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Cold thoughts of death: the role of ICE proteases in neuronal cell death
L
. REVIEW
Schwartz and C.E. Milligan – ICE proteasesin neuronaldeath
Cold thoughts of death: the role of ICE proteases in neuronal cell death Lawrence M. Schwartz and Carolanne E. Milligan While there has been extensivework describingthe timing, location and probable signals responsiblefor regulatingprogrammedcelldeath(PCD) in the nervoussystem,relativelylittleis knownabout the molecularmechanismsthat mediatethis process.Severalinvestigatorshave demonstratedthat PCD in general,andneuronalPCD in particular,canbe inhibitedby drugsthat arrestRNA or proteinsynthesis. Thesedatahavebeeninterpretedassuggesting thatdenovogene expressionis requiredfor cellsto commit suicide. The generalpictureemergingfrom a number of experimentalsystemsis that a varietyof proteinscan mediatethe couplingof extracellular signalsto a residentcell-deathprogram.[nthismodel,someofthe componentsrequiredfor death are more or lessconstitutivelypresentin the cell and awaitlineage-specific signalsfor their activation.A recentfloodof papershaspresentedconvincingevidencethatthe residentprogram for apoptosisin numerouscelltypesworksvia a seriesof essentialproteasesbelongingto the CED-3/lCE family. Trends Neurosci. (1996) 19, 555-562
HE EXISTENCEof programmed cell death (PCD) was presumably noted in ancient times with the T observation that tadpoles lose their tails during metamorphosis. Serious examination of the phenomenon began much later. For example, a century ago Beard’ observedthat specific neurons were lost in a spatially and temporally reproduciblepattern during development. Fifty years later, Victor Hamburger and Rita Levi-Montalcini2published a landmark paper using quantitative methods to demonstrate that massive neuronal loss was a normal component of nervoussystemdevelopment.Since then it has been well documented that in most regions of the central and peripheral nervous systems the programmed death of both neurons and glia during discrete periods of development is a normal process (for reviews, see Refs 3–7; also see Box 1 for a discussion on the morphological types of PCD that occur in the developing nervous system). The identification of the inter- and intracellular events that mediate PCD will enhance our understandingof normal developmentgreatly.In addition, the identification of the key regulatorycomponents responsible for regulating PCD might allow us to rescue selectivelyvaluablebut condemned cells, such as neurons in Alzheimer’s disease or alternatively, to target the removal of deleteriouscells, such as malignant cells. The difficulty in studying the molecular events involvedin regulatingPCDin neurons,or in anyheterogeneous tissue, is that condemned cells are interspersedamong viable ones. Compounding this problem is the fact that while cell death might ultimately involve the loss of a significant number of neurons within a given population, the speed of the process and the absence of tight developmentalco-ordination might mean that at any given moment dying cells might represent less than 1% of the total cellular population’. For example, in chicks, approximately Copyright @ 1996, Elsevier Science Ltd. All rights reserved. 0166-
2236/96/$15.00
12000 of the normal 24000 lumbar spinal motoneurons die during 4-5 days of embryogenesiss. Nevertheless, the maximal number of pyknotic motoneurons that can be observedduring this period in serial sections of the spinal cord is about 300 (Ref.7). This apparentpaucityof dyingcellscomplicates biochemical analyses, such as the identification of differentiallyexpressedgene products, since it would be difficult to distinguishbetween the events associated with PCD versus those processestaking place in survivingcells. Consequently,most investigatorshave turned to in vitro systemsto investigatethe biochemical and molecular mechanisms involvedin regulating cell death. While this is a valuableapproachfor defining mechanisms that could be essential for regulating a differentiativeprocess,there is still a requirementfor in vivo studiesto confirm that the processunder invesLawrenceM. tigation is, in fact, an essential one. Changes ingeneexpression associated with neuronaldeath Where it has been examined, the ability of cells to undergoPCDappearsto be dependenton de novo gene expression (reviewedin Ref. 9). [It should be noted that the term ‘programmedcell death’ is used here with its originalmeaning: the spatiallyand temporally reproducibleloss of cells within a developmentalcontext10 (see Box l).] Data supporting this hypothesis were generated initially in experiments where condemned cells were treated with inhibitors of RNAor protein synthesis.Instead of being toxic to cells, these metabolic poisons were protective and prevented cell deathll-14.The simplest interpretation was that these drugsblocked the synthesis of essential components of the cell-death machinery. Teleologically,this was a very appealinghypothesisas one wouldassumethat a variety of fail-safe mechanisms exist within cells to prevent inappropriateactivation of a suicideprogram. PII: S0166-2236(96)1
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Schwartzis at the Deptof Biologyand Programin Moiecularand CeZkdarBiology, Universityof Massachusetts, Amherst, MA01003, USA, and CarolanneE. Milliganis at the Dept of Neurobiologyand Anatomy,Bowman GraySchool of Medicineof Wake Forest University, Winston-Salem, NC 27157, USA.
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ICE proteases in neuronzl death
Box 1. Programmed
cell death, apoptosis and non-apoptotic cell loss in the nervous system
The t(rm apcq]tc)sis describes the morphological and structural changes IIiat accompany most programmed cell deaths seen ciurinx development, including: mcmbrane blebbing, I)NA ft:i~];lerlt~ltiorl,marination of chromatin along the innel- aspcd of the nuclear envelope and phago cytosis of the apupii)tic bodies”. It is important to note, however, that sornc of the cells that die by programmed cell death (PCD) dLIIjng development dispkrv few if any of thew specific chan~es (reviewed in Refs b,c). These nonapoptotic CCIIdeaths, most of which arc seen in the nervous system, hiive been :cfcrred to in the older literature as ‘type 11’or ‘cytoplairnic’ degeneration’’”. Whether conp(mcmk of the cell-dc,athcascade, such as 1(X-1ikeprotetrses, arc conserved betwc(,:)these two types of cell cieath remains to I)(, dctcrm ined. iI is interesting to note that mature neurons, which are !~ighlydifferentiated cells that al-eunIikel},to re-enter th( cell cycle or serve as hosts for most viruses, might not Iw as dependent on DNAfragmentation m a fail-sale mechanism as are actively dividing cells. [t should also IWnotmf that most neurobiologists and immunologists end, IWthe term programmed cell death with ciifferent mcar in,qs. The term, as used initially by lock shin and Will!ims’, referred to the temporally and
spatially ]reproducibic loss of cells within a developmental context. implicit in this term is the expectation that death is triggered as a normal component of development by physiological triggers. Immunologists appear to have a broader definition for 1’(.;1)that includes any death that is dependent on a cellular pro,grarn, even if the trigger for death is non-physiological in nature (reviewed in Ref. g). Thcmfore while an immunologist would consider irradiation-induced tieath in lymphocytes an example of P([), a developmental neurobiolcrgist might not. References a Wyllie, A.H., Kerr, .l.k’.R. and Currie, A.R. (1980)Int. Rev. Cw’i. f)x, 2s 1–306 b Schwartz, L.M., Kosz, L. and Kay, JLK.(1990) Pm. Nd. .4(-[/[/..Sci. U. S. A. 87, 6594-6598 c Clarke, P.G. H. (1 990) A?ztit. Et)/bryd. 181, 195–21:3 d Pilar, G. and I.andmesser, L.J. (1976) J. L’ell JJiol. 68, 3:}%35(, e
(:hwWang, I-W. and Oppenheim, it.W. (1978)/. C’orrZp. Nef{rd.
f g
177, 3+58
t.oc.kshin,R.A.and Williams, (1.M.(1965)/. Inset-tPhysid. 1I, 12:-1–13:+ Schwartz, 1..M.and Osborne, B.A. ( 1993) /mtIIIIfm/. Today 14, 582!–590
-.
The newi to synthesize essential mediators of this pathway would prcwidesuch a safety feature. As will be discussed below, it now appears that the killing components of this pathway are already resident in the cell, and that tht>newly synthesized components are used to couple c.xternal signals to this lethal internal reach inery. Several cell-cycle lwgulators, immediate–early genes and proto-oncogene~ have been shown to be upreguIated with the comrrl itment of cells to die. One series of experiments focused on rat superior cervical ganglion (SCG) cells, wtlich survive well in culture when provided with NGF. in the absence of NGF, these cells die rapidly by apopt( Isis’ (See Box 1 for a description and terminology. ) of cell-death morphologies Coincident with this death is the enhanced accumulation of mRNAs tor the proto-oncogenes c-jw7, c-fi~.s,~i~sB and jut?-fl the transcription factor NGF-lA, the cell-cycle regulator cyclin Dl, transirr and collagenasei’. A simiiai timetable of events has been demonstrated for 1)(”12 cells when they die following the cieprivation of tr<)phic factor]”. In the case of c-jun, it has been shown Ihat neutralizing antibodies17 or dominant negative c-jun proteins18 could protect NGF-deprived sympathetic neurons. These experiments suggest that AP-1 activity is essential for this cell death. In contrast to tht studies of c-jun, the analysis of most cell death-associated genes has involved predominantly descriptive as opposed to functional stuciies. (consequently, i( is not clear if all of these genes actually serve essential roles in the cell-death process or are merely responsive to the same external signals that are used to regulate PCD. For example, it has been shown that expressi( )n of the transcription factor c-@ is upregulated in many dying tissues, most notably neurons’”. However, ~-ftisis also induced in neurons in response to non-pathi)logical stimuli, such as matingz(’. In fact, in mice cai-r) ing germ-line deficiencies in the 556
n,vs\,,1.[y ~<,.12,Iw<,
c.~~s/;erle,there is no discernible effect on the normal patterns of neuron loss during development]. Consequently, elevated levels of c-flos expression are not: (1) accurate indicators of cell death; (2) sufficient to induce cell death alone; or (3) essential for cell death to occur during development. Instead, c-fios probably serves as a signal-transduction molecule to couple a variety of extracellular stimuli to specific cellular responses, some of which might include death. ICE-family proteases are involved in neuronal death
Many diverse signals can initiate cell death in neurcms but which, if any, are essential components of the death machinery? While a complete pathway has yet to be defined, it is clear that at least one essential component is the CED-3/ICE (interleukin-1~ converting enzyme) family of cysteine proteaseszz.The presence of ced-.3is essential for the occurrence of cell death in nematodes because loss-of-function mutations in this gene blocks all PCD in wormsz:; (see Box 2). Recently, CED-3 was shown to have both structura12425 and functiona1222<’similarity with the mammalian enzyme ICE, a cysteine protease that cleaves substrates after aspartate residuesz7.ICE was initially identified as the protease responsible for the proteolytic activation of pro-ILl~ into the active cytokine. It is one member of a multigene family of cysteine proteases that share the pwrtapeptide moiety QACRG, which includes the active-site cysteinez7. (It should be noted, however, that two new ICE-family members have just been described that have a QACQG motif in the active sitez8.) As shown in ‘l-able 1, sequence analysis of this protease family allows one to segregate these enzymes into three major classes of molecules: (1) the ICE family ([CEII/TX, ICE-relII, ICE-relIII); (2) the CED-3 family (CPP32/prICE/YAMA/apopain, ICE-LAP3,MCH2, MCH3, MCH4 and MCH5); and (3) the NEDD-2/
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L.M. Schwartz and C.E. Milligan - ICE proteases in neuronal death
Box 2.The free-livingnematode C. eleganshaselucidatedessential conservedfeaturesof PCD Most of our insight into the genes that mediate programmed cell death (PCD) comes from the work of Horvitz and co-workers examining the free-living nematode Caerrorhabditis elegans. These genes fall into several classes(for review,see Ref. a). At the top of the regulatory cascadeare genes that act to specifythe celIuIarlineages that are capableof respondingto asyet undefinedsignals in sucha wayasto allowPCD to be one of the developmental options chosen by the celL Asexamples,the celldeath specification genes include ces-1 (dominant mutations prevent the loss of four specific pharyngeal neurons) and ces-2 (which appears to be a negative regulator of ces-1). The sequence of ces-2 has just been reportedand shownto be a memberof the PAR(prolineand acid-rich) subfamilyof the bZIP family of transcription factorsb.A human homolog of ces-2, the E2A-HLF (hepatic leukemiafactor) chimeric protein, which is associated with acute lymphoblasticleukemia,can also sup-
cell death in an apparentlynon-lineage-specificmanner, while gain-of-function mutations rescue otherwise condemned cells. The ced-9 gene functionsto antagonize the activityof two additionalgenes, ced-3 and ced-4 . Both of these genesareessentialfor the cell-deathprocess,as lossof-function mutations in either of these genes resultsin the blockade of PCD in C. elegarrs. It has been demonstrated that ced-4 functions upstreamof ced-3, although the nature of this interaction is currently unknownd. Once cell death has been initiated, the products of another seven genes appear to function in the phagocytosisof the corpse.Loss-of-functionmutationsin these geneshave no impact on death,but do interferewith the subsequentremovalof the corpse. Lastly,the activity of the rruc-1gene appearsto facilitatethe digestionof corpse genomic DNAwithin the phagocyticcell. References
a Ellis,R., Yuan,J. and Horvitz,H.R.(1991)Annu. Rev. Cell
pressapoptosisin mammals’.Functionally, thenextgene in theC.eZegans cell-deathcascadeis ced-9, whichbehaves
Biol. 7, 663-698
b Metzstein,M.M.et al. (1996)Nature 382, 545-547 c Inaba,T. et al. (1996)Nature 382, 541-544 d Yuan,J. and Horvitz,H.R.(1990)Dev.BioZ.138,33-41
as a dominant-negative regulator of PCD. Loss-offunction mutations in this gene resultin massiveectopic
ICH-1 family. While the specific family member(s) responsible for cell killing has not been determined, attention has focusedon the more CED-3-likeproteases such as CPP32. For example, many lymphocytes can be induced to undergo apoptosiswhen the APO-I/Fas membrane receptor is crosslinked by either monoclinal antibodies or by the natural Fas ligand (for review,see Ref. 39). Induction of apoptosisin this system involves the rapid transient activation of an ICElike proteasefollowedby a delayedand sustainedactivation of a CPP32-like protease40.In this cascade, it appearsthat the CPP32-likefamily member(s) might play a more active role in the actual induction of apoptosis. In agreement with this hypothesis is the observationthat the triggeringof apoptosisis followed rapidly by the hydrolysis of PARP [poly(ADP-ribose) polymerase](116kDa) at a specific aspartateresidueto generate two cleavageproducts of 85 kDa and 25 kDa
(Ref.41). PARPis a DNArepairenzyme,and its hydrolysis with apoptosis is thought to block both attempts at DNArepair and reductions in the cellular pools of NAD (nicotinarnide adenine dinucleotide). While CPP32readilyhydrolyzesPARP,ICE displaysa limited ability to cleave this substrate3233’41. A recent paperby Troy and co-workers further suggests that different members of the ICE family might be involved in cell death dependingon the initiating signa142.These data indicate that: (1) ICE-familymembers displaydistinct substratespecificities;and (2) CPP32-likeactivityis more directly associatedwith the induction of apoptosis. Celldeath-associated substrates for ICE-family proteases In additionto PARP,severalother cellularsubstrates have been identified that are cleaved following the initiation of apoptosis,and in some cases, it has been
TABLE I. IdentifiedICE-familymembers Gene
Identitywith ICE protein(%)
identitywith CPP32 (%)
Source
Refs
ICE ced-3 Nedd-2 ICH-I CPP32
100 29 29 27 30
30 35 30 29 100
ICH-2 ICEn,ll ICER,III
67 53 50 50 29 27 26 ND 23
32 32 30 32 38 58 53 35 39
THP. I cells (human monocyte cell line) Nematode Mouse developing CNS Human developing CNS Jurket cells (human T-cell line) THP. I cells Human umbilical vein endothelial cells Human thymus Human peripheral blood monocytes Human peripheral blood monocytes Human neutrophils Jurket cells Human neutrophils Jurket cells Jurket cells jurket cells
27 22 29 30 31 32 33 34 35
TX MCH2 ICE-LAP3 MCH3 MCH4 MCH5
34 36 37 38 28 28
Abbreviations:ICE,interleukin-1~ convertingenzyme;ND, not determined.
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L.M. Schwartz and C.E. Milligan – ICE proteases in neuronal death
Toxin Receptor engagement Hormone Trophic-factor withdrawal
signal
~)
PA fd
W
I S~ecific substrates I
Addingto the complexity of this process is the observation that cleavage of ICE-family members generates two protein subunits that are approximately 10 kDa and 20kDa in size. These proteins then assemble into a tetramer, composed of two p10 subunits and two p20 subunits, to generate the active holoenzymez~. As multiple ICE-family members have been detected simultaneously in the same cell linezs+’{”there exists the possibility that active tetramers might form with subunits from different family members, thus adding to both the number of available proteases and the potential for varied substrate specificities. For example, functional proteases can be generated with p17 subunits from CPP32 and p12 subunits from the CPP32Iike protease MCH3, and vice versaJ*. ICE-like proteases are involved in cell death in many taxa
While the strongest evidence for a role for ICE proteases in cell death was provided by genetic studies in Ccrermrhobditi.s elegatls, there is now Fig. 1. Model of rreuroncd cell death mediated by the /CE-protecme krmily. A variety of physio/ogica/ and potho/ogical stimuli can activate xignol-iranxduction pathways that result in the sequential proteolytic activation of ICE-family ample evidence suggestingthat these members. In addition, the signa/ cascade that normally induces apoptosis can be bypossed by gronzyme IB,a serine proproteases are essential mediators of tease that can be introduced into target cells by cytotoxic T lymphocytes. The activation of ICEprotecrse:ycon be inhibapoptosis in other organisms as well. ited by severa/ me/ecu/es, including the proto-oncagene product Be/-2, viro/ proteins crmA and p35, ancl small peptide The first demonstration of a role for oldehydes that target the active-site cysteine of specific /CE-fami/y members. (Note: crmA is o weak inhibitor of CPP32the proteases in mammals was gen/ike proteases relative to /CE-fike proteases27.) On(”e activated, /CE proteases can both activate other /CE-fomi/y memerated by Yuan and colleagues by bers and hydro/yse a discrete set of cellular targets. It is thought thot the specific cleavage of these substrates is responinjecting Ratl fibroblasts with either sible for the actual ‘kiRing’ of cells. Abbreviatiorw ICE, intedeukin- J/3 converting enyzme; PARP, pcfy(ADP-ribose) ICE mRNA or ICE-expressing plaspo/ymerase; SRAfP,small ribonuc/ear protein. midszb.Transected cells rapidly and These cells also underciemcmstratect that this hydrolysis is mediated by ICE- selectively underwent apoptosis. . . family members (se(>Fig. 1 and Table 2). Some of these went apoptosis when they were injected with ced-3 exapoptosis-associated substrates are cytoskeletal, such pression vectors or mRNA. The specificity of this effect as actin, fcxtrin and t ;AS2, and might play roles in the was demonstrated by the observation that co-expression of the viral serpin c-rw-A, a known inhibitor of these condensation and hlebbing of the cell that accompanies apoptosis. Ot I\crputative substrates are nuclear cysteine proteases (see Box 3) inhibited fibroblast deathz”. proteins, such as nuclear lamin B and PARP, and ICE-induced death could also be inhibited by prior might play roles in c!~rornatin condensation and DNA expression of Be/-2,suggesting that a functional relationfragmentation. lnter{:sting]y, the cleavage of actin not ship exists between these two prc)teins much as has been seen with C-cd-9and ced3 in nematodeszd (see only limits its abil iiy to polymerize into filaments, Box 2:). ICE-like proteases have also been implicated in which might play a i()Ie in the morphological changes that accompany apt}ptosis, but also blocks its ability the death of severalother cell types including Fas-induced to bind and inhibit DNAseI (Ref. 48). Because actin cell deathszsx,bepatocyte apoptosiss’),human atheroma(’(’ hydrolysis occurs ~lrly in apoptosis, it has been and mammary cpithelium cell death induced by disruption of the extracellular matrix’”. hypothesized that i~ might play a role in liberating The role of ICE-like proteases and their potential endogenous nucleas~’s that in turn mediate genomic substrates in neuronal death is beginning to be DNA cleavagc~xs+. exp[clred. The first dernonstration that ICE-family proActivation of ICE-family proteases teases play a role in neuronal cell death was provided using an immortalized nigral neuronal cell line transICE proteases normally reside in cells as inactive pro-enzymes that require proteolytic activation by ICE ected with the baculovirus ICE-prc)tease inhibitor p35 proteasc’s for their t~wn regulation (Fig. 1). ICE pro- (see Box 3; Ref. 62). In these cells, p35 was able to enzymes appear to lmssess cleavage sites similar to prevent death induced by glucose withdrawal, Ca2+ those found in othtr cellular substratesz’$is”. ft7 vitro ionophore or serum deprivation. At the time it was data have provided strong evidence that ICE-family published, it was not known where p35 exerted its members are autmatalytic, which results in their activity, so a role for I(”;Eproteases in this process was enzymatic activatiol]~’s(’. ICE proteases can also trans- not appreciated initially. The same is true for the studdemonstrating that p.35 could block activate other family members by selective hydrolysis, ies of Hay et(?I. cell death in the eye of .Qrasophila tnelfz}lc~
‘I~IV.\ \,}i.Ic),N,).
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LJ’4. Schwartz and C.E. Milligan – ICE proteasesin neuronaldeath
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TABLE 2. Potentialsubstratesfor ICE-familyproteasesduring apoptosis Substrate
Function
Cellularlocalization
Refs
Pro-lLl 13 ICE CED-3 CPP32 Fodrin (non-erythroid spectrin) 70kDa component of the UI SRNP p35 GAS2 Actin Sterol regulatory elementbinding proteins I and 2 PARP Nuclear Iamin BI Retinoblastoma Protein kinaseC-8
Cleavedto yield active cytokine Cleaved to become active proteases Cleaved to become active proteases Cleaved to become active proteases Cytoskeletal
Cytoplasm Cytoplasm Cytoplasm Cytoplasm Membrane
27 24 4s 31,33 43
RNA processing Baculovirusanti-apoptotic protein Microfilament stability Cytoskeletal component Regulation of LDL receptor expression
Cytoplasm Cytoplasm Cytoplasm Cytoplasm and nucleus Membrane
44 45,46 47 48,49 50
DNA repair Chromatin structure Cell cycle Signaltransduction
Nucleus Nucleus Nucleus Cytoplasm
41 51 S2 53
Abbreviations:ICE,interleukin-1~ conferring enzyme;LDL, low-densitylipoprotein; PARP,poly(ADP-ribose)polymerase;SRNP,smallribonuclear protein.
first direct demonstration of a role for ICEproteasesin neuronal apoptosis was provided by Yuan and colleagueswhen they demonstratedthat chicken dorsalroot ganglion (DRG)cells in vitro could survivein the absence of NGF if they were microinjected with crrnA or BcZ-2(Ref. 64). Under these conditions, otherwise condemned neurons survived, strongly supporting a role for ICE-like proteases in NGF-deprived DRG death. When the complementaryexperimentwasperformed, injecting DRGs with the murine ICE cDNA under the control of the chicken p-actin promoter, neurons died within one day, even in the presence of NGF(Ref. 64). Similardata were obtainedwith Nedd-2,
a mouseICE-familymemberthat couldinduceapoptosis in the mouseneuroblastomacell line N18 (Ref.29). This cell death was preventedif cells were also transected withBcZ-2(Ref.29). The Nedd-2 genewasclonedinitially as a transcript that is expressedat high levels in the embryonic mouse brain but is then subsequentlyrepressedduringdevelopment?9. Thispatternof expression temporallycorrelateswith the bulk of naturallyoccurring neuronal cell death in the nervous systemcs. While these in vitro manipulationssupporta role for ICEproteasesin neuronalapoptosis,little experimental work has been conducted in vivo. Germ-line deletion of the ICE gene in mice fails to alter the patterns of
Box 3. Viruses and the regulationof apoptosis In the samewaythat viruseshave ‘learned’to manipulate the cell cycle in order to facilitate their own needs for replication, so too have they learned how to regulate apoptosis to ensure that their hosts do not commit suicide and thus thwart viral replication. In fact, it has been hypothesized that the DNA fragmentation that
accompanies apoptosisaroseduringevolutionasan antiviraldefense,muchlikethe restriction-enzyme systemof bacteria,onlymorealtruistic’.Atpresent,severalviruses in particularhaveprovento be valuablein this regard. TheE1B19Kproteinfromadenovirus hasbeenshownto
is mediatedhave not been determined.Recently,cellular homologs of the IAPshave been identifiedin Drosofrkilai and havebeen shownto inhibit apoptosisin the eye, and three IAP homologs were isolatedfrom mouse that protected HeLa cells from induced deathj. Intriguingly, a human homolog of IAPwasrecentlyidentifiedas a can-
didategenefor spinalmuscularatrophy(SMA),termed NIAP(neuronalinhibitorof apoptosis)k. Teleologically, the lossof this genecouldmakeneuronsmoresusceptibleto stimulithat initiateapoptosis,thus resultingin thepreferential lossof thiscellularpopulationwithSMA.
rescueNGF-deprivedsympatheticneuronsb.It is interest- Clearlythis is an areaof intense investigation. ing to note that E1B19Kshareshomology with the BH1 and BH2 domains of Bc1-2 (Ref. c). The herpes simplex References virusneurovirulencegene and the Epstein–BarrvirusEIB a Clouston,W.M. and Kerr,J.F. (1985)Med.Hypotheses 18, proteinshave alsobeen shownto protect cells from apop399-404 tosisc,d.Cowpox and baculovirus produce proteins crmA b Martinou,I. et aL (1995)J. Cell BioL 128,201-208 c Chou, J. and Roizman,B. (1992) l+oc. hktl. Acad. .SCi. andp35, respectively,which specificallyblock membersof U.S. A. 89, 3266-3270 the ICE-proteasefamil~g. In the case of p35, it is bound d Rae, L. et al. (1992) PToc.Natl. Acad. Sci. U. S. A. 89, by and cleavedby what appearsto be ICE proteasesat a 7742-7746 specific aspartate residue. It seems that these cleavage e Xue, D. andHorvitz,H.R.(1995)Nature 377, 248-251 products remain bound by the protease and act with a 1:1 f Bump, N.J. et al. (1995)Science 269, 1885-1888 g Ray,C.A.et al. (1992)Cell69, 597-604 stoichiometry to block further enzyme activity. At present h Clem, R.J. and Miller, L.K. (1994) Mol. Cell. BioL 14, putative cellular homologs of these anti-apoptosisgenes 5212-5222 have not been identified but, by analogy, they mimic i Hay,B.A.,Wassarman,D.A.and Rubin, G.M.(1995)Cell some of the propertiesof Bc1-2. 83, 1253-1262 In additionto p35, baculovirusalso produces””a 94 kDa j Uren,A.G. et al. (1996)Proc. NatL Acad. Sci. U. S. A. 93, IAP(inhibitor of apoptosis)proteinthat can inhibit apop4974-4978 k Roy,N. et aZ. (1995)Cell80, 167-178 tosish.Atpresent,the mechanismsby whichthis inhibition
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ICE proteases in neuronal death
1 *
1 22
20)
L (6) Y
~ n
a > >
mtrol
peL de inhi tors Fig. 2.
hrhibition
m;taneuron
death
of /CE-fike proteases
prevents
per inh
naturally
tors occurring
in vivo. Chick embryos were treated with peptide
inhibitors of /CE (interleukn~ 7P converting enzyme), contro/ protease irrhib!tors or a vehicle control on day 8 of development (time of maximum motoneuron death), and pyknotic motoneurom were subsequently counted on day 9. Resultsare expressedas mean *m. Multiple Student’s t tests were prlforrned with the 8onferroni correction. P voluex were the same fol- comparisons of YVAD-CHO (aldehyde) or YVAD-CMK (ch/oromethy/ h,?tone) with control, /eupeptin or Tos-LysCMK. The number in pare fltheses represents the sample size for each *Ps(I. OT,Data taken from Ref. 68. treatment group,
neuronal cell death’’” ‘7.These mice did, however, display a deficit in F’as-induced cell death. This might not be surprising, because: (1) members of the ced-.? ‘subfamily’ are implicated more strongly as mediators of apoptosis than [(L’; and (2) the presence of multiple family members might provide functional redundancy in regulating cell cieath. ICE-null animals were, howekrer, shown to be deficient in both IL1P production and the ability [o initiate toxic shock following exposure to bacterial super-antigens; phenotypes that would be anticipateci by the loss of IL1~ (Refs 66,67). One study that ha~ supported a role for ICE proteases in vertebrate tIcuronal cell death both i~~vitro and i/7 vivo examineci the loss of motoneurons in the developing embryonic chick spinal cord”8.This system is the classic model of Hamburger and colleagues described above, where approximately 50(%, of the lumbar spinal motoneurons in chicks die between embryonic days 6–10. If these neurons are deprived of muscle targets by limb extirpation, almost all of the rnotoneurons die(’(~~(’. In contrast, if neurons are provided with ectopic targets via limb grafts or treatment with an extract made from the limb musculature, most of these motoniurons survive the normal period of cell 10ss7i;Z. These ~iataprovided much of the foundation for the troph~c-factor theory of neuron selection during development. The dependence of chick rnotoneurons on targc)t-derived factors can be demonstrated it~ vitro, as motoneurons plated with muscle extract survive, while’ those lacking this component in the media die~{”’. ‘I”his death of motoneurons in vitro requires new gene c~pression and occurs by apoptosis; +.Using this mtxfel, the role of ICE proteases in motoneuron cell death has been examined(’x. A cellpermcable inhibitor of the ICE-like proteases, YVADaldehyde, effective! blocked motoneuron death i~]vitro following trophic-factor deprivation. in complemental> experiments, it was also demonstrated that this ICE inhibitor could block the naturally occurring rnotoneuron death il] vivo as well. In this experiment, peptide ICE-proteasc inhibitors were administered
systemically to chick embryos i}r ovo on day 8 of development, the time of maximum naturally occurring motoneuron death. By 24 h after treatment with the pept:ide inhibitors of ICE proteases, there was approximately a SO(H)reduction in the number of pyknctic cells and a corresponding increase in the number of healthy rnotoneurons (Fig. 2). interestingly, treatment of chick embryos with ICE inhibitors also prevented the PCD of interdigital cells, suggesting a potential role for ICE-like proteases in this cell loss as well”~.It should be noted, however, that the peptide inhibitors used in this study failed to prevent all forms of neuronal death. For example, the~e inhibitors had no effect on: motoneuron death following limb-bud remm;al; the target-independent death of cervical motoneurons; or on cell death in the early neural tube(’s. ‘l-his could be due to: (1) experimental problems ;associated with delivering an effective dose of inhibitor to the target cells at the required time in development; (2) the involvement of other ICE-family members, such as CPP32, which are poorly inhibited by YV”AD-aldehyde;or (3) possible differences in the molecular mechanisms of PCD among cell populations, particularly those between target-dependent and target-independent neurons. In experiments in vitro, these same authors demonstrated that inhibition of ICE protmses only delayed motoneuron death, rather than prevent it, and a subsequent ‘survival’ signal was necessary for apparent reversal of the cell-death processfx. Some considerations and future directions
It is also worth mentioning what we do not know about the role of ICE-family proteases in cell death in general and neuronal death in particular. First (as noted above) while there is substantial evidence supporting a role for ICE-like proteases in cell death ill vitra, only limited data exist to support this hypothesis irzvivo. In addition, the identities of the specific proteases required for PCD during development have yet to be identified. Data obtained with C. ele,yczrl.s provide the most conclusive data for the role of a specific Asp-ase in cell death during development. Although the data using p35 in flies”:; and YVAD-aldehyde in chicks<’xargue that ICE-family proteases play a role in PCD iIZvivo, they do not identify the relevant family member(s). Indeed, the limited data available examining germ-line knockouts in mice have not provided a significant apoptosis phenotype, with the exception of Fas-mediated cell loss. The use of germ-line deletion of spelcific ICE-family members could provide a powerful tool for examining the role(s) of ICE proteases in vivo. However, there are several potential problems, inducting lack of a phenotype due to protease redundancy or embryonic lethality due to disrupted development. Given that apoptosis occurs even at the earliest stages of development, for example during cavitations, there is the real possibility that global loss of these proteases might block full-term development. A second general area of ignorance is the determination of which ICE-protease substrates are essential mediators of death and which happen to be irrelevant targets. F’orexample, while the observation that actin is hydrolyses during apoptosis provides several attractive and testable hypotheses, it does not directly account for the death of the cell. In addition, while PAR]>appears to be a logical candidate, its role in physiological cell death is not conclusive, as apoptosis
-
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.
appears to occur normally in PARP-deficientmammalian cells76.Further complicating this picture is the recent finding by Yuan and co-workersthat while ICElike proteasesmight be a common effecter mechanism for neuron death, different suppressionmechanisms might be in place depending on the specific neurotrophic factor to which the cell has been exposed77.A correlatedissue is the developmentalstate of the cell, because some regulatory factors are apoptosis mediators only at specific stages of development. For example, motoneurons appear to be initially targetindependent, and as they mature further develop a dependence on target-derived trophic support78. Therefore, it appears that cells must mature to an appropriate stage of differentiation before they become competent to respond to developmental death signals, such as the removal of trophic support. A third consideration is the observationthat not all programmed cell deaths display the morphology associatedwith apoptosis (see Box 1). This raises the possibility that different cell-death morphologies are due to: (1) the existence of both ICEfamily-dependent and -independent pathways; (2) the utilization of differentICEproteasesin specificcell-deathsituations; or (3) the availabilityor utilization of different groups of protease substratesin different cell deaths. A fourth and important issueis whether the activity of ICE proteasescan be manipulated in vivo to afford any clinical benefits, that is, can ICE proteasesbe activated or inhibited in a tissue-specificmanner to facilitate the loss of deleterious cells or the retention of doomed but valuable cells? Clearly this is the goal of many investigatorswho hope to be able selectivelyto induce the non-inflammatory loss of malignant cells or the retention of neurons in neurodegenerativedisorders.While there is much enthusiasm for the possibilities of administering inhibitors of ICE in pathological conditions, such as following spinal-cord injuries to prevent the death of motoneurons, a better understandingof the specific protease(s)involved in the death process is necessary. For example, while specific inhibitors of ICE might prevent cell death, they might also block normal microphage function. Activated macrophages exhibit a large repertoire of actions, includingproduction and secretion of IL1 (for review, see Ref. 79). It is well documented that macrophages respond to injury sites and might be a necessary component of recovery8081.Inhibition of their function might, therefore, inhibit recovery followinginjury or in pathological conditions. Last is the issue of whether there are other proteolytic cascadesthat are essentialregulatorsof cell death. In recent studies with thymocytesxzand sympathetic neurons deprivedof NGF (Ref. 83) it has been demonstratedthat selectiveinhibition of the proteasomecan prevent apoptosis initiated by a wide variety of noxious stimuli. In both of these studies,not only wascell death prevented, but so too was PARP hydrolysis, suggestingthat the proteasome was acting upstream of ICE proteases in the apoptosis cascade. Specific questions, however,remain regardingthe specificrole of the proteasome in cell death and how it regulates the activity of ICE proteases. The discoverythat CED-3/ICEproteases are essential mediatorsof apoptosisrepresentsa profound leap in our understandingof the mechanisms that mediate PCD. Given the rapid progress in defining how ICE
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. REVIEW
Schwartz and C.E. Milligan – ICE proteasesin neuronaldeath
Cold thoughts of death: the role of ICE proteases in neuronal cell death Lawrence M. Schwartz and Carolanne E. Milligan While there has been extensivework describingthe timing, location and probable signals responsiblefor regulatingprogrammedcelldeath(PCD) in the nervoussystem,relativelylittleis knownabout the molecularmechanismsthat mediatethis process.Severalinvestigatorshave demonstratedthat PCD in general,andneuronalPCD in particular,canbe inhibitedby drugsthat arrestRNA or proteinsynthesis. Thesedatahavebeeninterpretedassuggesting thatdenovogene expressionis requiredfor cellsto commit suicide. The generalpictureemergingfrom a number of experimentalsystemsis that a varietyof proteinscan mediatethe couplingof extracellular signalsto a residentcell-deathprogram.[nthismodel,someofthe componentsrequiredfor death are more or lessconstitutivelypresentin the cell and awaitlineage-specific signalsfor their activation.A recentfloodof papershaspresentedconvincingevidencethatthe residentprogram for apoptosisin numerouscelltypesworksvia a seriesof essentialproteasesbelongingto the CED-3/lCE family. Trends Neurosci. (1996) 19, 555-562
HE EXISTENCEof programmed cell death (PCD) was presumably noted in ancient times with the T observation that tadpoles lose their tails during metamorphosis. Serious examination of the phenomenon began much later. For example, a century ago Beard’ observedthat specific neurons were lost in a spatially and temporally reproduciblepattern during development. Fifty years later, Victor Hamburger and Rita Levi-Montalcini2published a landmark paper using quantitative methods to demonstrate that massive neuronal loss was a normal component of nervoussystemdevelopment.Since then it has been well documented that in most regions of the central and peripheral nervous systems the programmed death of both neurons and glia during discrete periods of development is a normal process (for reviews, see Refs 3–7; also see Box 1 for a discussion on the morphological types of PCD that occur in the developing nervous system). The identification of the inter- and intracellular events that mediate PCD will enhance our understandingof normal developmentgreatly.In addition, the identification of the key regulatorycomponents responsible for regulating PCD might allow us to rescue selectivelyvaluablebut condemned cells, such as neurons in Alzheimer’s disease or alternatively, to target the removal of deleteriouscells, such as malignant cells. The difficulty in studying the molecular events involvedin regulatingPCDin neurons,or in anyheterogeneous tissue, is that condemned cells are interspersedamong viable ones. Compounding this problem is the fact that while cell death might ultimately involve the loss of a significant number of neurons within a given population, the speed of the process and the absence of tight developmentalco-ordination might mean that at any given moment dying cells might represent less than 1% of the total cellular population’. For example, in chicks, approximately Copyright @ 1996, Elsevier Science Ltd. All rights reserved. 0166-
2236/96/$15.00
12000 of the normal 24000 lumbar spinal motoneurons die during 4-5 days of embryogenesiss. Nevertheless, the maximal number of pyknotic motoneurons that can be observedduring this period in serial sections of the spinal cord is about 300 (Ref.7). This apparentpaucityof dyingcellscomplicates biochemical analyses, such as the identification of differentiallyexpressedgene products, since it would be difficult to distinguishbetween the events associated with PCD versus those processestaking place in survivingcells. Consequently,most investigatorshave turned to in vitro systemsto investigatethe biochemical and molecular mechanisms involvedin regulating cell death. While this is a valuableapproachfor defining mechanisms that could be essential for regulating a differentiativeprocess,there is still a requirementfor in vivo studiesto confirm that the processunder invesLawrenceM. tigation is, in fact, an essential one. Changes ingeneexpression associated with neuronaldeath Where it has been examined, the ability of cells to undergoPCDappearsto be dependenton de novo gene expression (reviewedin Ref. 9). [It should be noted that the term ‘programmedcell death’ is used here with its originalmeaning: the spatiallyand temporally reproducibleloss of cells within a developmentalcontext10 (see Box l).] Data supporting this hypothesis were generated initially in experiments where condemned cells were treated with inhibitors of RNAor protein synthesis.Instead of being toxic to cells, these metabolic poisons were protective and prevented cell deathll-14.The simplest interpretation was that these drugsblocked the synthesis of essential components of the cell-death machinery. Teleologically,this was a very appealinghypothesisas one wouldassumethat a variety of fail-safe mechanisms exist within cells to prevent inappropriateactivation of a suicideprogram. PII: S0166-2236(96)1
OO67-9
TINS Vol. 19,
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12, 1996
Schwartzis at the Deptof Biologyand Programin Moiecularand CeZkdarBiology, Universityof Massachusetts, Amherst, MA01003, USA, and CarolanneE. Milliganis at the Dept of Neurobiologyand Anatomy,Bowman GraySchool of Medicineof Wake Forest University, Winston-Salem, NC 27157, USA.
555
RFVIE\Y
L.M. Schwartz and C.E. Milligan
ICE proteases in neuronzl death
Box 1. Programmed
cell death, apoptosis and non-apoptotic cell loss in the nervous system
The t(rm apcq]tc)sis describes the morphological and structural changes IIiat accompany most programmed cell deaths seen ciurinx development, including: mcmbrane blebbing, I)NA ft:i~];lerlt~ltiorl,marination of chromatin along the innel- aspcd of the nuclear envelope and phago cytosis of the apupii)tic bodies”. It is important to note, however, that sornc of the cells that die by programmed cell death (PCD) dLIIjng development dispkrv few if any of thew specific chan~es (reviewed in Refs b,c). These nonapoptotic CCIIdeaths, most of which arc seen in the nervous system, hiive been :cfcrred to in the older literature as ‘type 11’or ‘cytoplairnic’ degeneration’’”. Whether conp(mcmk of the cell-dc,athcascade, such as 1(X-1ikeprotetrses, arc conserved betwc(,:)these two types of cell cieath remains to I)(, dctcrm ined. iI is interesting to note that mature neurons, which are !~ighlydifferentiated cells that al-eunIikel},to re-enter th( cell cycle or serve as hosts for most viruses, might not Iw as dependent on DNAfragmentation m a fail-sale mechanism as are actively dividing cells. [t should also IWnotmf that most neurobiologists and immunologists end, IWthe term programmed cell death with ciifferent mcar in,qs. The term, as used initially by lock shin and Will!ims’, referred to the temporally and
spatially ]reproducibic loss of cells within a developmental context. implicit in this term is the expectation that death is triggered as a normal component of development by physiological triggers. Immunologists appear to have a broader definition for 1’(.;1)that includes any death that is dependent on a cellular pro,grarn, even if the trigger for death is non-physiological in nature (reviewed in Ref. g). Thcmfore while an immunologist would consider irradiation-induced tieath in lymphocytes an example of P([), a developmental neurobiolcrgist might not. References a Wyllie, A.H., Kerr, .l.k’.R. and Currie, A.R. (1980)Int. Rev. Cw’i. f)x, 2s 1–306 b Schwartz, L.M., Kosz, L. and Kay, JLK.(1990) Pm. Nd. .4(-[/[/..Sci. U. S. A. 87, 6594-6598 c Clarke, P.G. H. (1 990) A?ztit. Et)/bryd. 181, 195–21:3 d Pilar, G. and I.andmesser, L.J. (1976) J. L’ell JJiol. 68, 3:}%35(, e
(:hwWang, I-W. and Oppenheim, it.W. (1978)/. C’orrZp. Nef{rd.
f g
177, 3+58
t.oc.kshin,R.A.and Williams, (1.M.(1965)/. Inset-tPhysid. 1I, 12:-1–13:+ Schwartz, 1..M.and Osborne, B.A. ( 1993) /mtIIIIfm/. Today 14, 582!–590
-.
The newi to synthesize essential mediators of this pathway would prcwidesuch a safety feature. As will be discussed below, it now appears that the killing components of this pathway are already resident in the cell, and that tht>newly synthesized components are used to couple c.xternal signals to this lethal internal reach inery. Several cell-cycle lwgulators, immediate–early genes and proto-oncogene~ have been shown to be upreguIated with the comrrl itment of cells to die. One series of experiments focused on rat superior cervical ganglion (SCG) cells, wtlich survive well in culture when provided with NGF. in the absence of NGF, these cells die rapidly by apopt( Isis’ (See Box 1 for a description and terminology. ) of cell-death morphologies Coincident with this death is the enhanced accumulation of mRNAs tor the proto-oncogenes c-jw7, c-fi~.s,~i~sB and jut?-fl the transcription factor NGF-lA, the cell-cycle regulator cyclin Dl, transirr and collagenasei’. A simiiai timetable of events has been demonstrated for 1)(”12 cells when they die following the cieprivation of tr<)phic factor]”. In the case of c-jun, it has been shown Ihat neutralizing antibodies17 or dominant negative c-jun proteins18 could protect NGF-deprived sympathetic neurons. These experiments suggest that AP-1 activity is essential for this cell death. In contrast to tht studies of c-jun, the analysis of most cell death-associated genes has involved predominantly descriptive as opposed to functional stuciies. (consequently, i( is not clear if all of these genes actually serve essential roles in the cell-death process or are merely responsive to the same external signals that are used to regulate PCD. For example, it has been shown that expressi( )n of the transcription factor c-@ is upregulated in many dying tissues, most notably neurons’”. However, ~-ftisis also induced in neurons in response to non-pathi)logical stimuli, such as matingz(’. In fact, in mice cai-r) ing germ-line deficiencies in the 556
n,vs\,,1.[y ~<,.12,Iw<,
c.~~s/;erle,there is no discernible effect on the normal patterns of neuron loss during development]. Consequently, elevated levels of c-flos expression are not: (1) accurate indicators of cell death; (2) sufficient to induce cell death alone; or (3) essential for cell death to occur during development. Instead, c-fios probably serves as a signal-transduction molecule to couple a variety of extracellular stimuli to specific cellular responses, some of which might include death. ICE-family proteases are involved in neuronal death
Many diverse signals can initiate cell death in neurcms but which, if any, are essential components of the death machinery? While a complete pathway has yet to be defined, it is clear that at least one essential component is the CED-3/ICE (interleukin-1~ converting enzyme) family of cysteine proteaseszz.The presence of ced-.3is essential for the occurrence of cell death in nematodes because loss-of-function mutations in this gene blocks all PCD in wormsz:; (see Box 2). Recently, CED-3 was shown to have both structura12425 and functiona1222<’similarity with the mammalian enzyme ICE, a cysteine protease that cleaves substrates after aspartate residuesz7.ICE was initially identified as the protease responsible for the proteolytic activation of pro-ILl~ into the active cytokine. It is one member of a multigene family of cysteine proteases that share the pwrtapeptide moiety QACRG, which includes the active-site cysteinez7. (It should be noted, however, that two new ICE-family members have just been described that have a QACQG motif in the active sitez8.) As shown in ‘l-able 1, sequence analysis of this protease family allows one to segregate these enzymes into three major classes of molecules: (1) the ICE family ([CEII/TX, ICE-relII, ICE-relIII); (2) the CED-3 family (CPP32/prICE/YAMA/apopain, ICE-LAP3,MCH2, MCH3, MCH4 and MCH5); and (3) the NEDD-2/
REVIEW
L.M. Schwartz and C.E. Milligan - ICE proteases in neuronal death
Box 2.The free-livingnematode C. eleganshaselucidatedessential conservedfeaturesof PCD Most of our insight into the genes that mediate programmed cell death (PCD) comes from the work of Horvitz and co-workers examining the free-living nematode Caerrorhabditis elegans. These genes fall into several classes(for review,see Ref. a). At the top of the regulatory cascadeare genes that act to specifythe celIuIarlineages that are capableof respondingto asyet undefinedsignals in sucha wayasto allowPCD to be one of the developmental options chosen by the celL Asexamples,the celldeath specification genes include ces-1 (dominant mutations prevent the loss of four specific pharyngeal neurons) and ces-2 (which appears to be a negative regulator of ces-1). The sequence of ces-2 has just been reportedand shownto be a memberof the PAR(prolineand acid-rich) subfamilyof the bZIP family of transcription factorsb.A human homolog of ces-2, the E2A-HLF (hepatic leukemiafactor) chimeric protein, which is associated with acute lymphoblasticleukemia,can also sup-
cell death in an apparentlynon-lineage-specificmanner, while gain-of-function mutations rescue otherwise condemned cells. The ced-9 gene functionsto antagonize the activityof two additionalgenes, ced-3 and ced-4 . Both of these genesareessentialfor the cell-deathprocess,as lossof-function mutations in either of these genes resultsin the blockade of PCD in C. elegarrs. It has been demonstrated that ced-4 functions upstreamof ced-3, although the nature of this interaction is currently unknownd. Once cell death has been initiated, the products of another seven genes appear to function in the phagocytosisof the corpse.Loss-of-functionmutationsin these geneshave no impact on death,but do interferewith the subsequentremovalof the corpse. Lastly,the activity of the rruc-1gene appearsto facilitatethe digestionof corpse genomic DNAwithin the phagocyticcell. References
a Ellis,R., Yuan,J. and Horvitz,H.R.(1991)Annu. Rev. Cell
pressapoptosisin mammals’.Functionally, thenextgene in theC.eZegans cell-deathcascadeis ced-9, whichbehaves
Biol. 7, 663-698
b Metzstein,M.M.et al. (1996)Nature 382, 545-547 c Inaba,T. et al. (1996)Nature 382, 541-544 d Yuan,J. and Horvitz,H.R.(1990)Dev.BioZ.138,33-41
as a dominant-negative regulator of PCD. Loss-offunction mutations in this gene resultin massiveectopic
ICH-1 family. While the specific family member(s) responsible for cell killing has not been determined, attention has focusedon the more CED-3-likeproteases such as CPP32. For example, many lymphocytes can be induced to undergo apoptosiswhen the APO-I/Fas membrane receptor is crosslinked by either monoclinal antibodies or by the natural Fas ligand (for review,see Ref. 39). Induction of apoptosisin this system involves the rapid transient activation of an ICElike proteasefollowedby a delayedand sustainedactivation of a CPP32-like protease40.In this cascade, it appearsthat the CPP32-likefamily member(s) might play a more active role in the actual induction of apoptosis. In agreement with this hypothesis is the observationthat the triggeringof apoptosisis followed rapidly by the hydrolysis of PARP [poly(ADP-ribose) polymerase](116kDa) at a specific aspartateresidueto generate two cleavageproducts of 85 kDa and 25 kDa
(Ref.41). PARPis a DNArepairenzyme,and its hydrolysis with apoptosis is thought to block both attempts at DNArepair and reductions in the cellular pools of NAD (nicotinarnide adenine dinucleotide). While CPP32readilyhydrolyzesPARP,ICE displaysa limited ability to cleave this substrate3233’41. A recent paperby Troy and co-workers further suggests that different members of the ICE family might be involved in cell death dependingon the initiating signa142.These data indicate that: (1) ICE-familymembers displaydistinct substratespecificities;and (2) CPP32-likeactivityis more directly associatedwith the induction of apoptosis. Celldeath-associated substrates for ICE-family proteases In additionto PARP,severalother cellularsubstrates have been identified that are cleaved following the initiation of apoptosis,and in some cases, it has been
TABLE I. IdentifiedICE-familymembers Gene
Identitywith ICE protein(%)
identitywith CPP32 (%)
Source
Refs
ICE ced-3 Nedd-2 ICH-I CPP32
100 29 29 27 30
30 35 30 29 100
ICH-2 ICEn,ll ICER,III
67 53 50 50 29 27 26 ND 23
32 32 30 32 38 58 53 35 39
THP. I cells (human monocyte cell line) Nematode Mouse developing CNS Human developing CNS Jurket cells (human T-cell line) THP. I cells Human umbilical vein endothelial cells Human thymus Human peripheral blood monocytes Human peripheral blood monocytes Human neutrophils Jurket cells Human neutrophils Jurket cells Jurket cells jurket cells
27 22 29 30 31 32 33 34 35
TX MCH2 ICE-LAP3 MCH3 MCH4 MCH5
34 36 37 38 28 28
Abbreviations:ICE,interleukin-1~ convertingenzyme;ND, not determined.
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L.M. Schwartz and C.E. Milligan – ICE proteases in neuronal death
Toxin Receptor engagement Hormone Trophic-factor withdrawal
signal
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I S~ecific substrates I
Addingto the complexity of this process is the observation that cleavage of ICE-family members generates two protein subunits that are approximately 10 kDa and 20kDa in size. These proteins then assemble into a tetramer, composed of two p10 subunits and two p20 subunits, to generate the active holoenzymez~. As multiple ICE-family members have been detected simultaneously in the same cell linezs+’{”there exists the possibility that active tetramers might form with subunits from different family members, thus adding to both the number of available proteases and the potential for varied substrate specificities. For example, functional proteases can be generated with p17 subunits from CPP32 and p12 subunits from the CPP32Iike protease MCH3, and vice versaJ*. ICE-like proteases are involved in cell death in many taxa
While the strongest evidence for a role for ICE proteases in cell death was provided by genetic studies in Ccrermrhobditi.s elegatls, there is now Fig. 1. Model of rreuroncd cell death mediated by the /CE-protecme krmily. A variety of physio/ogica/ and potho/ogical stimuli can activate xignol-iranxduction pathways that result in the sequential proteolytic activation of ICE-family ample evidence suggestingthat these members. In addition, the signa/ cascade that normally induces apoptosis can be bypossed by gronzyme IB,a serine proproteases are essential mediators of tease that can be introduced into target cells by cytotoxic T lymphocytes. The activation of ICEprotecrse:ycon be inhibapoptosis in other organisms as well. ited by severa/ me/ecu/es, including the proto-oncagene product Be/-2, viro/ proteins crmA and p35, ancl small peptide The first demonstration of a role for oldehydes that target the active-site cysteine of specific /CE-fami/y members. (Note: crmA is o weak inhibitor of CPP32the proteases in mammals was gen/ike proteases relative to /CE-fike proteases27.) On(”e activated, /CE proteases can both activate other /CE-fomi/y memerated by Yuan and colleagues by bers and hydro/yse a discrete set of cellular targets. It is thought thot the specific cleavage of these substrates is responinjecting Ratl fibroblasts with either sible for the actual ‘kiRing’ of cells. Abbreviatiorw ICE, intedeukin- J/3 converting enyzme; PARP, pcfy(ADP-ribose) ICE mRNA or ICE-expressing plaspo/ymerase; SRAfP,small ribonuc/ear protein. midszb.Transected cells rapidly and These cells also underciemcmstratect that this hydrolysis is mediated by ICE- selectively underwent apoptosis. . . family members (se(>Fig. 1 and Table 2). Some of these went apoptosis when they were injected with ced-3 exapoptosis-associated substrates are cytoskeletal, such pression vectors or mRNA. The specificity of this effect as actin, fcxtrin and t ;AS2, and might play roles in the was demonstrated by the observation that co-expression of the viral serpin c-rw-A, a known inhibitor of these condensation and hlebbing of the cell that accompanies apoptosis. Ot I\crputative substrates are nuclear cysteine proteases (see Box 3) inhibited fibroblast deathz”. proteins, such as nuclear lamin B and PARP, and ICE-induced death could also be inhibited by prior might play roles in c!~rornatin condensation and DNA expression of Be/-2,suggesting that a functional relationfragmentation. lnter{:sting]y, the cleavage of actin not ship exists between these two prc)teins much as has been seen with C-cd-9and ced3 in nematodeszd (see only limits its abil iiy to polymerize into filaments, Box 2:). ICE-like proteases have also been implicated in which might play a i()Ie in the morphological changes that accompany apt}ptosis, but also blocks its ability the death of severalother cell types including Fas-induced to bind and inhibit DNAseI (Ref. 48). Because actin cell deathszsx,bepatocyte apoptosiss’),human atheroma(’(’ hydrolysis occurs ~lrly in apoptosis, it has been and mammary cpithelium cell death induced by disruption of the extracellular matrix’”. hypothesized that i~ might play a role in liberating The role of ICE-like proteases and their potential endogenous nucleas~’s that in turn mediate genomic substrates in neuronal death is beginning to be DNA cleavagc~xs+. exp[clred. The first dernonstration that ICE-family proActivation of ICE-family proteases teases play a role in neuronal cell death was provided using an immortalized nigral neuronal cell line transICE proteases normally reside in cells as inactive pro-enzymes that require proteolytic activation by ICE ected with the baculovirus ICE-prc)tease inhibitor p35 proteasc’s for their t~wn regulation (Fig. 1). ICE pro- (see Box 3; Ref. 62). In these cells, p35 was able to enzymes appear to lmssess cleavage sites similar to prevent death induced by glucose withdrawal, Ca2+ those found in othtr cellular substratesz’$is”. ft7 vitro ionophore or serum deprivation. At the time it was data have provided strong evidence that ICE-family published, it was not known where p35 exerted its members are autmatalytic, which results in their activity, so a role for I(”;Eproteases in this process was enzymatic activatiol]~’s(’. ICE proteases can also trans- not appreciated initially. The same is true for the studdemonstrating that p.35 could block activate other family members by selective hydrolysis, ies of Hay et(?I. cell death in the eye of .Qrasophila tnelfz}lc~
‘I~IV.\ \,}i.Ic),N,).
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LJ’4. Schwartz and C.E. Milligan – ICE proteasesin neuronaldeath
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TABLE 2. Potentialsubstratesfor ICE-familyproteasesduring apoptosis Substrate
Function
Cellularlocalization
Refs
Pro-lLl 13 ICE CED-3 CPP32 Fodrin (non-erythroid spectrin) 70kDa component of the UI SRNP p35 GAS2 Actin Sterol regulatory elementbinding proteins I and 2 PARP Nuclear Iamin BI Retinoblastoma Protein kinaseC-8
Cleavedto yield active cytokine Cleaved to become active proteases Cleaved to become active proteases Cleaved to become active proteases Cytoskeletal
Cytoplasm Cytoplasm Cytoplasm Cytoplasm Membrane
27 24 4s 31,33 43
RNA processing Baculovirusanti-apoptotic protein Microfilament stability Cytoskeletal component Regulation of LDL receptor expression
Cytoplasm Cytoplasm Cytoplasm Cytoplasm and nucleus Membrane
44 45,46 47 48,49 50
DNA repair Chromatin structure Cell cycle Signaltransduction
Nucleus Nucleus Nucleus Cytoplasm
41 51 S2 53
Abbreviations:ICE,interleukin-1~ conferring enzyme;LDL, low-densitylipoprotein; PARP,poly(ADP-ribose)polymerase;SRNP,smallribonuclear protein.
first direct demonstration of a role for ICEproteasesin neuronal apoptosis was provided by Yuan and colleagueswhen they demonstratedthat chicken dorsalroot ganglion (DRG)cells in vitro could survivein the absence of NGF if they were microinjected with crrnA or BcZ-2(Ref. 64). Under these conditions, otherwise condemned neurons survived, strongly supporting a role for ICE-like proteases in NGF-deprived DRG death. When the complementaryexperimentwasperformed, injecting DRGs with the murine ICE cDNA under the control of the chicken p-actin promoter, neurons died within one day, even in the presence of NGF(Ref. 64). Similardata were obtainedwith Nedd-2,
a mouseICE-familymemberthat couldinduceapoptosis in the mouseneuroblastomacell line N18 (Ref.29). This cell death was preventedif cells were also transected withBcZ-2(Ref.29). The Nedd-2 genewasclonedinitially as a transcript that is expressedat high levels in the embryonic mouse brain but is then subsequentlyrepressedduringdevelopment?9. Thispatternof expression temporallycorrelateswith the bulk of naturallyoccurring neuronal cell death in the nervous systemcs. While these in vitro manipulationssupporta role for ICEproteasesin neuronalapoptosis,little experimental work has been conducted in vivo. Germ-line deletion of the ICE gene in mice fails to alter the patterns of
Box 3. Viruses and the regulationof apoptosis In the samewaythat viruseshave ‘learned’to manipulate the cell cycle in order to facilitate their own needs for replication, so too have they learned how to regulate apoptosis to ensure that their hosts do not commit suicide and thus thwart viral replication. In fact, it has been hypothesized that the DNA fragmentation that
accompanies apoptosisaroseduringevolutionasan antiviraldefense,muchlikethe restriction-enzyme systemof bacteria,onlymorealtruistic’.Atpresent,severalviruses in particularhaveprovento be valuablein this regard. TheE1B19Kproteinfromadenovirus hasbeenshownto
is mediatedhave not been determined.Recently,cellular homologs of the IAPshave been identifiedin Drosofrkilai and havebeen shownto inhibit apoptosisin the eye, and three IAP homologs were isolatedfrom mouse that protected HeLa cells from induced deathj. Intriguingly, a human homolog of IAPwasrecentlyidentifiedas a can-
didategenefor spinalmuscularatrophy(SMA),termed NIAP(neuronalinhibitorof apoptosis)k. Teleologically, the lossof this genecouldmakeneuronsmoresusceptibleto stimulithat initiateapoptosis,thus resultingin thepreferential lossof thiscellularpopulationwithSMA.
rescueNGF-deprivedsympatheticneuronsb.It is interest- Clearlythis is an areaof intense investigation. ing to note that E1B19Kshareshomology with the BH1 and BH2 domains of Bc1-2 (Ref. c). The herpes simplex References virusneurovirulencegene and the Epstein–BarrvirusEIB a Clouston,W.M. and Kerr,J.F. (1985)Med.Hypotheses 18, proteinshave alsobeen shownto protect cells from apop399-404 tosisc,d.Cowpox and baculovirus produce proteins crmA b Martinou,I. et aL (1995)J. Cell BioL 128,201-208 c Chou, J. and Roizman,B. (1992) l+oc. hktl. Acad. .SCi. andp35, respectively,which specificallyblock membersof U.S. A. 89, 3266-3270 the ICE-proteasefamil~g. In the case of p35, it is bound d Rae, L. et al. (1992) PToc.Natl. Acad. Sci. U. S. A. 89, by and cleavedby what appearsto be ICE proteasesat a 7742-7746 specific aspartate residue. It seems that these cleavage e Xue, D. andHorvitz,H.R.(1995)Nature 377, 248-251 products remain bound by the protease and act with a 1:1 f Bump, N.J. et al. (1995)Science 269, 1885-1888 g Ray,C.A.et al. (1992)Cell69, 597-604 stoichiometry to block further enzyme activity. At present h Clem, R.J. and Miller, L.K. (1994) Mol. Cell. BioL 14, putative cellular homologs of these anti-apoptosisgenes 5212-5222 have not been identified but, by analogy, they mimic i Hay,B.A.,Wassarman,D.A.and Rubin, G.M.(1995)Cell some of the propertiesof Bc1-2. 83, 1253-1262 In additionto p35, baculovirusalso produces””a 94 kDa j Uren,A.G. et al. (1996)Proc. NatL Acad. Sci. U. S. A. 93, IAP(inhibitor of apoptosis)proteinthat can inhibit apop4974-4978 k Roy,N. et aZ. (1995)Cell80, 167-178 tosish.Atpresent,the mechanismsby whichthis inhibition
TINSVO1. 19, No. 12,1996
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L.M. Schwartz and C.E. Milligan
ICE proteases in neuronal death
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inhibitors of /CE (interleukn~ 7P converting enzyme), contro/ protease irrhib!tors or a vehicle control on day 8 of development (time of maximum motoneuron death), and pyknotic motoneurom were subsequently counted on day 9. Resultsare expressedas mean *m. Multiple Student’s t tests were prlforrned with the 8onferroni correction. P voluex were the same fol- comparisons of YVAD-CHO (aldehyde) or YVAD-CMK (ch/oromethy/ h,?tone) with control, /eupeptin or Tos-LysCMK. The number in pare fltheses represents the sample size for each *Ps(I. OT,Data taken from Ref. 68. treatment group,
neuronal cell death’’” ‘7.These mice did, however, display a deficit in F’as-induced cell death. This might not be surprising, because: (1) members of the ced-.? ‘subfamily’ are implicated more strongly as mediators of apoptosis than [(L’; and (2) the presence of multiple family members might provide functional redundancy in regulating cell cieath. ICE-null animals were, howekrer, shown to be deficient in both IL1P production and the ability [o initiate toxic shock following exposure to bacterial super-antigens; phenotypes that would be anticipateci by the loss of IL1~ (Refs 66,67). One study that ha~ supported a role for ICE proteases in vertebrate tIcuronal cell death both i~~vitro and i/7 vivo examineci the loss of motoneurons in the developing embryonic chick spinal cord”8.This system is the classic model of Hamburger and colleagues described above, where approximately 50(%, of the lumbar spinal motoneurons in chicks die between embryonic days 6–10. If these neurons are deprived of muscle targets by limb extirpation, almost all of the rnotoneurons die(’(~~(’. In contrast, if neurons are provided with ectopic targets via limb grafts or treatment with an extract made from the limb musculature, most of these motoniurons survive the normal period of cell 10ss7i;Z. These ~iataprovided much of the foundation for the troph~c-factor theory of neuron selection during development. The dependence of chick rnotoneurons on targc)t-derived factors can be demonstrated it~ vitro, as motoneurons plated with muscle extract survive, while’ those lacking this component in the media die~{”’. ‘I”his death of motoneurons in vitro requires new gene c~pression and occurs by apoptosis; +.Using this mtxfel, the role of ICE proteases in motoneuron cell death has been examined(’x. A cellpermcable inhibitor of the ICE-like proteases, YVADaldehyde, effective! blocked motoneuron death i~]vitro following trophic-factor deprivation. in complemental> experiments, it was also demonstrated that this ICE inhibitor could block the naturally occurring rnotoneuron death il] vivo as well. In this experiment, peptide ICE-proteasc inhibitors were administered
systemically to chick embryos i}r ovo on day 8 of development, the time of maximum naturally occurring motoneuron death. By 24 h after treatment with the pept:ide inhibitors of ICE proteases, there was approximately a SO(H)reduction in the number of pyknctic cells and a corresponding increase in the number of healthy rnotoneurons (Fig. 2). interestingly, treatment of chick embryos with ICE inhibitors also prevented the PCD of interdigital cells, suggesting a potential role for ICE-like proteases in this cell loss as well”~.It should be noted, however, that the peptide inhibitors used in this study failed to prevent all forms of neuronal death. For example, the~e inhibitors had no effect on: motoneuron death following limb-bud remm;al; the target-independent death of cervical motoneurons; or on cell death in the early neural tube(’s. ‘l-his could be due to: (1) experimental problems ;associated with delivering an effective dose of inhibitor to the target cells at the required time in development; (2) the involvement of other ICE-family members, such as CPP32, which are poorly inhibited by YV”AD-aldehyde;or (3) possible differences in the molecular mechanisms of PCD among cell populations, particularly those between target-dependent and target-independent neurons. In experiments in vitro, these same authors demonstrated that inhibition of ICE protmses only delayed motoneuron death, rather than prevent it, and a subsequent ‘survival’ signal was necessary for apparent reversal of the cell-death processfx. Some considerations and future directions
It is also worth mentioning what we do not know about the role of ICE-family proteases in cell death in general and neuronal death in particular. First (as noted above) while there is substantial evidence supporting a role for ICE-like proteases in cell death ill vitra, only limited data exist to support this hypothesis irzvivo. In addition, the identities of the specific proteases required for PCD during development have yet to be identified. Data obtained with C. ele,yczrl.s provide the most conclusive data for the role of a specific Asp-ase in cell death during development. Although the data using p35 in flies”:; and YVAD-aldehyde in chicks<’xargue that ICE-family proteases play a role in PCD iIZvivo, they do not identify the relevant family member(s). Indeed, the limited data available examining germ-line knockouts in mice have not provided a significant apoptosis phenotype, with the exception of Fas-mediated cell loss. The use of germ-line deletion of spelcific ICE-family members could provide a powerful tool for examining the role(s) of ICE proteases in vivo. However, there are several potential problems, inducting lack of a phenotype due to protease redundancy or embryonic lethality due to disrupted development. Given that apoptosis occurs even at the earliest stages of development, for example during cavitations, there is the real possibility that global loss of these proteases might block full-term development. A second general area of ignorance is the determination of which ICE-protease substrates are essential mediators of death and which happen to be irrelevant targets. F’orexample, while the observation that actin is hydrolyses during apoptosis provides several attractive and testable hypotheses, it does not directly account for the death of the cell. In addition, while PAR]>appears to be a logical candidate, its role in physiological cell death is not conclusive, as apoptosis
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appears to occur normally in PARP-deficientmammalian cells76.Further complicating this picture is the recent finding by Yuan and co-workersthat while ICElike proteasesmight be a common effecter mechanism for neuron death, different suppressionmechanisms might be in place depending on the specific neurotrophic factor to which the cell has been exposed77.A correlatedissue is the developmentalstate of the cell, because some regulatory factors are apoptosis mediators only at specific stages of development. For example, motoneurons appear to be initially targetindependent, and as they mature further develop a dependence on target-derived trophic support78. Therefore, it appears that cells must mature to an appropriate stage of differentiation before they become competent to respond to developmental death signals, such as the removal of trophic support. A third consideration is the observationthat not all programmed cell deaths display the morphology associatedwith apoptosis (see Box 1). This raises the possibility that different cell-death morphologies are due to: (1) the existence of both ICEfamily-dependent and -independent pathways; (2) the utilization of differentICEproteasesin specificcell-deathsituations; or (3) the availabilityor utilization of different groups of protease substratesin different cell deaths. A fourth and important issueis whether the activity of ICE proteasescan be manipulated in vivo to afford any clinical benefits, that is, can ICE proteasesbe activated or inhibited in a tissue-specificmanner to facilitate the loss of deleterious cells or the retention of doomed but valuable cells? Clearly this is the goal of many investigatorswho hope to be able selectivelyto induce the non-inflammatory loss of malignant cells or the retention of neurons in neurodegenerativedisorders.While there is much enthusiasm for the possibilities of administering inhibitors of ICE in pathological conditions, such as following spinal-cord injuries to prevent the death of motoneurons, a better understandingof the specific protease(s)involved in the death process is necessary. For example, while specific inhibitors of ICE might prevent cell death, they might also block normal microphage function. Activated macrophages exhibit a large repertoire of actions, includingproduction and secretion of IL1 (for review, see Ref. 79). It is well documented that macrophages respond to injury sites and might be a necessary component of recovery8081.Inhibition of their function might, therefore, inhibit recovery followinginjury or in pathological conditions. Last is the issue of whether there are other proteolytic cascadesthat are essentialregulatorsof cell death. In recent studies with thymocytesxzand sympathetic neurons deprivedof NGF (Ref. 83) it has been demonstratedthat selectiveinhibition of the proteasomecan prevent apoptosis initiated by a wide variety of noxious stimuli. In both of these studies,not only wascell death prevented, but so too was PARP hydrolysis, suggestingthat the proteasome was acting upstream of ICE proteases in the apoptosis cascade. Specific questions, however,remain regardingthe specificrole of the proteasome in cell death and how it regulates the activity of ICE proteases. The discoverythat CED-3/ICEproteases are essential mediatorsof apoptosisrepresentsa profound leap in our understandingof the mechanisms that mediate PCD. Given the rapid progress in defining how ICE
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