abnormal cell survival or death either for specific cells or for a variety of different cells. Second, at least this one model of cell death can be understood as a multistep cascade of events: determination, triggering, killing, engulfment and degradation. Third, mutations disrupting the function of either "suicide" genes (which potentially encode "killer" proteins) or inhibitory genes, or overexpression of genes apparently not normally involved in the death pathway, lead to dysregulation of the pathway. Fourth, morphologically divergent forms of cell death may nonetheless all be under genetic control. Fifth, abnormal neuronal death or survival may alter, albeit subtlely, the behavior of an organism.

Activation of Programmed Cell Death by an ExtraceUular Signal Selection of T lymphocytes, important regulators of the immune system, occurs in the thymus. Those cells that bind to self-antigens are destroyed (Blackman et al 1990; Golstein et al 1991; McConkey and Orrenius 1991), whereas cells recognizing foreign antigens are positively selected and leave the thymus to serve as immunologic defenders of

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the host (Golstein et al 1991; McDuffie et al 1986). To prevent host attack upon itself---autoimmune disease--T cells that recognize host tissue antigens must be eliminated (negatively selected). This occurs when, on binding to the T-cell receptor (TCR) complex, an antigen initiates a complex set of events ultimately leading to the demise of the cell (Golstein et al 1991; McConkey and Orrenius 1991). This process is the prototypic example of the form of programmed cell death often known as apoptosis. Remarkably, the TCR also mediates positive selection of T cells; the molecular mechanisms by which one receptor complex may have such diametrically opposite effects on the fate of a cell provide intriguing leads about the regulation of cell life and death. In work dating back over 50 years (Seyle 1936), cell death of T lymphocytes has been investigated through examination of the glucocorticoid-induced death of immature T cells (thymocytes). Glucocorticoids increase thymocyte cytosolic calcium levels (McConkey et al 1989c); the rise is mediated by glucocorticoid receptors (Compton and Cidlowski 1992; Muller and Renkawitz 1991; Wyllie et al 1984) and requires new macromolecular synthesis. The rise in calcium is essential for cell death, and the process appears dependent on the influx of external calcium, perhaps entering through newly synthesized calcium pores (McConkey et al 1989c). Calcium appears to mediate cell death, at least in part, by binding to calmodulin, a protein activated by calcium and responsible for the transduction of at least some calcium-mediated phenomena (McConkey et al 1989c; Means et al 1991). Glucocorticoids increase the amount of calmodulin messenger ribonucleic acid (mRNA) (Dowd et al 1991), suggesting a coordination in the levels of intracellular calcium and calmodulin during glucocorticoid-induced cell death. Although extracellular calcium entering via newly synthesized pores and acting through calmodulin is an essential component of thymocyte programmed cell death, too much calcium induces cell death with features of necrosis (Wyllie et al 1984). DNA fragmentation, a near-terminal event in thymocyte programmed cell death, is produced by endonuclease(s), nuclear enzymes that specifically attack DNA (Arends et al 1990). This process appears necessary for thymocyte programmed cell death (McConkey et al 1989b), and probably requires specific activation of a latent endonuclease (Schwartzman and Cidlowski 1993). Protein kinase A (PKA) and protein kinase C (PKC), enzymes that each regulate numerous cellular functions by phosphorylating other proteins, also regulate thymocyte programmed cell death (McConkey et al 1990). Activation of PKA leads to DNA cleavage independent of increases in calcium concentration (McConkey et al 1990), and to phosphorylation of the giucocorticoid receptor, thereby enhancing the impact of glucocorticoids (McConkey and Orrenius 1991). Activa-

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tion of PKC blocks calcium ionophore-stimulated or glucocorticoid-stimulated DNA fragmentation and subsequent cell death (McConkey et al 1989a) and also inhibits PKAinduced endonuclease activity (McConkey et al 1990), an interesting example of "cross-talk" between these two major enzyme systems. This evidence has led to the demonstration that activation of PKC, with a concomitant rise in calcium concentration, induces proliferation, whereas calcium influx or PKC activation alone each triggers cell death (Cairns et ai 1993). This type of interaction may help explain how T cell receptor activation mediates both cell proliferation and cell death; the dominant process depending on differences in the quantitative, qualitative, and temporal activation of other receptors and intercellular messengers. Several genes selectively expressed during thymocyte programmed cell death have ~'~w been identified. The pro. tein encoded by one of these, RP-2, likely migrates to a cellular membrane, whereas the product of another induced gene, RP-8, has a zinc finger domain and hence may function as a transcription factor (Owens et al ! 991). Expression of another transcription factor, the protooncogene c-myc, is required for activation-induced, but not glucoconicoid-induced, programmed cell death in a human T cell line (Shi et al 1992). Overexpressiea of the protooncogene bcl-2 has been linked to the inhibition of programmed cell death in a number of systems, including human interleukin-3 dependent lymphoid and myeloid cell lines, a rat neuroendocrine cell line (PCI2), and immature thymocytes, but not in the negative selection ofT cells with potentially adverse immunologic properties (Mah et al 1993; Oren 1992). The role of protooncogenes is therefore complicated and variable from one system to another. A schematic summary of some of the pathways known to regulate programmed cell death in the thymocyte is portrayed in Figure 2. This diagrammatic depiction vastly oversimplifies the mechanism of thymocyte apoptosis, excluding, for example, adenosine triphosphate (ATP) activation of the process (Zbeng et al 1991) and the interaction between calcium and PKC. it emphasizes the role of extracellular stimuli and some of the diverse pathways and regulatory mechanisms involved in this system, however, and leads to questions pertaining to programmed cell death in other systems. What stimuli promote and block programmed cell death? Do these stimuli include ubiquitous molecules such as ATP? Can the same stimulus differentially trigger both protective (or proliferative) and death pathways, and if so, what factors lead the cell to respond in one direction and not the other? The biochemistry of thymocyte programmed cell death suggests that the n s w e ~ ~,.~ these questions will require attention to quantitative and temporal aspects of the initiating stimuli, specific receptor subtypes, calcium concentration, regulation of protein phorphorylation, and control of gene synthesi-

R.L. Margoliset al

Programmed Cell Death Following Withdrawal of a Trophic Factor Recognition that neuronal survival may depend on factors extrinsic to the cells themselves began over 100 years ago with the work of Wilhelm Roux, and was elaborated in the pioneering work of Hamburger and Levi-Montalcini (Jacobson 1991). This work led to the isolation and purification of neuronal growth factor (NGF) (Angeletti and Bradshaw 1971; Bothwell and Shooter 1977; Edwards et al 1988; Greene et al 1971), and then to other related neurotrophic compounds, including brain-derived neurotrophic factor (BDNF), neurotrophin (NT.3), and most recently NT-4 (Maisonpierre et al 1990', Persson and Ibanez 1993). NGF and other neurotropins act by binding to specific high affinity receptors, now recognized as products of the trk (tyrosine kinase) protooncogene family (Persson and Ibanez 1993). After binding, the receptor.ligand complex is taken into the neuron by endocytosis and transported back to the cell soma (Johnson et al 1987; Palmatier et a11984), where it ultimately alters at least some aspects of gene transcription (Greene and Shooter 1980), The role of NGF during normal development has been demonstrated in vivo. Within hours after injection of antiserum against NOF into neonatal mammals, sympathetic ganglion cells cease to transmit information (Larrabee 1969), and degenerative changes in morphology appear (Levi-Montalcini and Booker 1960; Sabatini et al 1965). Within 2 days after antiserum injection many neurons are dead, and after 7 days most neurons have died; most glial cells survive for another few weeks. Withdrawal of NGF from sympathetic neurons cultured from superior cervical ganglia of neonatal rats also leads to programmed cell death, accompanied by the ladder pattern of DNA cleavage (Edwards et ai 1991) characteristic of many types of programmed cell death, lnhibitors of mRNA or protein synthesis prevent cell death after NGF withdrawal; the pyknotic morphology of some of the dying cells is similar to the morphology originally used to define apoptosis (Martin et al 1988). lntracellular calcium concentration mediates neuronal dependence on NGF and hence, cell survival. At low internal calcium levels, sympathetic neurons require NGF to block cell death. At intermediate calcium concentrations NGF is not required for cell survival, whereas high levels of calcium may have direct toxic effects (Johnson et al 1992; Koike et ai 1989). Various pharmacologic manipulations that increase intracellular cAMP concentration (Martin et al 1992) also block the death of NGF-deprived neurons, though apparently not through inhibition of protein kinase activity. Johnson and colleagues (Johnson et al 1989) have proposed a model in which trophic factors, released from target tissue, bind to specific neuronal receptors and are transported from neuronal processes into the cell body. Once in the cell body, the trophic factors initiate the synthesis of

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FRAGMENTATION & CELL DEATH Figure 2. A speculative model of thymocyte apoptosis. Steroidsenter the cell and bind to specific cytoplasmic receptors (I). The ligand receptor complex enters the nucleus and binds to DNA (2), leading to the induction of gene transcription and hence RNA synthesis (3). After appropriate processing, mRNA is transported out of the nucleus to the endoplasmicreticulum, where it is translated into proteins (4). These proteins, including a calcium pore (5), caimodulin (CAM)(6),and perhaps transcriptional factors such as RP-8, are transported to appropriate locations within the ceil. Calcium (Ca2÷)enters the cell and activates calmodulin, which in turn activates membrane-bound adenylate cyclase(7) to,produce cAMP.The cAMP stimulatesPKA (8), which through a seriesof intervening steps, and along with various enzymes dependent on Ca=*-Calmodulinfor stimulation (9), further regulates gene transcription (3). RNA is again synthesized (4) and transported out of the nucleus to serve as the template for the formation of additional proteins. A few of these proteins return to the nucleus and through mechanisms yet to be determined activate an endonuclease (10). The endonuclease then cleaves the DNA between nucleosomes, resulting in nucleotide fragments that are multiplesof 200 nucleotide base pairs in length (1 !). Interleukin-1 and antigens, through surface receptor-mediated mechanisms, activate PKC (12), which negatively regulates the PKA-mediated pathway of DNA fragmentation (! 3). proteins necessary for cell growth and/or survival. Preventing this synthesis, at any step of the pathway, leads to the synthesis of a different set of proteins, termed "thanatins," which eventually kill the cell. Some recent, albeit controversial (Martin et al 1992) evidence suggests that the cell death process can be regulated even after the steps of gene transcription and protein translation have been completed (Edwards et al 1991). Perhaps NGF withdrawal does not lead to the induction of new genes that promote death, but rather frees a basally expressed toxin from NGF-mediated inhibition. Blocking protein or mRNA synthesis after NGF withdrawal would therefore save cells by limiting the continued expression of such a factor, but only

before significant levels of the endogenous toxin have accumulated. Once sufficient concentrations are present, only reinstatement of posttranstription/posttranslation inhibition such as protein.phosphorylation will save the cell.

T h e L i n k b e t w e e n P r o g r a m m e d Cell D e a t h a n d Neuropsychiatric Disorder: Future Directions

Excitotoxicity and Free Radicals The mechanisms by which excitotoxins and free radicals damage cells, and the consequent implications for neuro-

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psychiatric disease, have been extensively reviewed (Choi 1990; Coyle et al 1990; Lohr 1991, Meldmm and Garthwaite 1990; Rosen et al 1993). Induction of cell death by these mechanisms and by programmed cell death is not mutually exclusive. Rather, excitotoxicity and free radical toxicity, in some systems, function as the upstream initiators of programmed cell death. For instance, cultured cerebellar granule cells undergo morphologically defined apoptosis (as determined through electron microscopy) after reaching maturation (Ishitani et al 1993). Both glutamate and carbamazepine accelerate this process, as determined by an increase in DNA fragmentation and cell death. The response to both agents can be blocked by N-methyl-Daspartic acid (NMDA) and inhibition of endonuclease activity (Chuang et al 1992, Gao and Chuang 1992; Gao et al 1993; Marini and Paul 1992). As additional examples, MPP* (l-methyl-4-phenylpyridinium) neurotoxicity, potentially mediated by free radicals, is accompanied by DNA fragmentation (Dipasquale et al 1991), and bci-2 inhibition of neuronal death has recently been directly linked to its ability to decrease free radicals (Hockenbery et al 1993; Kane et al 1993). At least one neuropsychiatric disorder, that related to HIV infection, has been postulated to stem from excitotoxic induction of programmed cell death (Ameisen and Capron 1991). The possibility that this combination plays a role in other diseases deserves careful study, beginning with further elucidation oftbe mechanisms through which programmed cell death is induced within neurons.

Neural Plasticity A second approach to determining the role of programmed neuronal death in pathological systems is to examine the impact of cell death on central nervous system function. A number of systems exist in which this phenomenon may be explored. For instance, sexual dimorphism in the song of the zebra finch is related to endocrinologicaily mediated cell death in specific brain nuclei (Bottjer and Johnson 1992; Gurney 1981, 1982; Nordeen et al 1987). Kindling, a phenomenon in which an initially subconvulsant stimulus, given repeatedly, eventually induces electrographic and behavioral seizures (Ooddard et al 1969; Post et al 1975; Post and Weiss 1988), is also closely paralleled by neuronal death, in this case in the hilar polymorphic region of the hippocampus (Sutula 1991). The induction of neuronal death by stress is a third pbenomenon in which the relationship between cell death and brain function can be examined. Stress itself (Kerr and Harmon 1991) and exogenous glucocorticoids at levels similar to that induced by major stressors (Sapolsky et al 1985) decrease neuronal number in the rat hippocampus, primarily affecting CAI and CA3 pyramidal cells. This loss leads to even more production of glucocorticoids by elimi-

R.L. Margoliset al

nating a portion of the negative feedback to the adrenal gland. Handling neonatal rat pups reduces adult glucocorticoid levels and diminishes both the neuronal dropout and spatial learning deficits seen in older rats (Meaney et al 1988). Granule cells oftbe hippocampal dentate gyms, in a manner paradoxically opposite to hippocampal pyramidal neurons, actually require adrenal steroids for survival (McEwen et al 1992; Sloviter et al 1989). Understanding the effect of cell death on common mechanisms of neural system regulation, such as inhibitory processes (Margolis 1991), in the context of biological processes such as sexual dimorphism, kindling, and stress, will shed light on the pathologic implications of cell death in human disease.

Molecular Neurobiology and Programmed Cell Death Programmed cell death, by definition, involves activation of intrinsic cellular pathways, and specific molecules expressed during cell death have been identified. The presence, or overexpression, of these same molecules in neuronal tissue from patients with neuropsychiatric disorders would suggest that programmed cell death was part of the pathophysiology of these disorders. A search for death-associated molecules in pathological samples is underway (Michel et al 1992). For instance, the TRPM~ (testosteronerepressed prostate message) gene, and similar or identical homologues (clusterin, SGP-2, pTBI6, pADHC9 and others) are expressed during the programmed death of various cells, including the postcastration death of rat ventral prostate epithelial cells, from which TRPM2 was originally cloned (Kyprianou et ai 1988; Kyprianou and Isaacs 1988). Elevated levels of TRPM2 homologues have been identified in post mortem brain tissue from patients with several neuropsychiatric disorders (Danik et al 1991; Duguidet al 1989; May et al 1990) and following electrolytic, neurotoxic, and viral infection of rodent brain (Duguid et al 1989; May et al 1990). The point is not that TRPM2 itself is necessarily a crucial component or even a marker for programmed cell death in human neuropsychiatric disease. Rather, searching pathological tissue for the presence of genes known to be selectively expressed in models of cell death is a powerful tool for the molecular dissection of neuropsychiatric disease. The recent discovery of the genetic etiology of several neuropsychiatric diseases (Ross et al 1993) allows investigators to employ the opposite strategy for understanding the molecular biology of these disorders: using model systems of cell death to determine the function of genes known to be relevant to disease. The cause of Huntington's disease (Huntington's Disease Collaborative Research Group 1993) is an expansion of a trinucleotide consisting of the bases cytosine, adenosine, and guanosine repeated in order, leading to a protein with an abnormally long string of glutamate units. Curiously, a similar type of trinucleotide repeat

Programmed Cell Death

expansion causes other neuropsychiatric conditions, including fragile X syndrome (Fu et al 1991; Knight et al 1993). How such expansions cause disease is not known, but at least in Huntington's disease, neither localization of IT-15 expression nor alteration in the quantity of mutated IT-15 expressed can account for the selective neuronal loss characteristic of this disorder (Liet a11993). Analysis of the expression and function of IT-15 within model systems of cell death will be critical in defining the pathophysiology of Huntington's disease, and the same approach will be useful in other neuropsychiatric diseases caused by a specific mutation.

Conclusions Cell death can be brought about by the activation of diverse intracellular pathways through otherwise nontoxic stimuli, such as glucocorticoids, or through intrinsic mechanisms, as in the genetically controlled neuronal death during develop. ment of C elegans. Though programmed cell death probably follows different pathways depending on the triggering event and the specific cell type, these pathways tend to have certain features in common: the role of calcium as an intermediary, modulation by various protein kinases, and DNA

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cleavage by an endonuclease. Normal development of the nervous system depends on programmed cell death, and hence the appropriate function of these pathways. Experimental models clearly demonstrate the pathological consequences of abnormal programmed cell death, whether secondary to abnormalities in these pathways or as a result of inappropriate activation or inhibition of otherwise normal pathways of cell death. Application of the concept of programmed cell death to neuropsychiatric disease leads to several ultimately answerable questions: In models of human neuronal death, does the process depend on the transcription of genes specific to the death process? If so, what are the functions of the protein products? What specific signals are required to initiate the synthesis of the unique genes? Is either a qualitative or quantitative abnormality of a death-specific protein found in pathological states? Is some aberration of the initiating signal present in pathological states? What is the physiologic effect of such mutations, and how might these effects be reversed? What role do genes with mutations known to cause human neuropsychiatric disorder play in pathways of cell death? The evidence that abnormal programmed cell death may play a role in neuropsychiatric disorders makes such questions well worth pursuing.

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