Recent advances on neuronal caspases in development and neurodegeneration

Neurochemistry International 35 (1999) 195±220

www.elsevier.com/locate/neuint

Invited review

Recent advances on neuronal caspases in development and neurodegeneration Neville Marks*, Martin J. Berg Nathan S. Kline Institute for Psychiatric Research, and New York University, Division of Neurochemistry, 140 Old Orangeburg Road, Orangeburg, NY 10962, USA Received 4 January 1999; accepted 10 February 1999

Abstract In view of a large and growing literature, this overview emphasizes recent advances in neuronal caspases and their role in cell death. To provide historical perspective, morphology and methods are surveyed with emphasis on early studies on interleukin converting enzyme (ICE) as a prototype for identifying zymogen subunits. The unexpected homology of ICE (caspase-1) to Caenorhabditis elegans death gene CED-3 provided early clues linking caspases to programmed cell death, and led later to discovery of bcl-2 proteins (CED-9 homologs) and `apoptosis associated factors' (Apafs). Availability of substrates, inhibitors, and cDNAs led to identi®cation of up to 16 caspases as a new superfamily of unique cysteine proteinases targeting Asp groups. Those acting as putative death e€ectors dismantle neurons by catabolism of proteins essential for survival. Caspases degrade amyloid precursor protein (APP), presenilins (PS1, PS2), tau, and huntingtin, raising questions on their role in neurodegeneration. Brain contains `inhibitors of apoptosis proteins' (IAPs) survivin and NAIP associated also with some neuronal disorders. Apoptotic stress in neurons initiates a chain of events leading to activation of distal caspases by pathways that remain to be fully mapped. Neuronal caspases play multiple roles for initiation and execution of cell death, for morphogenesis, and in nonmitotic neurons for homeostasis. Recent studies focus on cytochrome c as pivotal in mediating conversion of procaspase-9 as a major initiator for apoptosis. Identifying signaling pathways and related events paves the way to design useful therapeutic remedies to prevent neuronal loss in disease or aging. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Apoptosis; Programmed cell death; bcl-2 proteins; Inhibitor of apoptosis proteins (IAPs); Caspase inhibitors; Caspase substrates; CED-3; CED-4; CED-9; Apoptosis associated factors (Apafs)

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

2.

Morphology as a tool to identify and quantify neuronal death . . . . . . . . . . . . . . . . . . . . . . 196

Abbreviations: IAPs, inhibitory associated proteins include; NAIP, `neuronal apoptosis inhibitory protein' and survivin; IERG, immediate early response genes; ICAD, inhibitor of caspase associated DNase; PARP, poly(ADP-ribose) polymerase, a DNA repair enzyme; DD, death domain; DED, death e€ector domain; CARD, caspase associated recruitment domain; Apaf, apoptosis associated factor; PCD, programmed cell death. * Corresponding author. Tel.: +1-914-398-5543; fax: +1-914-3985531. E-mail address: [email protected] (N. Marks) 0197-0186/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 1 9 7 - 0 1 8 6 ( 9 9 ) 0 0 0 6 1 - 3

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3.

Neurodegeneration and caspases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199

4.

PCD and neurochemical measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

5.

Isolated neurons as models to investigate PCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201

6.

Discovery of interleukin converting enzyme (ICE, caspase-1) and its implications . . . . . . . . . 203 6.1. Death e€ector domains (DD, DED, CARD) and their role in zymogen conversion . . 204

7.

Implications arising from studies on null mice and transgenics 7.1. Nematode CED-3-genes and mammalian counterparts . 7.2. Apoptosis associated factors (Apafs) . . . . . . . . . . . . . . 7.3. CED-9/bcl-2 proteins and neuronal PCD . . . . . . . . . .

8.

Structural and regulatory proteins as substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

9.

Caspase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

10.

Concluding comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

1. Introduction Death of irreplaceable neurons adversely impacts brain function directing attention to the need to identify relevant pathways as an aid to the design of neuroprotective agents or strategies. Post-mitotic neurons are `privileged' since they lack signaling pathways identi®ed in immune cells for self-destruction via receptor-mediated events. Identifying such pathways provides a rational basis not only for design of novel therapies but also for probes to gain insight in processes essential for homeostasis in non-mitotic cells. Ultimately, cell survival depends on an orchestration of events causing death counterbalanced by those of cytoprotection (Fig. 1). Here we focus on neuronal caspases as putative cell death e€ectors in immature and mature neurons. Caspases are a relatively new category of 016 cysteine proteinases having unusual speci®city since they target essential cytoskeletal or regulatory proteins containing appropriate aspartyl motifs (Table 1). 2. Morphology as a tool to identify and quantify neuronal death Apoptosis, a Greek term representing `dropping of leaves from trees', was coined to cover an orderly rather than a chaotic removal of cells without harming neighbors. Morphological changes were codi®ed in a systematic manner ®rst by Kerr and colleagues in 1970s on the basis of studies with malignant tissues, or cultures using light and electron microscopy (Kerr et

al., 1995). Despite phenotypic di€erences between cells it was apparent some are subject to removal by an `active' process that leads to shrinkage of cell cytoplasm, condensation of nuclear chromatin, blebs on the plasma membrane, and appearance of intracellular inclusions or `apoptotic bodies'. These changes facilitate phagocytosis by processes that remain to be clari®ed but which could involve alterations in rates of vesicular fusion or tracking such as those mediated by rabaptin 5, recently shown also to be a substrate for caspases (Cosulich et al., 1997a). The di€erential sensitivity of neuronal subsets in vivo to physical insults such as experimental ischemia is a property exploited in recent studies on CA1 hippocampal neurons (Nitatori et al., 1995; Chen et al., 1998; Gervais et al., 1999). Apoptotic changes that facilitate phagocytosis and removal of cellular debris appear to fail in many diseases associated with deposition of abnormal deposits in brain such as amyloidogenesis. Standard methods of cell biology apply to monitor manner of cell death such as uptake of ¯uorescein acetate or trypan blue to identity viable cells, uptake of propidium iodide, DAPI, or chromatin stains, the binding of annexin, or release of MTT/LDH to identify cell damage (Kerr et al., 1995; Marks et al., 1998a,b; Kuida et al., 1998; Hakem et al., 1998). Such methods demonstrate a remarkable r50% turnover of proliferating neurons during maturation and synaptogenesis, probably for the purpose of removing excess neurons or ones with improper connections (Oppenheim, 1991; Pettman and Henderson, 1998). It is during these phases that deletion of caspase-3, -9, or Apaf-1 (see section 7.2 for de®nition) in null mice,

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Fig. 1. Schematic for proposed signaling pathways for activation of procaspases. Conversion of proximal or long prodomain zymogens initiates events leading to activation of distal e€ector forms. Events on the right-hand side are intended to illustrate counterbalancing mechanisms a€ecting survival initiated by neurotrophins or interactions with ion-channels. The top left-hand side illustrates a receptor-mediated pathway applicable to immune cells or proliferating neurons, and at the bottom left a cytochrome c-mediated pathway for conversion of procaspase-9 as a major initiator for activation of distal (e€ector) proteases. Translocation of cytochrome c may occur via pores formed by bcl-2 dimers in mitochondrial membranes (see section 7.3 and Adams and Cory, 1998). Activation of caspase-3 results in proteolysis of essential proteins required for cell integrity or for DNA degradation as illustrated for studies on cerebellar granule cells cultured in absence of survival factors to induce apoptosis (Ap). In this model, the psychoactive drug lithium (Li+) is potently neuroprotective as shown by diminution in DNA laddering and reduction in cytosolic caspase-3 with enhanced survival (Marks et al., 1998 and unpublished).

induce gross brain speci®c malformations probably because of unrestricted hyperplasia (see section 7). In other models, transcriptional events in¯uence neuronal maturation, as illustrated in recent studies on retinal cells and the role of caspase-2 mRNAs that yield alternatively spliced pro-and anti-apoptotic modulators (Kojima et al., 1998a, b). Maturation of brain is an outcome of an interplay between neurotrophins, often target derived, combined with nuclear components of cell progression in¯uencing G- and S-phase checkpoints; a full discussion of this is outside the scope of this review (Ross, 1996; Vaux and Korsmeyer, 1999).

Interestingly, E-14 embryonic motor neurons or proliferating cortical neurons express fas receptors and respond to fas antigen showing these have a high vulnerability to apoptotic signals. Such pathways are absent or downregulated in post-mitotic neurons probably in keeping with the need to preserve cells that are irreplaceable (Wood et al., 1993; Rensing-Ehl et al., 1996; Henderson et al., 1998; Cheema et al., 1999). Among provocative suggestions are that apoptosis represents an aberrant form of mitosis in response to mitogenic signals acting in the absence cell progression in mature neurons (Ross, 1996).

Alternative nomenclature

Recognition tetrapeptide(s)

b

Shown present in neuronal cytosols. Activated in neurons or transformed neuronal-like cells subjected to conditions inducing apoptosis. c Long prodomain forms with DED (death domain e€ector) (see Fig. 1). d Contains CARD (see Fig. 1).

a

Caspase subfamilies

Protein substrates

Table 1 Classi®cation of the family of proteases. Classi®cation of the caspase family into ICE and CED-3 phylogenic subgroups. Earlier trivial names and systematic nomenclature are included together with examples of preferred native or synthetic substrates (see Table 2 for expanded list of caspase-3 native substrates and Refs). Inhibitors prepared with -YVAD- acting on ICE, or -DEVD-derivatives acting on CED-3 subgroups can further characterize enzymes. Both groups are inhibited by the viral p35, or by CrmA acting at Ki of 10±1100 pM towards the ICE subgroup, compared to r1.6 mM for caspases -2, -3, and -7, and 0.3±17 nM for caspases -6, -8, -9, and -10. Tetrapeptide inhibitors, based on the preferred recognition sequences in the table (N-Ac-, C- CHO) yield Ki for caspase-1 of 56 pM with -WEHD-, caspase-3 of 230 pM with -DEVD-, caspase-6 of 5.6 nM with -IETD-, caspase-8 of 1 nM with -IETD- or -DEVD-, caspase-9 of 48 nM with -AEVD-. Z-VAD-fmk- is a broad spectrum, irreversible inhibitor of all caspases tested (Garcia-Calvo et al., 1998)

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3. Neurodegeneration and caspases There is interest in Alzheimer's Disease (AD) as a result of regional neuronal loss that may be mediated by apoptosis, although this remains controversial (Sugaya et al., 1997). One hypothesis considers amyloid peptides as a key etiological factor. Soluble or ®brillated forms of the fragment Ab40/42, or its 25-35 domain, are highly cytotoxic on presentation to cultures for most studies on neurons (Loo et al., 1993; Behl et al., 1994; Allen et al., 1995; Forloni et al., 1996; Cotman and Su, 1996; Suzuki, 1997; Neve and Robakis, 1998). Since apoptosis per se results in upregulation of distal caspases, it can be inferred that cytotoxicity is mediated via this pathway although direct proof is lacking. APP695 itself is a substrate for caspases and contains a vulnerable site at Asp664: mutation of this residue prevents proteolysis on transfection of this mutant in CPS-1 cells (Weidemann et al., 1999). The decrease in Ab cytotoxicity in PC12, or in hippocampal neurons by transfection or gene transfer of the viral caspase inhibitor, CrmA, cytokine response modi®er A, (Ivins et al., 1999), or following exposure to synthetic peptide inhibitors (Jordan et al., 1997) reinforces a role for caspases. Despite cytotoxicity in vitro, analyses of post-mortem AD temporal cortex rich in amyloid neuritic plaques do not show signi®cant caspase activity (Kitamura et al., 1998). Analyses of post-mortem AD temporal cortex, rich in amyloid neuritic plaques, do not show alterations in caspases (Kitamura et al., 1998). There is a report for increase in caspase-3 staining in dying pyramidal neurons of the hippocampal CA3 region probably re¯ecting regional di€erences (Gervais et al., 1999). It cannot be ruled out that Ab toxicity initiate alternative signaling events leading to cell loss including changes in an ER-associated binding protein (ERAB) leading to activation of alcohol dehydrogenase, the generation of reactive aldehydes and oxidative stress, thus indicating a need for further studies on mechanisms. Of particular interest is the over-expression of amyloid precursor protein (hAPP695) in PC-12 cells that induces dUTP nick-end labeling (TUNEL) and DNA fragmentation implicating apoptotic-like mechanisms (Zhao et al., 1997; Nishimura et al., 1998). Presenilin (PS-1), a gene associated with familial AD (FAD), also on transfection in PC12 cells promotes Ab cytotoxicity or cell loss following NGF-deprivation (Wolozin et al., 1996). However this e€ect is cell-type and stimulus-speci®c and cannot be demonstrated on transfection in embryonic cortical neurons exposed to etoposide or staurosporine (Bursztan et al., 1998). A detracting feature regarding the `amyloidogenic hypothesis' is the unexplained lack of neuronal loss or intracellular tangles, and behavioral de®cits in mice

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overexpressing APPs with a single-missense mutation. This is despite enhanced plaque formation within 4±9 months depending on the construct (Hsiao et al., 1996; Du€ et al., 1996). However a recent study using the double missense APP770 (K670N, M671L) transgenic shows a modest 10±15% neuronal loss in the CA1 hippocampal layer but again without neuro®brillary tangles (NFTs) (Calhoun et al., 1998). When crossed with one expressing hPS-1 (M146L) production of Ab42/43 plaques increases but without formation of NFTs (Holcomb et al., 1998). While increase in APP turnover during apoptosis infers a role for caspases (LeBlanc, 1995; Zhang et al., 1997; Galli et al., 1998) these cannot act as `secretases' in the manner described for TACE, a metalloendopeptidase (TNFa-associated converting enzyme) acting as an a-secretase (Buxbaum et al., 1998). Wild type or FAD mutations of APPs and presenilins are substrates for caspases implying a role in formation of intermediates for subsequent secretase processing. Secretases a, b, and g in brain still remain to be characterized and there are questions whether these act directly on the haloprotein or on intermediates (Loetscher et al., 1997; Kim et al., 1997a; Barnes et al., 1998; Gervais et al., 1999; Grunberg et al., 1998). That caspases increase during neuronal apoptosis in vitro may be signi®cant in terms of APP turnover. In recent studies on AD brain no discernable di€erences in the patterns of PS-1 or PS-2 metabolites were found compared to controls. This implies only intact presenilins act co-operatively on APP turnover in situ, or that presenilin breakdown is a late event and not a precipitating factor for pathology (Paul Mathews, Nathan Kline Institute, personal communication). FAD-PS-1 with a deletion of exon 9 containing a susceptible caspase cleavage site yields a mutant incapable of preventing the phenotype for the sel-12 mutant C. elegans (Steiner et al., 1999). However, other mutants lacking caspase-sites retain activity in notch signaling in a nematode model (Brockhaus et al., 1998). A number of studies in AD show increased Ab42 secretion in cells for FAD-PS-1 patients implying co-operative e€ects on APP turnover by mechanisms that are unexplained. Similar changes emerge on PS-1 transfection in cultured neuronal cells further illustrating a role in the production of `toxic' fragments (Borchelt et al., 1996; Du€ et al., 1996). Exogenous Ab42 is taken up by neuroblastoma cells with targeting as protease-resistant aggregates to lysosomal-like particulates based on uptake of acridine orange (Yang et al., 1999). At such sites the apparent lysomotrophic actions by amyloid aggregates may promote release of secondary e€ectors such as cathepsins that are capable of damaging organelles, or directly degrading APP or its fragments (Marks et al., 1994; Marks et al., 1995). While mechanisms for uptake of extracellular amyloid

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Table 2 Selected protein substrates for caspase-3 Component

Example(s)

Reference(s)

Cytoskeletal elements

Gelsolin Actin Fodrin Spectrin

Kothakota et al., 1997 (Kothakota et al., 1997) Janicke et al., 1998b Nath et al., 1996; Pike et al., 1998; Wang et al., 1998a Casciola-Rosen et al., 1996 Canu et al., 1998 Brancolini et al., 1997 Lazebnik et al., 1994; D'Amours et al., 1998 Liu et al., 1997; Enari et al., 1998; Sakahira et al., 1998 Wang et al., 1995 Walter et al., 1998 Casciola-Rosen et al., 1995; Emoto et al., 1995; Ghayur et al., 1996; Datta et al., 1997 Na-dependent kinase subunit Beyaert et al., 1997 Santoro et al., 1998 Wen et al., 1997; Gervais et al., 1998; Widmann et al., 1998a; Widmann et al., 1998b Cheng et al., 1997 Janicke et al., 1996 Erhardt et al., 1997 Wang et al., 1998a Loetscher et al., 1997 Barnes et al., 1998; Gervais et al., 1999 Goldberg et al., 1996; Wellington et al., 1998; Ellerby et al., 1999 Zhang et al., 1998c

DNA repair or degrading enzymes

Ribonucleoproteins (U1 70 kDa) Tau Catenin PARP ICAD/DFF45

Transcription factors Kinases, phosphatases, and regulatory proteins

SREBP p21-Activated kinase protein kinase C isoforms

Pka, PITSLRE Phosphatase2A RAsGAP,raf1, Akt-1,Cb1 and Cbl-b, MEKK1, focal adhesion kinase

Protease inhibitor(s) Neuropathology and in¯ammation

Bcl-2 Rb MDM2 Calpastatin Presenilin mutants APPs Huntingtin, androgen receptors Atrophin-1, ataxin-3 Pro-IL-16

peptides are unknown, the formation of stable Ab/ ApoE complexes provide one potential mode of entry via lipoprotein receptor proteins (LRP) that may account for detection of immunopostive complexes in AD brain, or in extracts of clathrin coated vesicles involved in tracking (Berg et al., 1997). Arguably, other senile plaque components including proteoglycans, cathepsins, protease inhibitors, complement, apolipoproteins, glycans (RAGE), and divalent metals potentially are also cytotoxic and initiate changes in caspases (Marks and Berg, 1997). Tau, a caspase substrate (Canu et al., 1998), is a component of intracellular PHF-tangles, and exists as a autosomal-dominant form in `Frontal Lobe Dementia' linked to chromosome 17 (FTDP-17) and neuronal atrophy in patients (Hutton et al., 1998; Spillantini and Goedert, 1998). Proteins with glutamine repeats linked to CAG-extension disorders and neuronal loss contain sites recognized by caspases (see section 8 and Table 2). A role for caspases in such diseases is suggested

from studies showing transfection of the huntingtin gene in striatal neurons induces cell loss by a mechanism blocked by the caspase inhibitor Ac-DEVD-CHO, or on cotransfection with an anti-apoptotic bcl-xL (see section 7.2 and Saudou et al., 1998). Schizophrenia, another leading form of dementia is not associated with neuronal loss in the prefrontal cortex associated with alterations in behavior, although losses are reported in thalamus using an unbiased stereological technique, but the signi®cance of this in disease is unclear (Pakkenberg, 1990). There is speculation, however, that aberrant PCD in the second trimester during active phases of brain remodeling constitutes a risk factor for psychosis later in life (Bunney et al., 1995). Necrosis or `passive cell death' leads to cell swelling, loss of cell contents via leaky membranes, and random breakdown of DNA but mechanisms accounting for these changes are not known (Kerr et al., 1995). Necrotic damage arises on brief exposure of neurons

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to a number of EAAs or agents altering Ca2+¯ux (Oberto et al., 1996; Armstrong et al., 1997; Du et al., 1997a,b; Marks et al., 1998a). `Necrosis' and `apoptosis' are operational terms covering gross changes in cell ultrastructure and do not cover other forms of cell damage that might represent a continuum between the two extremes before a commitment to a de®ned pathway. This indicates scope to re®ne methods further and to supply acceptable criteria for damage elicited during ischemia or other forms of trauma. Morphology may not be sucient to de®ne mode of cell death leading to interest in use of biochemical markers. 4. PCD and neurochemical measurements Two biochemical or neurochemical markers for identifying apoptosis include: (1) nuclease fragmentation of DNA; and (2) activation of caspases. Damage to DNA exposes 3'phosphate end-groups enabling detection of double stranded breaks using TUNEL and similar stains, single-stranded breaks using the Klenow assay, or evidence of chromatin condensation using bisbenzamide. Oligonucleosomes on extractions form ladders of high and low Mr products on gels consisting of multiples of 0200 nucleotides (Wyllie et al., 1984) as illustrated in Fig. 1 for apoptosis in cerebellar neurons and neuroprotective e€ects of lithium. TUNEL detects 3 '-phosphate groups but underestimates DNA damage as a result of action by tissue phosphatases, and inability to detect 5 '-end-groups resulting from cleavage by alternative nucleases (BenSasson et al., 1995). DNA laddering occurs in neurons but not for cells that lack a functional caspase-3 gene. Examples include human breast carcinoma MC7, mutants lacking DFF45/ICAD, or in mutant FM3A cells (Janicke et al., 1998a; Yamauchi et al., 1998; Zhang et al., 1998b). Despite interest in DNA integrity as an biochemical index for apoptosis, the analyses of post-mortem human AD brain do not show any consistent change in the patterns for TUNEL in AD, or do not correlate well with incidence of cell loss or plaques: this has raised questions on using such criteria to assess neuronal damage in AD (Su et al., 1994; Sugaya et al., 1997; Lucassen et al., 1997, Li et al., 1997c). Caspases provide a valuable new tool for monitoring cell damage and subsequent loss of many cell types including neurons. The leading candidates as e€ectors for neurons are caspases-2 and/or -3 but further progress depends on methods used for assay (Stefanis et al., 1996; Lynch et al., 1997). Transient changes in mRNAs in cultured neurons suggest transcriptional events contribute to de novo synthesis supplementing the pool of pre-existing procaspases (Schulz et al., 1996; Eldadah et al., 1997; Ni et al., 1997). In CAI

Fig. 2. Comparison of caspase-3 activation in cytosols and viability of rat cerebellar neurons after withdrawal of serum and lowering K+ from 25 ( . . . ) to 5 mM ( ), compared to a slower rise on inclusion of 1 mM staurosporine in presence of 25 mM K+. Basal levels are low and do not markedly increase with time in both models. Restoration of survival factors (± ±) at 4 but not 13 h prevents cell loss (see text for Refs).

neurons, a delay in transcriptional events may account for their di€erential sensitivity to damage from experimental ischemia (Ni et al., 1998). Temporal studies on cultured neurons subject to apoptotic stress show that activation of caspase-3 is predictive of cell loss. Activation of caspases for execution is supported by rescue of NGF-deprived sympathetic ganglia on microinjection of the viral inhibitor CrmA, or use of peptide inhibitors (Gagliardini et al., 1994; Milligan et al., 1995). In experimental cerebral ischemia the conversion of procaspase-3 correlates with neuronal atrophy, or a-spectrin and APP breakdown as an index of caspase activity (see Table 1 and Namura et al., 1998; Chen et al., 1998; Pike et al., 1998; Gervais et al., 1999). Moreover the reduction in edema and other consequences of hypoxia by administration of peptide inhibitors (Hara et al., 1997), or injection in the hippocampus, point to a therapeutic approach for treatment of stroke-related injuries (Himi et al., 1998).

5. Isolated neurons as models to investigate PCD Primary neurons from embyronic or newborn hippocampus, cortex, and cerebellum are established models to investigate e€ector proteases. On culture, these acquire many physiological properties of mature neur-

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Fig. 3. Conversion of procaspases to release small and large subunits capable of reassembling to form an active tetramer to degrade proteins participating in cell function and neurodegeneration are indicated. Release of subunits occurs on cleavage of zymogen by initiator caspases or other proteases acting as convertases: the tetrapeptide recognition sequences containing the Asp motifs adjacent to the linker regions are shown as examples for caspases-1, 2, 3 and 6. Mechanisms for conversion following dimerization are depicted in Fig. 1 or discussed in the text. An asterisk indicates the active cysteinyl group of the consensus sequence QACXG contained in the large subunit.

ons when supplied with appropriate survival factors. PC12 or neuroblastoma cells in vitro di€erentiate to yield neural-like cells suitable for transfection and propagation despite retention of a tumor phenotype: di€erent conditions for culture and inducing apoptosis make comparison to primary neurons dicult. Cerebellar granule cells provide models for primary neurons since they di€erentiate within 7 days in vitro (DIV) in presence media containing high [K+]o and fetal calf serum (D'Mello et al., 1993). High [K+]o is thought to promote the interaction of survival factors in media with relevant ion-channels by pathways that prevent procaspase conversion (Atabay et al., 1996; Schulz et al., 1996; Yu et al., 1997; Yan and Barrett,

1998). Removal of serum and low [K+]o induces a rapid and large time-dependent increase in cytosolic caspase-3 assayed with Ac-DEVD-amc (Fig. 2) and veri®ed by use of the speci®c procaspase-3 IgG for detection of active p20 subunit (see Fig. 3 and Marks et al., 1998a,b). Staurosporine induces a protracted but smaller increase in enzyme by a pathway insensitive to protein synthesis inhibitors or lithium (Fig. 2 and Krohn et al., 1998). Temporal studies show increase in caspase-3 is not necessarily lethal provided the pool can be reversed within 4 h by restoration of serum and [K+]o. This may be the result of turnover or release of factors such as IAPs to account for reduced activity before irreversible damage (see Fig. 2 and Marks et

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al., 1998a,b). The time delay between procaspase-3 conversion and neuronal loss may re¯ect tracking and/or translocation of activated protease, or activation of latent cytosolic e€ectors such as CAD, caspase-activated DNase (section 8). There is evidence in NGF-deprived sympathetic ganglia for time-related loss of cells connected to translocation of mitochondrial cytochrome c (Apaf-2) to the cytoplasm (see section 7.2 and Deshmukh and Johnson, 1998). Studies of this nature supply information on whether there are `windows of opportunity' for rapid intervention by neuroprotective agents before irreversible damage has occurred (Marks et al., 1998a,b; Saito et al., 1998). The chief limitation using isolated neurons as models is extrapolation of ®ndings to neuronal loss in vivo. It can be anticipated that advances may come using cells isolated from transgenics or with knockouts to demonstrate the role of individual cell death components (see section 7 and Kuida et al., 1996, 1998; Hakem et al., 1998). Among agents inducing apoptosis are: speci®c or non-speci®c inhibitors of protein kinases, H2O2, or other oxidants, viral coat proteins, etoposide, ethanol, Ara C, MPTP, butyric acid, thapsigargin, dexamethasone, cyclosporine, geranylgerianol, 6-hydroxydopamine, nigicerine, salsolinol, camptothecin, excitotoxins, ceramides, Zn2+, Pb2+, Cu2+, and gamma or UV radiation, etc. (Hakem et al., 1998; Saito et al., 1999; Koh et al., 1995; Oberto et al., 1996; McDonald et al., 1996; Zhang et al., 1997; Masuda et al., 1997; Nath et al., 1997; Perry et al., 1997; Oh et al., 1997a,b; Kim et al., 1997a; Woo et al., 1998; Gobbel et al., 1998; Tenneti et al., 1998; Yoshimura et al., 1998; Ochu et al., 1998; Keane et al., 1997). Interestingly, bisindolylmaleimide VIII, an inhibitor of PKC, targets killer Tcells linked to EAE demyelination suggesting use of agents targeting speci®c cells may have useful therapeutic applications (Zhou et al., 1999). This would o€set non-speci®c suppression of processes required for repair or homeostasis in peripheral tissues. Agents acting upstream or downstream in the putative cascades linked to procaspase conversion events provide tools to identify signaling pathways. Agents available for this purpose include protein synthesis inhibitors cycloheximide and actinomycin D, antisense nucleotides, Li+, lipids such as gangliosides or the semi-synthetic derivative Liga20, lysophosphatidic acid, sphingosine-1-phosphate, and peptide or viral protease inhibitors, IAPs or other endogenous factors in¯uencing formation of intracellular complexes required for transduction of apoptotic stimuli (Oberto et al., 1996; Ellerby et al., 1997; Saito et al., 1998; Stefanis et al., 1998; Deshmukh and Johnson, 1998; Nonaka et al., 1998). Caspase peptide inhibitors, or an antisense nucleotide block cell loss in NGF-deprived PC12 mediated by caspase-2 (Stefanis

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et al., 1997, 1998), but this also depends on the paradigm used to induce apoptosis. PC12, for example when exposed to ceramide or hypoxia, are rescued on NGF deprivation by inhibitors for caspase-3, or by transfection of anti-apoptotic bcl-2 (Yoshimura et al., 1998; Hart®eld et al., 1998). Peptides poorly penetrate neurons and require high concentrations to demonstrate signi®cant rescue. Because of overlapping speci®city, such inhibitors do not suciently discriminate between caspase family members when used at high concentration. Cell-free assays provide an alternative approach to identify `apoptosis associated factors' (Apafs) that on reconstitution convert procaspases to degrade the DNA of isolated nuclei or plasmids (see Fig. 1 and section 7.2; Lazebnik et al., 1993; Liu et al., 1996; Ellerby et al., 1997; Enari et al., 1998). 6. Discovery of interleukin converting enzyme (ICE, caspase-1) and its implications From an historical perspective, several developments ¯owed from the discovery and isolation of ICE a decade ago (Black et al., 1989; Kostura et al., 1989): 1. Identifying Asp sites in Pro IL-1b for synthesis of substrates and inhibitors (Fig. 3). 2. Homology of ICE with C. elegans death gene CED3 (Cohen, 1997). 3. Identifying a `cascade' of sequential events for processing procaspases involving adaptors and cofactors containing `death domains' (DD, DED, CARD, etc.) (see section 6.1 and Muzio et al., 1996; Boldin et al., 1996; Fernandes-Alnemri et al., 1996). 4. Discovery of 014±16 aspartyl proteinases with short and long prodomains (Mr of zymogens 30±50 kDa) (Table 1). 5. Identi®cation of vital protein substrates (Table 2). 6. Discovery of inhibitor of apoptosis proteins (IAPs) (Fig. 6). Pro-ICE provides a model for studies of subunit structure of caspase-zymogens: it contains an N-terminal domain linked to a large (020) and small (010 kDa) subunit, as depicted in Fig. 3. Cleavage at Asp motifs adjacent to these domains releases subunits which are capable of reassembly to form tetramers, now regarded as the active form of enzyme. Presence of a Cys/His diad at the active center but absence of inhibition by leupeptin and E-64 combined with cleavage at Asp motifs indicates these are novel cysteine proteinases providing a rationale for the term `caspases' (Alnemri et al., 1996). Actually the incentive for study of ICE arose from the need to clarify mechanisms for conversion of Pro IL-1b to release of IL-1b, a highly potent

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and pleiotrophic cytokine. Later discovery of homology to nematode CED-3 prompted inquiry on roles played in mammalian PCD (see section 7.1). In neurons, basal levels of ICE are low in keeping with low levels of cytokine, and importantly, are not altered as a result of apoptosis (Armstrong et al., 1997; Marks et al., 1998a). Neuronal IL-1b increase in in¯ammation may result from uptake by non-TrkA receptors of exogenous cytokine secreted by microglia or other cells (Bhat et al., 1996; Rothwell et al., 1997). In brain, the functional role of this cytokine is unknown. Conversion of Pro-IL-1b at the Asp motifs of human -His-Asp-Ala-Pro-, or rabbit -Cys-Asp-Ala-Val-, or bovine -Cys-Asp-Val-Pro provides the basis to synthesize tetrapeptides for N- and C-terminal modi®cations for use as inhibitors or substrates. Combinatorial or classical methods have provided substrates for analysis of speci®city. Caspases-1, -4 and -5 preferentially hydrolyze N-Ac-WEHD- or Ac-YVADamc at high eciency (Km/Kcat for WEHD, 3.3  10ÿ6 M/s) while caspases -2, -3 and -7 act on Ac- DEXDor DEVD-amc, and caspases -6, -8, and -9 act on L/V/ EXD (Talanian et al., 1997; Thornberry et al., 1997). `Preferred' substrates still require partial or full sequencing of the relevant enzyme or use of antisera for the unequivocal identi®cation of the hydrolases. Caspases -1 and -3 were puri®ed from cellular sources, with the structure of others deduced from their cDNAs, and are available as recombinant forms (Wi et al., 1997; Zhou et al., 1997b). Caspases 11±14 belong to the ICE-subgroup with little information available about their function(s), or if present in neurons (Hu et al., 1998a; Humke et al., 1998). Recently described forms have the following properties: procaspase-13 (ERICE) is a substrate for caspase-8; procaspase-14 (MICE) has an unusually short prodomain; procaspases 13 and 14 induce apoptosis in human breast cancer cells MC7; both associate readily with long prodomain caspases-1, -2, -4, -8 and -10. 6.1. Death e€ector domains (DD, DED, CARD) and their role in zymogen conversion The ®rst evidence for existence of an ordered pathway for procaspase conversions came from studies of antigen-ligated pathways in immune cells. This lead to identi®cation of a domain then termed FADD/ MORT-1 found in `proximal' procaspases -8 or -10 and essential for binding cognate regions of the receptor and adaptors via homophilic interactions (see Fig. 1 and Muzio et al., 1996; Boldin et al., 1996; Nagata, 1997; Aravind et al., 1999). Speci®city exists for each class of procaspase: procaspase-2 adaptor RAIDD, for example, interacts with RIP, a serine threonine kinase (Boldin et al., 1996; Nagata, 1997; Kumar and Colussi, 1999). Among the most studied domains is

the DD-DED-CARD triad found in animal tissues. Others now include cysteine-rich motifs found in RING and TRAF (CART), the Toll-interleukin receptor TIR, and the meprin and TRAF-homology domain MATH that are structurally unrelated to the triad. The roles of these recently discovered forms in neurons are presently unknown. Post-mitotic neurons lack receptor-ligated mechanisms mediated by DD-type domain and do not express the Fas/APO-1 receptors capable, for example, of binding fasL or TNFa as noted earlier for these receptors in proliferating neurons. There is little evidence in post-mitotic neurons for receptors DR 1,2,4,5 binding TRAIL, or receptors TRAMP/DR3 binding Apo-3 etc. Thus, mature neurons appear to be highly privileged and immune to antigens initiating apoptosis in peripheral cells. This may re¯ect the need to preserve the number of `irreplaceable' neurons again emphasizing the importance of preventing unscheduled apoptosis. Similar comments apply to DD-type adaptors required for transduction of antigen-mediated events. Data on presence of TRADD (TNF-associated-death domain), FADD ( fas-associated-) in post-mitotic neurons is sketchy as is the case for TRAF 1±5, RIP, RAIDD, FAN, FLIP, FAF, etc emphasizing the need for more detailed studies on post-mitotic rather than embryonic cells (Boldin et al., 1996; Baker and Reddy, 1996; Naismith and Sprang, 1998; Ashkenazi and Dixit, 1998). The absence of receptor-mediated events places importance on alternative pathways for processing procaspases since this clearly occurs in neurons subject to apoptosis. Foremost among these are roles for three `apoptosis associated factors' or Apafs (see section 7.2; Cecconi et al., 1998; Yoshida et al., 1998; Green, 1998; Kuida et al., 1998; Hakem et al., 1998). Apaf-1 was found to be a CED-4 homolog with a putative ATPase domain, Apaf-2 was shown to be cytochrome c, and ®nally Apaf-3 was found to be procaspase-9. The presence of `caspase recruitment domain' (CARD) in Apafs 1 and 3 is important since this has functionality assigned to death domains (see Fig. 1 and Hofmann et al., 1997; Pan et al., 1998; Adams and Cory, 1998). Such domains have conformational features found in solution that promote protein-protein interactions via presence of antiparallel-amphipathic a-helices surrounding a hydrophobic core (Kumar and Colussi, 1999). Conversion of procaspase-8, a `proximal' DED-containing member of the putative caspase cascade in immune cells, initiates events leading to activation of distal e€ector caspases (Nagata, 1997; Thornberry and Lazebnik, 1998; Stennicke et al., 1998). Its conversion is required to initiate an ordered pathway in immune cells and results from autocatalysis following dimerization (see Fig. 1 and Muzio et al., 1998; MacCorkle et

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al., 1998; Yang et al., 1998b; Butt et al., 1998b). Autocatalysis may account for conversion of other procaspases rather than direct activation following cleavage of relevant Asp sites by activated caspases acting as convertases themselves. Autocatalysis occurs on rearrangement of small and large subunits of procaspase-3 and -6 prepared from cDNA constructs (Srinivasula et al., 1998b; Butt et al., 1998a), or on association of long prodomain (proximal) zymogens with short prodomains augmenting autocatalysis (Colussi et al., 1998). Cell membranes themselves also provide catalytic surfaces for autocatalysis of procaspase-8 conversion stressing the importance of biophysical events (Martin et al., 1998). However activation by serine protease acting as a convertase recognizing Asp groups is unlikely in neurons since these lack Granzyme B associated with the perforin pathway in cytotoxic T-cells, or other serine proteases such as A-24 (Podack, 1995; Harris et al., 1998). This does not rule out presence of novel convertases in neurons other than caspases, but these remain to be identi®ed. Other events in¯uencing neuronal loss are mediated by TrkA-C receptors binding neurotrophins such as NGF, BDNF and NT 1-3. These act via separate adaptors and can upregulate cytoprotective IERG `immediate early response genes' c-jun or c-fos, NF-kB transcription factor, and protein kinases/phosphatases (see Fig. 1 and Lindsay et al., 1994; Bredesen and Rabizadeh, 1997; Dobrowsky and Carter, 1998). Removal of NGF from cultures of sympathetic ganglia or PC12 a€ects cytochrome c release from mitochondria and activates caspases 2 or 3 (see also section 7.2 and Deshmukh and Johnson, 1998). Neurotrophin receptors vary considerably in neurons: neuroblastoma (NIE-15) cells and PC-12 express p55-TNF-R but not p75-TNF-R (Sipe et al., 1996) and sympathetic ganglia bind NGF. The death domain of p55 (Naismith and Sprang, 1998) can itself induce loss of PC12 by pathways prevented by expression of bcl-2 or sensitive to caspase inhibitors dependent on their state of di€erentiation (Haviv and Stein, 1998). The p75 neurotrophin receptor associates with TRAF 6 in Schwann cells with translocation of p65 subunit of NF-kB, and in T9, NIH3T3 and PC12 cells it transiently increases ceramide and activates early response genes (Khursigara et al., 1999). There is evident need to further clarify the role of neurotrophins and cell death. 7. Implications arising from studies on null mice and transgenics Null mice are models useful to identify signaling events linked to apoptosis. Deletion of caspase-3, -9 or Apaf-1 in mice results in gross malformations speci®c to brain because of defective apoptosis and hyperplasia

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(Kuida et al., 1996, 1998; Cecconi et al., 1998; Yoshida et al., 1998; Hakem et al., 1998). Neuronal cytosols of caspase-9 ÿ/ÿ mice do not hydrolyze AcDEVD-amc or yield procaspase-3 subunits on blots indicating a probable defect upstream. There is evidence for considerable di€erences in cell type and response to apoptotic signals indicating bifurcation of pathways of importance to developing therapeutic agents (see Fig. 1 and Woo et al., 1998; Hakem et al., 1998). Procaspase-2 exists as alternatively spliced forms having pro- and anti-apoptotic properties but deletion of both in animals does not signi®cantly alter brain development (Bergeron et al., 1998; Haviv et al., 1998). Deletion of ICE-related caspase-1 or -11 also is without ultrastructural consequences for brain though such animals show increased tolerance to endotoxic shock because of cytokine de®ciency (Li et al., 1995). Transgenics expressing mutant caspase-1 (Cys285Gly) show cytokine de®ciency and crossing these with SOD ÿ/ÿ mice, a model for motor neuron degeneration, reduces neuronal atrophy. Microinjection of the mutant gene in embryonic dorsal root ganglia (DRG) attenuates apoptosis on removal of trophic factors (Friedlander et al., 1997). DRG isolated from such transgenics resist apoptosis for reasons not fully understood (Friedlander et al., 1997) unless the caspase-1 prodomain acts independently as suggested in recent studies following its translocation to nuclei (Mao et al., 1998). Mice lacking cystatin B, an animal model for human Unverricht-Lundborg disease, leads to loss of cerebellar granule cells by a process morphologically similar to apoptosis (Pennacchio et al., 1998). Further developments are awaited using animals de®cient in DNA fragmentation factor CAD/DFF45 and the adaptor FADD (Zhang et al., 1998a,b). 7.1. Nematode CED-3-genes and mammalian counterparts Gain or loss of function provides evidence for the profound e€ects that genes have on maturation of worms and ¯ies (Hengartner, 1997). Pivotal studies on C. elegans demonstrate that 010 genes direct loss of 131 cells of which 105 are neuronal from 1090 somatic cells in hermaphrodite larvae during development. Many of these genes have mammalian counterparts (summarized in Fig. 4). Cell death speci®cation CES 2 and 3 are genes that determine the fate of (serotonergic-like) motor neurons and encode basic region leucine zipper transcription factor (bZip) proteins with homology to HLF (human leukemia factor) (Metzstein et al., 1996; Inaba et al., 1996). CED-3 is pro-apoptotic, and is notable for homology to ICE that provided early clues on a role for caspases in mammalian cell death. In nematodes, the binding of a protein encoded

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Fig. 4. C. elegans death (CED ) genes linked to number and death of neuronal progenitor cells on maturation of C. elegans. Counterparts of proteins (solid line) or domains encoded by mammalian genes are indicated. (See text for abbreviations and see sections 7.1±3.)

by EGL-1 containing a single BH3 domain (Fig. 5) to CED-9, a homolog lacking this domain results in uncoupling and activation of CED-3 caspase from the inactive CED-4/CED-3 complex (del Peso et al., 1998). Gain of function of EGL-1 causes hermaphroditespeci®c neurons to undergo PCD, whereas use of a gene with loss of function prevents somatic cell death (Conradt and Horvitz, 1998; del Peso et al., 1998). CED-5±7 genes in¯uence the engulfment and removal of cell debris or corpses: CED-5 shows similarity to mammalian DOCK 180, CED-6 has a phosphoryl tyrosine binding domain (PTB), and CED-7, an ATP binding cassette protein (ABC) similar to many animal proteins (Hengartner, 1997; Wu and Horvitz, 1998; Liu and Hengartner, 1998; Savill, 1998). Nematodes possess MEC 4/10 and DEG genes for removal of neurons by necrotic-like processes which encode proteins having domains resembling those of some mammalian acetylcholine channel subunits (Berger et al., 1998). In Drosophila melanogaster, three genes known as rpr, htd, and grm determine the fate of neurons (Zhou et al., 1997a). Drosophila also contains dcp-1 and drICE that resemble procaspases and are capable of autocatalysis to provide enzyme acting on a DEVD- substrate, p35, and lamin (Song et al., 1997). In ¯ies, overexpression of p35, a viral inhibitor of caspases, prevents blindness in a mutant strain that normally has retinal degeneration (Davidson and Steller, 1998). Studies on nematodes and ¯ies continue to pave

the way for PCD cofactors, and have gained in importance with recent publication of the full genomic sequence for C. elegans (The C. elegans sequencing consortium, 1998). 7.2. Apoptosis associated factors (Apafs) Reconstitution in vitro of three Apafs with ATP results in activation of procaspase-9 and subsequently procaspase-3 (Fig. 1). Apaf-1, a mammalian homolog of CED-4 (Zou et al., 1997), contains CARD and a putative ATPase domain in addition to other motifs (Hu et al., 1998b). The ATPase domain represented in CED-4 and hAPAF-1 may represent a new class of apoptotic regulators, highly conserved in di€erent species, and recently referred to as Ap-ATPases (Aravind et al., 1999). Apaf-2 was identi®ed as cytochrome c of mitochondria, while Apaf-3 was found to be procaspase-9 (Liu et al., 1996; Li et al., 1997b; Green, 1998). The CARD sequences (Hofmann et al., 1997) on Apafs -1 and -3 facilitate formation of a selfactivating complex by a process dependent on the presence of WD-40 repeats within Apaf-1. The removal of WD-40 results in procaspase-9 conversion by a cytochrome c independent pathway (Srinivasula et al., 1998a; Levkau et al., 1998; Fig. 1; Hu et al., 1998b). Seol and Billiar (1999) identi®ed a naturally occurring caspase-9 S variant missing most of the catalytic sequence with a dominant-negative inhibition of apop-

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Fig. 5. Homology domains (BH) of selected bcl-2 proteins of mammalian cells compared to nematode CED-9 and EGL-1 (see Fig. 4). The ®rst three examples act anti-apoptotically with mammalian members, and are thought to form oligomers via BH 1-3, and pores via BH 1-2 in mitochondrial membranes (Fig. 1) as compared to pro-apoptotic properties of the remainder (Adams and Cory, 1998; Matsuyama et al., 1998). Coalescence of a-helical regions indicated by bars creates structures or clefts for the BH3 region of other members facilitating formation of dimers (Sattler et al., 1997). Hatched regions depict domain for anchoring to mitochondrial or other membranes.

tosis in MC7 induced by expression of caspase-9 itself providing a strategy that may be applicable to neuroprotection. Slee et al. (1999) report that procaspases-2, -3, -6, -7, -8, and -10 are processed in cell-free extracts in presence of cytochrome c by activated caspase-9, indicating that cytochrome c acted as an upstream regulator. It is paradoxical that cytochrome c, a mitochondrial component essential for oxidative phosphorylation and survival, on translocation to cytosol is so highly cytotoxic ultimately triggering the conversion of an e€ector protease (Green and Reed, 1998). The ®rst indication for a role for mitochondrial components was found in a cell-free system from Xenopus eggs showing bcl-2 prevented apoptosis (Newmeyer et al., 1994). The role for cytochrome c came as a surprise indicating it is pivotal, especially for neurons lacking receptor-ligated pathways for procaspase conversion. Mitochondria contain other factors thought to in¯uence translocation of cytochrome c (see section 7.3 and Hockenbery et al., 1990; Adams and Cory, 1998), including roles played by megachannels (PT pores), and membrane (Dcm) or redox potentials (Stridh et al., 1998). Increase in mitochondrial permeability by mastoparan or atractyloside promotes apoptosis in cultured neural-

like cells (Ellerby et al., 1997), whereas in the case of pheochromocytoma PC-6, staurosporine induces cell death with a decrease in Dcm and translocation of cytochrome c (Heiskanen et al., 1999). AIF, an `apoptosis inducing factor' released from the intermembrane space of mitochondria, once thought to be a protease was recently sequenced and shown to be a ¯avoprotein homologous to many bacterial oxidoreductases (Susin et al., 1999b). AIF addition to nuclei causes peripheral condensation and large-scale fragmentation of chromatin insensitive to caspase-inhibitors. Studies by the same group establish mitochondria from some sources are reservoirs for caspases-2 and -9 along with another nuclease distinct from CAD (Susin et al., 1999a). Few studies exist on anatomical and subcellular localization of procaspases in brain similar to those available for other tissues (Li et al., 1997a,b,c; Mancini et al., 1998; Sanghavi et al., 1998; Chandler et al., 1998; Samali et al., 1998). Cytosolic caspases potentially amplify mitochondrial damage by a feedback mechanism with release of other e€ectors (Marzo et al., 1998). Studies show changes in permeability and cytochrome c release contributes to the development of multi-drug resistance (MDR) in tissues (Kojima et al., 1998a). Microinjection of cytochrome c is cytotoxic for sym-

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pathetic ganglia and other cells by a pathway sensitive to caspase-inhibitors (Zhivotovsky et al., 1998; Deshmukh and Johnson, 1998). Cytochrome c microinjection in human breast cancer MC7 that lack caspase-3, is non-toxic but restoration of the gene restores this property, thereby elegantly con®rming a role for Apaf-2 in procaspase-3 conversion (Li et al., 1997a). In Jurkat T-cells staurosporine or other chemical agents can bypass cytochrome c-mediated caspase-3 activation (Sun et al., 1999). Treatment of SH-SY5Y cells with NO donor NOC18 induces translocation of cytochrome c with increase in caspase-3 and reduction in procaspase-2 (Uehara et al., 1999). Few studies are available on structure-activity relationships for mammalian cytochrome c, but cytoxicity is expressed only by the haloprotein possessing an intact hinge region and a free Lys (Ellerby et al., 1997; Okazaki et al., 1998). 7.3. CED-9/bcl-2 proteins and neuronal PCD A family of 015 proteins referred to as bcl-2-like (Bcell lymphocyte oncogene) contain one or more domains with homology to nematode CED-9. While terminology varies, these include pro-apoptotic Bax, Bak, Bik, Blk, BAD, BID, Bim/BOD, Hrk, Bok, and anti-apoptotic Bcl XL, Bcl-w, McH-1, B¯-1, and Diva. A recent survey in AD brain showed an increase in Bcl-XL, Bak, and BAD in membranes of temporal cortex (Kitamura et al., 1998). Deletion or overexpression of bcl-2 proteins profoundly in¯uences brain maturation, the ®nal number of neurons, and their response to injury (Allsopp et al., 1993; Martinou et al., 1994; Yang et al., 1995; Xiang et al., 1998; White et al., 1998). Bcl-2 proteins participate in release of cytochrome c and events triggering conversion of procaspase-9 mediated within the Apaf 1±3 complex (Fig. 1). These relationships are illustrated in recent studies on peripheral cells where Blk is blocked by a dominant negative procaspase-9 (Garcia-Calvo et al., 1998), and binding of Diva to Apaf-1 prevents apoptosis (Inohara et al., 1998a). Chronic treatment of cerebellar granule cells with lithium prevents glutamate-induced release of proapoptotic Bax, p53, lamin-B degradation, and cytochrome c translocation, but upregulates anti-apoptotic Bcl-2, providing a possible basis for its pleiotrophic neuroprotective properties (Marks et al., 1998a,b; Chen and Chuang, 1999). Structurally, Bcl-2 resembles nematode CED-9 and EGL-1 proteins that contain one or more `homology domains' BH, and several other domains with a-helixes (Figs. 4 and 5) (Zha and Reed, 1997; Adams and Cory, 1998; Matsuyama et al., 1998). Localization of bcl-2 proteins on the cytoplasmic face of the mitochondrial outer membrane favors formation of pores with

homodimers promoting cytochrome c translocation. However, formation of homodimers does not account for pro-apoptotic e€ects in all cases as illustrated for recombinant forms in studies on dopaminergic neurons (Oh et al., 1997a,b). It is not established if the BH3 domain of Blk is essential (Hegde et al., 1998) although it is required for Diva, a developmentally regulated component in the CNS (Inohara et al., 1998a). Further studies on the BH and helical regions could reveal relationships useful for design of new categories of neuroprotective agents. In nematodes, binding of EGL-1 to CED-9 promotes activation of CED-3, a caspase-3 like enzyme, present in the CED3/CED-4 complex (Conradt and Horvitz, 1998; del Peso et al., 1998). By analogy to mammalian tissues, the translocation of cytochrome c mediated by pro-apoptotic bcl-2 dimers activates procaspase-9, and then procaspase-3 as shown in Fig. 1, and compared to the pathway for nematodes in Fig. 4 (Adams and Cory, 1998). For this purpose, CARD of Apaf-1 and-3 facilitates binding and formation of the Apaf-3 dimer as depicted. In sympathetic ganglia, cytochrome c toxicity is prevented by boc-aspartyl-fmk, indicating mediation by caspases, and on deletion of BAX implying a role for this bcl-2 protein in forming an autocatalytic complex (Deshmukh and Johnson, 1998). Apoptosis due to overexpression of Bax or Bak can be prevented by z-VAD-fmk, a non-speci®c caspase inhibitor, reinforcing a role in conversion of initiator procaspases (Martinou et al., 1998). Degradation of BID, an antiapoptotic BH3 component, by caspase-8 provides a potential feedback mechanism for fragments interacting with mitochondria (Hampton et al., 1998; Luo et al., 1998; Li et al., 1998b; Finucane et al., 1999; Gross et al., 1999). In Xenopus eggs, the BH3 domains alone of Bak and Bax initiate events leading to activation of caspase-3 (Cosulich et al., 1997b). 8. Structural and regulatory proteins as substrates Degradation of cytoskeletal or regulatory proteins irreversibly damages cells leading to their self-destruction by cleavage at Asp motifs (Tables 1 and 2). The list is large, raising questions on whether there are overlap in speci®city, or if any one substrate alone plays a rate-limiting role. Caspase-3 is the leading contender as the primary e€ector protease in neurons but studies in human carcinoma breast MC7 lacking this enzyme demonstrate redundancy since extracts hydrolyze the proteins PARP, Rb, DNA-PKcs, gelsolin and ICAD/DFF-45 (Inhibitor of caspase-activated DNase/ DNA fragmentation factor p45 ) (Janicke et al., 1998b). In one scenario the cleavage of lamins by caspase-6 at a -VEID-motif (Table 1 and Orth et al., 1996; Takahashi et al., 1996), could damage nuclear integ-

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rity, permitting access of caspases and resultant breakdown of nuclear components as bystanders. Degradation of PARP prevents normal DNA repair and turnover thereby crippling the cell. Processed caspases do not contain a `nuclear localization sequence' NLS for translocation. Some prodomains, however, have NLS that may be pro-apoptotic on translocation (Mao et al., 1998). In a second scenario, caspases may activate latent cytosolic e€ectors such as the nuclease CAD followed by its translocation to nuclei following cleavage/inactivation of an inhibitory subunit ICAD/ DFF45 (Fig. 1). This mechanism has analogy to the activation and translocation of NF-kB following degradation of its Ik-B inhibitory subunit (Baeuerle and Baltimore, 1996; Liu et al., 1997; Barkett et al., 1997; Enari et al., 1998). The CAD/DFF45 complex consists of two subunits, a 40 kDa nuclease, and a 45 kDa inhibitor containing the caspase-3 sensitive Asp site. The nuclease contains a C-terminal catalytic and a regulatory N-terminal sequence recently shown to be homologous to the N-terminal domain of a DFF45-like CIDE (Inohara et al., 1998b, 1999). DFF45 knockouts develop normally, although cells from null animals following apoptosis show no DNA fragmentation (Zhang et al., 1998b). Other substrates for caspase-3 include: SREBP, a sterol-regulatory element binding protein, PITSLRE (CDk2), Rb, Cb1, Cbl-b, Raf-1, Akt-1, MEKK1 p21, Cd, CdMK-IV kinase, and MDM2 that are linked to signaling pathways, ProIL-16, an in¯ammatory cytokine, and STAT1, a factor mediating the action of interferon and cytokines (see Table 2; Wang et al., 1995; Erhardt et al., 1997; Zhang et al., 1998c; King and Goodbourn, 1998; McGinnis et al., 1998; Widmann et al., 1998a,b). Native substrates participating in cell progression are particularly relevant to PCD during remodeling of embryonic neurons. Wild or mutated APPs, presenilins, and tau also are caspase-3 substrates but it is unclear if caspase-degradation is a factor in pathology (see section 3, Table 2 and Loetscher et al., 1997; Cheng et al., 1997; Kim et al., 1997b; Gervais et al., 1999; Chandler et al., 1998; Kitamura et al., 1998). Neuronal NT2 cells produce elevated levels of amyloid b-peptides as a result of apoptosis induced by serum deprivation: this production is attenuated by inclusion of a nonselective irreversible caspase inhibitor Z-VAD (OMe)-Ch2F. Truncated APP751 lacking the major caspase-3 Asp720 site on transfection in B103 cells results in an increase in Ab production, suggesting this favors the amylodogenic or pathogenic pathway for reasons that are poorly understood (Gervais et al., 1999). The Swedish APP mutant (Met651/Leu652 instead of wild type Arg/Met) on transfection in K562 cells promotes cleavage at the putative b-secretase site at the N-terminus of Ab within the

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haloprotein. The altered site appears to be a consensus domain for recognition by caspase-6, since it is not vulnerable to caspase-1, -3, -7, -8, or granzyme B. Secondary sites that are recognized within APP751 by caspase-3 include Asp197 and Asp219. Caspases play a hitherto unrecognized role in APP catabolism forming intermediates, especially in FAD mutants, that can be trimmed by `secretases' or other processing enzymes. Whether this is solely the result of apoptosis is unclear since some procaspases are reported to have low-grade catalytic activity contributing to constitutive processing over time. Phosphorylation of PS-2 at sites adjacent to those recognized by caspases blocks cleavage thereby enhancing the anti-apoptotic properties of the stabilized C-terminal fragment (Vito et al., 1997; Walter et al., 1999). There are eight genetic neurodegenerative disorders associated with CAG expansions that result in proteins rich in polyglutamine repeats some of which are substrates for caspase-3. These include huntingtin (Huntington's chorea, HD), androgen receptor (Kennedy's Disease), atrophin-1 (dentatorubropallidoluysian atrophy, DRPLA), and ataxin-3 (MachadoJoseph disease, MJD) (Kim et al., 1997b; Vito et al., 1997; Wellington et al., 1998). Cleavage of the androgen receptor (AR) results in perinuclear aggregates associated with neural death but it is unknown if this is an etiological factor in pathology (Ellerby et al., 1999). Cytoskeletal or regulatory protein substrates are fodrin, gelsolin, gas-2, actin, spectrin, nuclear sca€old proteins, catenin, and focal adhesion protein (see Table 2 and Kothakota et al., 1997; Canu et al., 1998; Gervais et al., 1999; Wang et al., 1998a; Schmeiser et al., 1998; Tesco et al., 1998). Detection of spectrin or actin fragments in diseased brains may provide an alternative method for identifying neuronal atrophy or damage in neurodegeneration (Kothakota et al., 1997; Yang et al., 1998a). Breakdown of the calpain inhibitor calpastatin or fodrin may a€ect calcium homeostasis (Nath et al., 1996; Wang et al., 1998b). 9. Caspase inhibitors N- and C-terminal modi®cation of peptides or Asp itself yield reversible or irreversible inhibitors (Table 1). These result from modifying tetrapeptide substrate motifs with C-terminal aldehyde, ketone and nitrile, CO-NH2-X (X=cloromethylketones, ¯uoromethylketones, diazomethylketones, acyloxymethoketones, and phosphinyloxymethoketones) using standard procedures available for other proteases (Marks and Berg, 1992; Dolle et al., 1994; Mashima et al., 1995; Nicholson and Thornberry, 1997; Lynch et al., 1997; Garcia-Calvo et al., 1998). Therapeutic applications

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Fig. 6. Comparison of inhibitors of apoptosis proteins (IAPs) containing baculoviral inhibitory repeats (+) in di€erent species (Uren et al., 1998). Other regions include RING, a putative Zn2+-binding domain (q) and a domain with homology to CARD (Q). IAPs bind and inhibit caspases 3 and 7 (Deveraux et al., 1997). NAIP and survivin are present in human tissue and are linked to some neurodegnerative disorders (see section 9).

depend on speci®city and penetration of inhibitors to tissue target sites. Currently, high concentrations are required to demonstrate e€ects on intact neurons (Milligan et al., 1995), or in the case of CrmA, by direct microinjection (Gagliardini et al., 1994). Use of pan inhibitors such as Z-VAD-fmk, or others in ischemia may act on both cytokine production and death e€ector proteases (Hara et al., 1997). Non-speci®c inhibitors may pose a danger by a€ecting other tissue rather than a neuronal subset at risk. Viral proteins inhibiting caspases include poxvirus (CrmA) a 38 kDa protein, and baculoviral p35 (Komiyama et al., 1994; Bump et al., 1995; GarciaCalvo et al., 1998). Mammalian tissues contain inhibitor of apoptosis proteins' (IAPs) containing BIRs `baculoviral inhibitory repeats' (see Fig. 6 and Uren et al., 1996; Roy et al., 1997) that are a family of conserved proteins. CrmA inhibits ICE-like caspases in the pM range, and somewhat higher for CED-3 proteases (see

Table 1; Zhou et al., 1997b; Garcia-Calvo et al., 1998). In tissues, viral CrmA preserves infectivity by suppressing cytokine release and recruitment of viral destroying macrophages (Ray et al., 1992). Transfection of mitotic immune cells with CrmA indicates this inhibitor acts upstream to Bik and Bak (Orth and Dixit, 1997). The viral inhibitor p35 selectively and irreversibly inhibits caspases-3 and -7, Ki <1 nM (Roy et al., 1997; Deveraux et al., 1997), and transfection in NGF deprived sympathetic neurons blocks apoptosis (McCarthy et al., 1997). IAPs di€er in number of BIRs, and can contain CARD and RING (Zn+ binding) domains (see Fig. 6 and Uren et al., 1998). IAPs in Drosophila include DIAP-1, DIAP-2/dILP (Hay et al., 1995; Uren et al., 1996; Duckett et al., 1996; Vucic et al., 1998a). Human tissues contain cIAP-1 and 2, XIAP, survivin and NAIP (human neuronal apoptosis inhibitory protein). Recombinant c-IAPs tightly bind caspases-3 and -7, but not the proximal caspase-8, nor

N. Marks, M.J. Berg / Neurochemistry International 35 (1999) 195±220

caspases-1 or -6. NAIP does not tightly bind to caspase-3, -6, or -7, implying this inhibitor acts intracellularly on other unidenti®ed e€ector protease(s) (Roy et al., 1997). Inhibition by recombinant c-IAPs with Ki 30±120 nM is several orders lower than CrmA (Ki 500 nM) compared to picomolar for XIAP: since Ki varies for each caspase, there is considerable selectivity by IAPs. IAPs function upstream of p35, a non-speci®c inhibitor with similarities to action of CrmA, or amacroglobulin. A study on structure-activity relationships show hXIAP that lacks RING retains its potency, and that the second of its three BIR is the active domain (Takahashi et al., 1998) similar to the action of Orgyia psuedotsugata nuclear polyhedrosis virus Op-IAP (Vucic et al., 1998b). NAIP is linked to spinal muscular atrophy (SMA), a childhood disease with muscle weakness and spinal cord pathology: this malady occurs on deletion of this gene and apparent unrestricted apoptosis (Roy et al., 1995; Morrison, 1996). The manner it acts is unclear since NAIP does not interact with caspases-3 or -7. Transfection in non-neural cells prevents apoptosis while viral vector introduction in brain reduces injury to hippocampal neurons in experimental ischemia (Liston et al., 1996; Xu et al., 1997a,b). Survivin, a 16.4 kDa protein, is an IAP with only one BIR, interestingly, is highly expressed in fetal brain and in some human tumors suggesting roles in mitosis (Ambrosini et al., 1998; Li et al., 1998a). IAPs are not metabolized but bind TRAF-1 and 2 adaptors making it unlikely they can act as `suicide inhibitors' similar to that proposed for CrmA (Rothe et al., 1995). 10. Concluding comments There has been an exponential increase in studies on caspases demonstrating presence of 016 members of this new superfamily although not all are found in primary neurons, or believed to act as ®nal e€ectors of cell death. Many play multiple roles in the initiation and execution of cell death. There is interest in Alzheimer's and some evidence linking caspases to CAG repeat disorders associated with neuronal atrophy. In AD, regional losses occurring over decades must be reconciled to rapid forms of cell loss observed in vitro models for apoptosis, or in the case of necrotic damage elicited by EAA's. Evidence linking procaspase-3 conversion to neuronal atrophy in experimental ischemia/hypoxia point to a role for activated enzyme in stroke-related damage suggesting possible therapeutic applications for speci®c inhibitors. This is provided such agents target neurons at risk and not cells in other tissues. The loss of mature neurons as stressed in this overview appears to have no self-evident purpose in adults.

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In contrast, neuronal turnover is critical for brain remodeling and synaptogenesis in embryos. There is a recent focus on alternative pathways in post-mitotic neurons lacking receptor-mediated events with emphasis on mitochondrial c (Apaf-2). Among the many unanswered questions relevant to neurons are: (1) what constitutes a valid apoptotic signal in situ and how is it transduced; (2) whether pathways for apoptosis di€er in proliferating neurons; (3) identify of e€ector caspases in neuronal subsets or in transformed cells; (4) do neurons contain cofactors and adaptors similar to those of peripheral cells; (5) the identity of native substrates and their hierarchical breakdown; (6) the role of IAPs or other endogenous factors interfering with signaling pathways; (7) if inhibitors can be used for clinical purposes; (8) on selection of appropriate probes to unravel signaling events; and (9) on the further use of transgenics or knockouts to examine the role of apoptosis in neuronal development and function. References Adams, J.M., Cory, S., 1998. The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322±1326. Allen, Y.S., Devanathan, P.H., Owen, G.P., 1995. Neurotoxicity of beta-amyloid protein: cytochemical changes and apoptotic cell death investigated in organotypic cultures. Clin. Exp. Pharmacol. Physiol. 22, 370±371. Allsopp, T.E., Wyatt, S., Paterson, H.F., Davies, A.M., 1993. The proto-oncogene bcl-2 can selectively rescue neurotrophic factordependent neurons from apoptosis. Cell 73, 295±307. Alnemri, E.S., Livingston, D.J., Nicholson, D.W., Salvesen, G., Thornberry, N.A., Wong, W.W., Yuan, J., 1996. Human ICE/ CED-3 protease nomenclature. Cell 87, 171±171. Ambrosini, G., Adida, C., Sirugo, G., Altieri, D.C., 1998. Induction of apoptosis and inhibition of cell proliferation by survivin gene targeting. J. Biol. Chem. 273, 11,177±11,182. Aravind, L., Dixit, V.M., Koonin, E.V., 1999. The domains of death: evolution of the apoptosis machinery. Trends Biochem. Sci. 24, 47±53. Armstrong, R.C., Aja, T.J., Hoang, K.D., Gaur, S., Bai, X., Alnemri, E.S., Litwack, G., Karanewsky, D.S., Fritz, L.C., Tomaselli, K.J., 1997. Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J. Neurosci. 17, 553±562. Ashkenazi, A., Dixit, V.M., 1998. Death receptors: signaling and modulation. Science 281, 1305±1308. Atabay, C., Cagnoli, C.M., Kharlamov, E., Ikonomovic, M.D., Manev, H., 1996. Removal of serum from primary cultures of cerebellar granule neurons induces oxidative stress and DNA fragmentation: protection with antioxidants and glutamate receptor antagonists. J. Neurosci. Res. 43, 465±475. Baeuerle, P.A., Baltimore, D., 1996. NF-kappa B: ten years after. Cell 87, 13±20. Baker, S.J., Reddy, E.P., 1996. Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12, 1±9. Barkett, M., Xue, D., Horvitz, H.R., Gilmore, T.D., 1997. Phosphorylation of IkappaB-alpha inhibits its cleavage by caspase CPP32 in vitro. J. Biol. Chem. 272, 29,419±29,422. Barnes, N.Y., Li, L., Yoshikawa, K., Schwartz, L.M., Oppenheim,

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