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Neuronal cell dea th : w h e n , w hy and how

Lee L RubinEisat London Research Laboratories Ltd, University College London, London, UK

Apoptosis is recognised increasingly as a prominent event in nervous systemdevelopment and disease.This form of death appears to obey the same rules inneurones as in othe r cells, in that it is initiated by similar extracellular perturba tionsand distinguished by similar morphological and biochemical changes. Whenneurones die after survival factor w ithd raw al, gene transcription is importan t, withthe transcription factor c-jun and the cytoplasmic signalling cascade that regulatesit being particu larly significant in at least some types of cells. However, death canbe induced in a trans cription-independ ent manner by agents such as

staurosporine. Both types of death involve activation of members of the ICE fam ilyof proteases but, surprisingly, the pa rticular protease involved seems to depe ndvery much on the manner in which death is initiated.

Correspondence toLee L Rubin

Eisai London ResearchLaboratories Ltd

Bernard Katz BuildingUniversity College

London Gower StreetLondon W O E 66T, UK

Cell death in the nervous system occurs under three sets ofcircumstances, with perhaps three sets of underlying mechanisms.During embryonic and early postnatal development, a large percentage(perhaps 50% or so) of neurones in each region of the nervous system dieby programm ed cell death. The timing varies from region to region, but,

in each case, this death is though t to be by apoptosis and to be similar tothat occurring in other tissues, in that it is the result of competition for alimited amount of one or more extracellular survival factors, generallypolypeptide in nature. One difference for neurones may be that thesefactors are often target-derived and, hence, produced at a distance andtransported back to the neuronal cell body. A second phase of cell deathaccompanies a variety of neurodegenerative disorders, such as Alzhei-mer's disease. In these cases, the cell death may be quite significant inamount, but it may occur over a period of years, so that at any point intime, there are only a small number of dying cells. The causes of deathassociated with degenerative diseases are often not known and, untilrecently, there was little data concerning the types of death thatoccurred. A final instance of neuronal cell death occurs after the hypoxiathat accompanies stroke. In this case, a large amount of neuronal celldeath m ay take place over a period of days, with the type of death beinga current topic of debate.

This article will focus on recent studies of neuronal cell death, with anemphasis on apoptosis. Death of cultured neurones in differentsituations will be discussed first. Cell death modulators and pathways

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will be described, with particular regard to intracellular events thatinitiate and that retard death. These events will be seen to be similar tothose taking place in other types of cells. Finally, there will be anextensive account of how the different types of neuronal cell deathshould be classified, and of whether it is meaningful, from a mechamsticpoint of view, to make an absolute distinction between necrosis andapoptosis.

Neu rona l cell de ath in culture

The fact that developmental or programmed cell death of neurones hadbeen studied in some detail led to the establishment of useful cell culturemodels of these types of death. The types of cells utilised most often

include: rat superior cervical ganglion (SCG) neurones, which are nervegrowth factor (NGF)-dependent sympathetic neurones, PC12 cells, anNGF-dependent neurone-like cell line, and cerebellar granule neurones(CGNs), normally grown in the presence of high extracellular K+, tosupport survival1-3. When deprived of NGF or, in the case of CGNs,when K+ is lowered, these cells die by classical apoptosis, withmembrane blebbing, neurite fragmentation, chromatin condensation,formation of apoptotic bodies, a decrease in dehydrogenase activity(measured by the MTT reaction) and DN A laddering, all taking place ata time at which the plasma membrane remains relatively intact (non-leaky to dyes such as trypan blue and propidium iodide). The death isrelatively slow (the comm itment time is 15-20 h, and 50 % cell deathtakes place in 448 h, although CGN death is somewhat faster) andasynchronous; this presents problems for certain types of biochemicaland molecular biological experiments. One clear observation, pertainingat least to SCG and CGN cell death, is that the process is blocked byinhibitors of mRN A or protein synthesis, as is again typical of apoptosis.Thus, death of neurones in response to survival factor withdrawal isdependent on gene transcription and subsequent protein synthesis.

Following the observation that high concentrations of the non-specificprotein kinase inhibitor staurosporine initiate apoptosis in many cell

types4

, this drug was also applied to different kinds of cultured neurones.Staurosporine-induced death was typically apoptotic, at least usingmorphological criteria5, and the sequence of events seemed roughly thesame as with survival factor withdrawal. Mechanistically, however,there was one major difference from survival factor withdrawal death inthat the staurosporine type was not blocked by RNA or protein synthesisinhibitors. Thus, neurones, like other cells, appear to have a set of deatheffectors even when present, m cell culture, in a seemingly healthy state.

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While the death initiated by these two procedures was thought toreproduce some aspects of programmed cell death and, perhaps of thedeath that accompanies neurodegenerative disease, investigators soughtto derive a model tha t might reveal more about the type of cell death tha toccurs after stroke. Numerous groups have examined cultured rat ormouse cortical neurones m aintained for a brief period in an hypoxic andhypoglycaemic state6'7. Under these conditions, cells swell rapidly anddie by a process normally agreed to represent necrotic death. Themechanism is presumed to be release of glutamate from depolarisedneurones, followed by excess Ca2+ entry into the cells8. Another way ofstudying this type of death is simply to apply high concentrations ofglutamate directly to the neurones9'10. Again, many cells die rapidly,mostly by necrosis. How ever, Choi et al 7 found that blocking NMDAand AM PA/kainate receptors together slowed much of the rapid necroticdeath, leaving the cells to die by apoptosis at later times. Thus, there isan underlying apoptotic component to this glutamate-induced death.Recent work also suggests the existence of a similar underlyingcomponent in stroke brain itself (see below).

Intracellular changes during neu ronal cell dea th

The type of neuronal cell death brought on by survival factorwithdrawal can be thought of as potentially consisting of three phases.In the first phase, the cell 'senses' the absence of the factor. This isaccomplished by activation or inactivation of cytoplasmic signallingpathways. Following these cytoplasmic changes, there is a phase ofrequired gene expression. Finally, there is the appearance or activationof the death effectors themselves (meant here as the molecules thatproduce the changes that define death). It is also necessary to unders tandthat staurosporine, and related initiators of apoptosis, bypass the firsttwo phases and produce direct activation of extant cytoplasmiceffectors.

Cytoplasmic signalling, transcription factors a ndneu rona l cell de ath

NGF activates multiple signalling pathways, including the MAP kinasecascade, that are important for survival, neurite elongation, and otherprocesses associated with differentiation in PC 12 cells and sympatheticneurones see, for example, Nobes and Tolkovsky11 and Xia et al 12 .When NGF is removed from these cells, and they begin to lose their

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differentiated phenotype and die, these signalling pathways turn off, andat the same time, other signalling pathways turn on. Since genetranscription plays an important role in the initiation of apoptosisfollowing NGF removal, it should be the case that the activity ofparticular transcription factors is altered early in the death process. It islogical to assume that this is achieved via one or more of these signallingevents.

This possibility has been examined by several groups. Cultured SCGneurones dying following NGF withdrawal have again been mostcarefully studied. One of the earliest events in these cells observablewithin 4 h or so of NG F withdraw al was the appearance of thephosphorylated, active, form of the transcription factor c-jun and anincrease in its mRNA and protein levels13-14. Both in situ hybridisationand immunocytochemistry revealed an increase in c-jun in mostneurones, even before they adopted an apoptotic morphology. Levels

of other members of the AP-1 family of transcription factors did notchange; in particular, c-fos, once thought to be essential in the deathprocess, only appeared in relatively high concentration in a smallnumber of frankly apoptotic neurones. Nonetheless, because c-jun hadbeen implicated in many types of cellular changes, it was necessary toshow that it was functionally important in the onset of death. In oneseries of experiments, neuronal c-jun was blocked by microinjection ofan anti-c-jun antibody13. In another, a similar result was obtained bymicromjection of an expression plasmid for a transcriptionally inactive,dominant negative, c-jun variant14. In both experiments, the rate of celldeath was noticeably slowed. Thus, blocking c-jun regulated genetranscription blocks death. It was also important to see what wouldhappen if c-jun levels were increased in the presence of NGF. When thiswas achieved, again by microinjecting neurones with an expressionvector, the rate of death was accelerated. Therefore, high levels of c-junare sufficient to induce death even in the presence of NGF14.

Recent work has suggested that levels of c-jun might increase in otherapopto tic situations as well. For example, treatment of cortical neuroneswith amyloid-EJ-peptide produced an increase in c-jun mRNA and anaccumulation of nuclear c-jun protein, early in the death process15. Ourlaboratory has also shown recently that c-jun increases rapidly when

CGNs undergo apoptosis (A. Watson et al, manuscript submitted forpublication).Since apoptosis is associated with an increase in the phosphorylated,

transcriptionally active, form of c-jun, it might be expected that theactivity of JNK, the kinase that phosphorylates c-jun, would increaseafter NGF withdrawal. In fact, Greenberg et aln have shown this to betrue in PC12 cells, and our laboratory has similar data for sympatheticneurones16. A further prediction is that over-expression of catalytically

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active variants of upstream kinases should lead to death, even in thepresence of NGF. This was also tested by Greenberg et a l, who foundthat transient transfection of PC 12 cells with constitutively activeMEKK1 (which phosphorylates and activates SEK1, which phosphor-ylates and activates JNK) kills them . This death was blocked by

simultaneous over-expression of dominant-negative c-jun. Again, ourlaboratory has obtained similar results w ith sympathetic neurones16.Finally, Greenberg et al showed that activation of another parallelsignalling pathw ay that for p38 MAP kinase can also lead to celldeath. Thus, it can be concluded that, under at least certaincircumstances, apoptosis is due to the induction of signal transductionpathways that regulate particular transcription factors. Remaining to beestablished is the generality of these observations. That is, are the p38MAP kinase/JNK pathways the initiators of death in all kinds ofneurones under all circumstances, at least when apoptosis is involved?

Gen e activity and neu ronal apoptosis

The experimental observation that survival factor withdrawal-inducedneuronal apoptosis can be blocked by actinomycin D and cycloheximidewas w idely interpreted to mean that cell death initiated following factorwithdrawal was based on the appearance of new proteins that causedeath. These were meant to be proteins tha t previously w ere either notpresent at all or were present at very low levels when survival factorswere still available17. While this is not the only interpretation of theseexperimental data, a wide variety of experiments have been designed toaddress this possibility. Certainly, it has been the case that mRNA levelsof a number of potentially interesting proteins, such as cyclin Dl insympathetic neurones18, have been found to increase early in the deathprocess, but still no particular transcriptional event has been implicatedin apoptosis. This work will not be described in detail here. However,the identification of particular functionally imp ortant transcriptionfactors may assist in the search for neuronal death genes.

Cytoplasmic effectors of neu rona l cell dea thIn this discussion, it is important to distinguish between cytoplasmicchanges that accompany or are upstream initiators of the death processand those that actually produce the changes that we associate withdeath. Sometimes, this can be difficult, though. For example, the role ofreactive oxygen species in neuronal cell dea th is controversial. Althoughsome types of cells clearly are able to die in the total absence of reactive

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oxygen19'20, some investigators still feel that reactive oxygen is anessential part of the death process in neurones. We favour the view that,in certain instances, oxygen radicals may be upstream initiators of death,possibly acting via a pathway such as the JNK cascade.

Discussions of death effectors invariably centre around the role ofICE-like proteases (now termed caspases), which undoubtedly arefeatured prominently in many articles in this issue. In that regard,neurones behave like all other cells in tha t a cascade of caspases seems tobe activated during death, as judged by various criteria, including: (i)initiation of neuronal cell death by over-expression of ICE; (ii) use offluorescent enzyme substrates; (iii) cleavage of know n caspase substrates;and (iv) inhibition of neuronal cell death by caspase inhibitors.

Much of the work concerning caspases and neurones has been carriedout on NGF-dependent neurones dying following NGF withdrawal.Gagliardini et a/21 microinjected cultured sensory neurones with an

expression vector for either wild-type murine ICE or enzymaticallyinactive ICE. Wild-type ICE killed cells maintained in NGF, whileinactive ICE had no effect. This suggests tha t unregulated ICE activity issufficient to kill cells even when they are maintained under normalsurvival conditions.

There is also evidence that induction of a caspase cascade occursduring apop tosis and is functionally significant. Direct proof forincreased caspase activity in dying neurones has been provided bySchulz et alu, who showed cleavage of a fluorescent caspase substrateduring the death of CGNs. Further evidence was provided byGagliardini et aln, who microinjected sensory neurones with anexpression vector for the viral ICE inhibitor crmA. These cells wereconsequently less likely to die following NGF withdrawal. Similarstudies were done by Martinou et aP 3 who over-expressed, again bymicroinjection, the baculovirus caspase inhibitor p35 in SCG neurones.They found that this inhibitor also offered a significant degree ofprotection against death due to NGF withdrawal.

A variety of peptide-type caspase inhibitors of varying specificitiestowards individual members of the caspase family have also beenapplied to dying neurones. The general finding has been that several ofthese inhibitors block growth factor withdrawal death, although they

vary in effectiveness to some degree. zVAD-fmk, a somewhat generalinhibitor of caspases, has blocked cell death in several different systems,including PC12 cells24 and sympathetic neurones (McCarthy, Rubin andPhilpott, submitted for publication). However, it was ineffective inblocking low K+-death of CGNs58. YVAD-based inhibitors, which blockat least ICE itself, were found to block motor neuron apoptosis inculture and during normal avian embryo development15 and to blocklow K+-death of CGNs22. DEVD-type inhibitors, derived from the

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cleavage sequence of PARP, a CPP-32 substrate, blocked death of SCGneurones (McCarthy, Rubin and Philpott, submitted for publication).However, for this inhibitor to be effective, it was necessary tomicroinject it into the cells because it has very limited membranepermeability.

Since staurosporine induces neuronal apoptosis by direct activation ofdeath effectors, it was important to determine if it acted via caspases.Evidence that this is true again comes from several different studies.Philpott et al s found that lamin, a known caspase substrate26, is cleavedfrom 9 kD a to 46 kDa during death of PC12 and SCG cells. Taylor etal 5S found that the nuclear enzyme PARP, a caspase substrate, wasdegraded from a 116 kDa intact form to an 85 kDa proteolytic fragmentduring CGN death. Thus, caspases are activated during staurosporinedeath. That this is required for death was suggested by the work ofMcCarthy, Rubin and Philpott (submitted for publication) who found

that over-expression of baculovirus p35 in SCG neurones preventedstaurosporine-induced death, much as it did NGF-withdrawal death.Interesting differences were revealed, however, when the effects of

peptide ILP inhibitors were compared. DEVD-fmk was effective on bothstaurosporine and growth factor-withdrawal types of death in SCGneurones. On the other hand, zVAD-fmk was more effective in blockingNG F death than staurosporine death of these cells (Taylor et al submittedfor publication). Unexpectedly, in CGNs, zVAD-fmk was very effective inblocking staurosporine death, but no t very good a t blocking low K+-death.Differences in the effectiveness of these inhibitors was also found by Troyet at 14 who compared death of PC12 cells due to either down-regulationof superoxide dismutase or withdrawal of serum or NGF.

These results suggest that there are several ways of inducing neuronalapoptosis that involve the same types of morphological and biochemicalpathways. However, it seems that there is variability, both amongdifferent types of neurones and among different initiators of death in anindividual neuronal type, in the particular caspases that cause death. Itwill be very important to determine the source of this variability. Forinstance, are the very upstream activators of the caspase cascade in thedifferent types of cell death identical, with differences arising in at leastsome of the downstream proteases? Alternatively, do the different

stimulators of death generate different intracellular cascades at theironset, with perhaps some overlap of downstream caspases?

Bcl-2, ba x , and neuronal cell death

Entry into the death cascade in most cell types involves the participationof bcl-2 and bax-like proteins27. Bcl-2 over-expression appears to block

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death, probably by inhibiting entry into the caspase cascade, whereas baxseems to stimulate the onset of the cascade. It has been clear for some timethat bcl-2 over-expression is anti-apoptotic in neurones. Martinou et at18

injected sympathetic neurones with an expression plasmid for bcl-2 andfound that survival of these cells in the absence of NGF was improved.Allsopp et al

29

obtained similar results for other types of neurotrophin-dependent neurones, but found, surprisingly, that bcl-2 over-expressiondid not block death of neurones dependent on ciliary neurotrophic factor(CNTF). This group also found that antisense bcl-2 constructs decreasedthe ability of neurotrophins to support survival30. However, theseconstructs failed to affect the activity of CNTF. This might mean thatCNTF promotes survival by a mechanism dependent on a bcl-2-relatedprotein . It is clearly the case that other m embers of the bcl-2 family, suchas bcl-x, support survival31. This is reasonable since many types of adultneurones have undetectably low levels of bcl-2 with levels of bcl-x being

more substantial. An important role for such related proteins is confirmedby the observation that there is some neuronal cell death in bcl-2knockout mice, but not massive malformation of the nervous system.

Bax and related pro-death proteins are important regulators ofneuronal apoptosis. NGF addition to PC12 cells causes a substantialdecrease in their bax levels, suggesting that this is a normal part ofdifferentiation. This is consistent with the finding that bax is high inneurones at developmental times when there is significant programmedcell death, but decreases substantially afterwards59. Interestingly, baxlevels remain high in certain neuronal populations, possibly those thatare particularly susceptible to dying. For instance, bax levels are high inadult cerebellar Purkinje neurones, which die most readily when thecerebellum is made ischaemic32. The important role of bax can beevaluated directly in several ways. One is to engineer its over-expression.When this is done in sym pathetic neurones, they die, even in the presenceof NGF. This death can be blocked by concomitant over-expression ofbcl-x and is also blocked by p35 over-expression, which indicates thatBax initiates death by activating the caspase cascade59.

Necrosis versus apo ptosis in neuronal cell de athOne of the most controversial topics amongst investigators focusing onneuronal cell death occurring under pathological conditions relates toclassification: is it necrosis or is it apoptosis? Everyone agrees that celldeath in development resembles apoptosis. Most investigators nowbelieve that neurodegenerative disorders, such as Alzheimer's disease,involve apoptosis to a substantial degree and, until recently, most

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probably accepted the theory tha t neuronal death in stroke ispredominantly by necrosis.

An important question is whether or not it is valuable to categoriseneuronal death as being of one type or the other. We will conclude th atthere is likely to be some sharing of intracellular pathways in the twoprocesses. However, although in extreme cases it is not difficult todistinguish between the two types, one very im portant issue must centreon the criteria which can be used in a given situation to distinguishbetween the two types of death.

The classical view is that necrotic cells are swollen, due to earlychanges in the permeability of their plasma membranes, and areassociated with inflammation in response to leaked cytoplasmicconstituents. Apoptotic cells have intact membranes, distorted orga-nelles, condensed chromatin and are not associated with inflammationsince apoptotic cells are generally engulfed early in the death process.

Ultrastructural analysis should be able to determine the death type, butthis is a time-consuming technique and not useful as a routineexperimental procedure. It is particularly difficult in studies that requirethe use of biopsied or post mortem human nervous tissue. As alreadymentioned, the rapid disappearance of apoptotic cells presents anotherproblem in trying to ascertain the type of cell death.

Thus, investigators have sought more convenient procedures, two ofwhich are now routinely used. The first is to look for labelling with theTUNEL technique, which is generally considered to be diagnostic forapoptosis, but can occur during necrosis as well. The second is to isolatetissue from damaged regions and look for evidence of DNA fragmentation.DNA from apoptotic cells is cleaved at 180 bp intervals and runs as aladder, while that from necrotic cells runs as a smear. However, thistechnique is also problematic from two points of view. It is not always easyto see DNA laddering even in cultured neurones undergoing apoptosis, andany tissue sample might contain only a small percentage of apoptotic cells,visible by TUNEL staining or, perhaps, by ultrastructural examination, butnot able to produce enough DNA to make laddering obvious. So, in theend, many studies simply describe whether or not TUNEL-positive cells orDNA laddering is seen. The presumption is that if either occurs, cell dea thby apoptosis is involved. However, it is clear that this type of information

is often not conclusive in deciding on the type of cell death.

Death in neurodegen erative diseases

Despite all of these problems, there has been a recent flurry of paperstrying to determine the extent of, especially apoptotic, cell death invarious degenerative diseases. These experiments are difficult, for the

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reasons just outlined, and compounded by having to use human biopsysamples, which themselves are often processed slowly, perhaps leadingto more cell death. Nonetheless, reasonable progress has been made.Using Alzheimer's disease human brain samples, several investigatorshave found evidence for TUNEL-positive cells, including cortical andhippocampal neurones33 35. However, there is some disagreement as towhether or not these cells have a typically apoptotic morphology.Further, there is some confusion as to whether there is a directcorrelation between the location of dying cells and the presence ofamyloid plaques and neurofibrillary tangles, the distinguishing patholo-gical features of the disease. In cell culture, the situation is somewhatclearer. It appears that addition of high concentrations of amyloid-P-peptide kills cortical neurones by apoptosis, with chromatin condensa-tion, DNA fragmentation and surface blebbing15'36.

Other neurodegenerative diseases have also been studied. In

Huntington's disease samples, TUNEL-positive cells were seen in thestriatum, and there was some indication that these cells were dying byapoptosis, although it was difficult to find DNA fragmentationconsistently37-39. In status epilepticus in rodents40 '41 and scrapie42, therehave been descriptions of apoptosis. Finally, a very interesting case isthat of spinal-muscular atrophy, which is associated with extensiveapoptosis of motor neurones43-44. The gene affected in this disease istermed NAIP, neuronal apoptosis inhibitor protein, and is homologousto a baculovirus inhibitor of apoptosis. When over-expressed in differentcell types, not yet including neurones, NAIP inhibits apoptosis, asexpected45.

Cell death in stroke

Of all disorders of the nervous system, stroke has been examined mostcarefully for its cell death phenotype. One reason for this (other than theobvious prevalence and importance of the condition) is the relativeavailability of animal models (although the exact correspondencebetween the different models and the human disease is frequentlydebated). However, another reason is that the neuronal cell death isfairly extensive and relatively rapid, occurring in hours to days. This is atremendous experimental advantage when compared to the slowdegenerative disorders. The common view until recently was that celldeath in stroke was entirely by necrosis, with excess glutamate releasecausing Ca2+-overload, cell swelling and death8'9. However, when thefrequency of apoptosis as a death type became clear, investigators wereinterested in discovering if it accompanied stroke as well. Reviewing the

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now extensive literature w ould itself require a review of this size, but it ispossible to summarise the essential inform ation fairly succinctly. Themost important point is that there is an apoptotic component toneuronal death that follows stroke46 50. This has been established by: i)the appearance of TUNEL-positive cells; ii) DNA laddering in tissuesamples; iii) ultrastructure of dying cells; and (IV) partial inhibition ofdeath by cycloheximide. There is variability in the degree of apoptosis,depending on species, time and type of occlusion, time of reperfusion,and so on. The clearest example of apoptosis is in the CA1 region of thehippocampus, in which death is significantly delayed w ith respect to theonset of ischaemia51'52. Other significant indications that apoptosisoccurs in stroke include inhibition by agents normally thought of asbeing anti-apoptotic bcl-253*54 and caspase inhibitors55.

Necrosis and apoptosis: how different are they?

The concluding topic in this review will centre around the question ofhow important it is to divide types of death into discrete categories. Apragmatic position is that the important issue is not categorisation, butprevention. Of course , it seems logical to expect that know ing the type ofcell death underlying a particular disorder will be very important in thatregard, but this will be difficult for the following reasons. First, somedisorders are associated with dying cells tha t have some characteristicsnormally thought to be associated with apoptosis and some withnecrosis. In some situations, it is very difficult to decide on the dom inantpheno type. Second, the same type of stimulus hypoxia, glutam ate, thecalcium ionophore A23187 can lead to either necrosis or apoptosis orboth, depending on length of treatment, concentration of drug, etc.Third, certain agents neurotrop hins, bcl-2, caspase inhibitors normally categorised as anti-apoptotic seem to block types of deathoften thought of as necrotic.

The situation with A 23187 is, in a way, particularly instructive in tha tlow concentrations kill by apoptosis, high by necrosis56. This pre-sumably means that events underlying apoptosis must begin to occur

when A23187-treated cells are dying by necrosis and would becomeobvious if they survived for a long enough time. The same is true ofhypoxia-induced death of cortical cells that, as already mentioned,undergo delayed apoptosis made apparent if the 'necrotic-type' death isblocked pharmacologically.

A recent set of experiments carried out by Tsujimoto et al 57 is alsoextremely important. They placed three different types of cells underhypoxic conditions. The cell types all underwent a mixture of necrosis

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and apoptosis, with the ratio varying from one cell type to the next. Yet,bcl-2 blocked all types of death in these cells. These experiments suggestimmediately that at least some of the events underlying necrosis andapoptosis must be similar or even identical.

Conclusion

Apoptosis in neurones, resulting from growth factor withdrawal oroccurring in different neurodegenerative disorders, is fundamentallysimilar to tha t in other cell types. It is normally activated by cytoplasmicsignalling pathways, transmitted via transcription factors and alterationsin gene transcription, and carried out by the appearance or activation ofcytoplasmic effectors. There are cell-specific events, but ICE-likeproteases are key effectors of the death programme, and bcl-2 andbax-like proteins regulate entry into the pathway. While neuronal celldeath has two extreme phenotypes, apoptosis and necrosis, there may bemany forms of death that are not so simple to distinguish. Nonetheless,substantial progress has been made in understanding these processes andin blocking them, at least in experimental systems.

Acknowledgements

I would like to thank members of Eisai's cell death group (C. Bazenet,H. Desmond, A. Eilers, C. Gatchalian, J. Ham, G. Keen, M. McCarthy,M. Mota, S. Neame, K. Philpott, G. Rimon, C. Spadoni, J. Taylor,K. Vekrellis, A. W atson and J. Whitfield) for their hard work and helpfulcomments and Ms Helen Blake for help in preparing this article.

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