Neuronal cell death: when, why and how
Lee L Rubin
Eisat London Research Laboratories Ltd, University College London, London, UK
Apoptosis is recognised increasingly as a prominent event in nervous system
development and disease.This form of death appears to obey the same rules in
neurones as in other cells, in that it is initiated by similar extracellular perturbations
and distinguished by similar morphological and biochemical changes. When
neurones die after survival factor withdrawal, gene transcription is important, with
the transcription factor c-jun and the cytoplasmic signalling cascade that regulates
it being particularly significant in at least some types of cells. However, death can
be induced in a transcription-independent manner by agents such as
staurosporine. Both types of death involve activation of members of the ICE family
of proteases but, surprisingly, the particular protease involved seems to depend
very much on the manner in which death is initiated.
Correspondence to:
Lee L Rubin,
Eisai London Research
Laboratories Ltd,
Bernard Katz Building,
University College
London, Gower Street,
London W O E 66T, UK
Cell death in the nervous system occurs under three sets of
circumstances, 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 die
by programmed cell death. The timing varies from region to region, but,
in each case, this death is thought to be by apoptosis and to be similar to
that occurring in other tissues, in that it is the result of competition for a
limited amount of one or more extracellular survival factors, generally
polypeptide in nature. One difference for neurones may be that these
factors are often target-derived and, hence, produced at a distance and
transported back to the neuronal cell body. A second phase of cell death
accompanies a variety of neurodegenerative disorders, such as Alzheimer's disease. In these cases, the cell death may be quite significant in
amount, but it may occur over a period of years, so that at any point in
time, there are only a small number of dying cells. The causes of death
associated with degenerative diseases are often not known and, until
recently, there was little data concerning the types of death that
occurred. A final instance of neuronal cell death occurs after the hypoxia
that accompanies stroke. In this case, a large amount of neuronal cell
death may take place over a period of days, with the type of death being
a current topic of debate.
This article will focus on recent studies of neuronal cell death, with an
emphasis on apoptosis. Death of cultured neurones in different
situations will be discussed first. Cell death modulators and pathways
Bntith Medical Bulletin 1997;53 (No 3)617-o31
©The Bntuh Council 1997
Apoptosis
will be described, with particular regard to intracellular events that
initiate and that retard death. These events will be seen to be similar to
those taking place in other types of cells. Finally, there will be an
extensive account of how the different types of neuronal cell death
should be classified, and of whether it is meaningful, from a mechamstic
point of view, to make an absolute distinction between necrosis and
apoptosis.
Neuronal cell death in culture
The fact that developmental or programmed cell death of neurones had
been studied in some detail led to the establishment of useful cell culture
models of these types of death. The types of cells utilised most often
include: rat superior cervical ganglion (SCG) neurones, which are nerve
growth factor (NGF)-dependent sympathetic neurones, PC12 cells, an
NGF-dependent neurone-like cell line, and cerebellar granule neurones
(CGNs), normally grown in the presence of high extracellular K+, to
support survival1-3. When deprived of NGF or, in the case of CGNs,
when K+ is lowered, these cells die by classical apoptosis, with
membrane blebbing, neurite fragmentation, chromatin condensation,
formation of apoptotic bodies, a decrease in dehydrogenase activity
(measured by the MTT reaction) and DNA laddering, all taking place at
a time at which the plasma membrane remains relatively intact (nonleaky to dyes such as trypan blue and propidium iodide). The death is
relatively slow (the commitment time is 15-20 h, and 50% cell death
takes place in 24—48 h, although CGN death is somewhat faster) and
asynchronous; this presents problems for certain types of biochemical
and molecular biological experiments. One clear observation, pertaining
at least to SCG and CGN cell death, is that the process is blocked by
inhibitors of mRNA or protein synthesis, as is again typical of apoptosis.
Thus, death of neurones in response to survival factor withdrawal is
dependent on gene transcription and subsequent protein synthesis.
Following the observation that high concentrations of the non-specific
protein 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 using
morphological criteria5, and the sequence of events seemed roughly the
same as with survival factor withdrawal. Mechanistically, however,
there was one major difference from survival factor withdrawal death in
that the staurosporine type was not blocked by RNA or protein synthesis
inhibitors. Thus, neurones, like other cells, appear to have a set of death
effectors even when present, m cell culture, in a seemingly healthy state.
618
Bnhth Mtdical Bullttm 199723 (No 3)
Neuronal cell death
While the death initiated by these two procedures was thought to
reproduce some aspects of programmed cell death and, perhaps of the
death that accompanies neurodegenerative disease, investigators sought
to derive a model that might reveal more about the type of cell death that
occurs after stroke. Numerous groups have examined cultured rat or
mouse cortical neurones maintained for a brief period in an hypoxic and
hypoglycaemic state6'7. Under these conditions, cells swell rapidly and
die by a process normally agreed to represent necrotic death. The
mechanism is presumed to be release of glutamate from depolarised
neurones, followed by excess Ca2+ entry into the cells8. Another way of
studying this type of death is simply to apply high concentrations of
glutamate directly to the neurones9'10. Again, many cells die rapidly,
mostly by necrosis. However, Choi et al7 found that blocking NMDA
and AMPA/kainate receptors together slowed much of the rapid necrotic
death, leaving the cells to die by apoptosis at later times. Thus, there is
an underlying apoptotic component to this glutamate-induced death.
Recent work also suggests the existence of a similar underlying
component in stroke brain itself (see below).
Intracellular changes during neuronal cell death
The type of neuronal cell death brought on by survival factor
withdrawal can be thought of as potentially consisting of three phases.
In the first phase, the cell 'senses' the absence of the factor. This is
accomplished by activation or inactivation of cytoplasmic signalling
pathways. Following these cytoplasmic changes, there is a phase of
required gene expression. Finally, there is the appearance or activation
of the death effectors themselves (meant here as the molecules that
produce the changes that define death). It is also necessary to understand
that staurosporine, and related initiators of apoptosis, bypass the first
two phases and produce direct activation of extant cytoplasmic
effectors.
Cytoplasmic signalling, transcription factors and
neuronal cell death
NGF activates multiple signalling pathways, including the MAP kinase
cascade, that are important for survival, neurite elongation, and other
processes associated with differentiation in PC 12 cells and sympathetic
neurones (see, for example, Nobes and Tolkovsky11 and Xia et al12).
When NGF is removed from these cells, and they begin to lose their
Bntnh Medico/ BuH.fin 1997^3 (No 3)
619
Apoptosii
differentiated phenotype and die, these signalling pathways turn off, and
at the same time, other signalling pathways turn on. Since gene
transcription plays an important role in the initiation of apoptosis
following NGF removal, it should be the case that the activity of
particular transcription factors is altered early in the death process. It is
logical to assume that this is achieved via one or more of these signalling
events.
This possibility has been examined by several groups. Cultured SCG
neurones dying following NGF withdrawal have again been most
carefully studied. One of the earliest events in these cells — observable
within 4h or so of NGF withdrawal — was the appearance of the
phosphorylated, active, form of the transcription factor c-jun and an
increase in its mRNA and protein levels13-14. Both in situ hybridisation
and immunocytochemistry revealed an increase in c-jun in most
neurones, even before they adopted an apoptotic morphology. Levels
of other members of the AP-1 family of transcription factors did not
change; in particular, c-fos, once thought to be essential in the death
process, only appeared in relatively high concentration in a small
number of frankly apoptotic neurones. Nonetheless, because c-jun had
been implicated in many types of cellular changes, it was necessary to
show that it was functionally important in the onset of death. In one
series of experiments, neuronal c-jun was blocked by microinjection of
an anti-c-jun antibody13. In another, a similar result was obtained by
micromjection of an expression plasmid for a transcriptionally inactive,
dominant negative, c-jun variant14. In both experiments, the rate of cell
death was noticeably slowed. Thus, blocking c-jun regulated gene
transcription blocks death. It was also important to see what would
happen if c-jun levels were increased in the presence of NGF. When this
was achieved, again by microinjecting neurones with an expression
vector, the rate of death was accelerated. Therefore, high levels of c-jun
are sufficient to induce death even in the presence of NGF14.
Recent work has suggested that levels of c-jun might increase in other
apoptotic situations as well. For example, treatment of cortical neurones
with amyloid-EJ-peptide produced an increase in c-jun mRNA and an
accumulation of nuclear c-jun protein, early in the death process15. Our
laboratory has also shown recently that c-jun increases rapidly when
CGNs undergo apoptosis (A. Watson et al, manuscript submitted for
publication).
Since apoptosis is associated with an increase in the phosphorylated,
transcriptionally active, form of c-jun, it might be expected that the
activity of JNK, the kinase that phosphorylates c-jun, would increase
after NGF withdrawal. In fact, Greenberg et aln have shown this to be
true in PC12 cells, and our laboratory has similar data for sympathetic
neurones16. A further prediction is that over-expression of catalytically
620
Bntnh Mmdml Bulletin 1997^3 (No 3)
Neuronal cell death
active variants of upstream kinases should lead to death, even in the
presence of NGF. This was also tested by Greenberg et al, who found
that transient transfection of PC 12 cells with constitutively active
MEKK1 (which phosphorylates and activates SEK1, which phosphorylates and activates JNK) kills them. This death was blocked by
simultaneous over-expression of dominant-negative c-jun. Again, our
laboratory has obtained similar results with sympathetic neurones16.
Finally, Greenberg et al showed that activation of another parallel
signalling pathway — that for p38 MAP kinase — can also lead to cell
death. Thus, it can be concluded that, under at least certain
circumstances, apoptosis is due to the induction of signal transduction
pathways that regulate particular transcription factors. Remaining to be
established is the generality of these observations. That is, are the p38
MAP kinase/JNK pathways the initiators of death in all kinds of
neurones under all circumstances, at least when apoptosis is involved?
Gene activity and neuronal apoptosis
The experimental observation that survival factor withdrawal-induced
neuronal apoptosis can be blocked by actinomycin D and cycloheximide
was widely interpreted to mean that cell death initiated following factor
withdrawal was based on the appearance of new proteins that cause
death. These were meant to be proteins that previously were either not
present at all or were present at very low levels when survival factors
were still available17. While this is not the only interpretation of these
experimental data, a wide variety of experiments have been designed to
address this possibility. Certainly, it has been the case that mRNA levels
of a number of potentially interesting proteins, such as cyclin Dl in
sympathetic neurones18, have been found to increase early in the death
process, but still no particular transcriptional event has been implicated
in apoptosis. This work will not be described in detail here. However,
the identification of particular functionally important transcription
factors may assist in the search for neuronal death genes.
Cytoplasmic effectors of neuronal cell death
In this discussion, it is important to distinguish between cytoplasmic
changes that accompany or are upstream initiators of the death process
and those that actually produce the changes that we associate with
death. Sometimes, this can be difficult, though. For example, the role of
reactive oxygen species in neuronal cell death is controversial. Although
some types of cells clearly are able to die in the total absence of reactive
Bnhth Medical Bu/I.hn 1997;53 (No. 3)
621
Apoptosii
oxygen19'20, some investigators still feel that reactive oxygen is an
essential 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 of
ICE-like proteases (now termed caspases), which undoubtedly are
featured prominently in many articles in this issue. In that regard,
neurones behave like all other cells in that a cascade of caspases seems to
be activated during death, as judged by various criteria, including: (i)
initiation of neuronal cell death by over-expression of ICE; (ii) use of
fluorescent enzyme substrates; (iii) cleavage of known caspase substrates;
and (iv) inhibition of neuronal cell death by caspase inhibitors.
Much of the work concerning caspases and neurones has been carried
out 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 enzymatically
inactive ICE. Wild-type ICE killed cells maintained in NGF, while
inactive ICE had no effect. This suggests that unregulated ICE activity is
sufficient to kill cells even when they are maintained under normal
survival conditions.
There is also evidence that induction of a caspase cascade occurs
during apoptosis and is functionally significant. Direct proof for
increased caspase activity in dying neurones has been provided by
Schulz et alu, who showed cleavage of a fluorescent caspase substrate
during the death of CGNs. Further evidence was provided by
Gagliardini et aln, who microinjected sensory neurones with an
expression vector for the viral ICE inhibitor crmA. These cells were
consequently less likely to die following NGF withdrawal. Similar
studies were done by Martinou et aP3 who over-expressed, again by
microinjection, the baculovirus caspase inhibitor p35 in SCG neurones.
They found that this inhibitor also offered a significant degree of
protection against death due to NGF withdrawal.
A variety of peptide-type caspase inhibitors of varying specificities
towards individual members of the caspase family have also been
applied to dying neurones. The general finding has been that several of
these inhibitors block growth factor withdrawal death, although they
vary in effectiveness to some degree. zVAD-fmk, a somewhat general
inhibitor of caspases, has blocked cell death in several different systems,
including PC12 cells24 and sympathetic neurones (McCarthy, Rubin and
Philpott, submitted for publication). However, it was ineffective in
blocking low K+-death of CGNs58. YVAD-based inhibitors, which block
at least ICE itself, were found to block motor neuron apoptosis in
culture and during normal avian embryo development15 and to block
low K+-death of CGNs22. DEVD-type inhibitors, derived from the
622
Bnhih Med.ro/Bu/Uhnl997;53 (No 3)
Neuronal cell death
cleavage sequence of PARP, a CPP-32 substrate, blocked death of SCG
neurones (McCarthy, Rubin and Philpott, submitted for publication).
However, for this inhibitor to be effective, it was necessary to
microinject it into the cells because it has very limited membrane
permeability.
Since staurosporine induces neuronal apoptosis by direct activation of
death effectors, it was important to determine if it acted via caspases.
Evidence that this is true again comes from several different studies.
Philpott et als found that lamin, a known caspase substrate26, is cleaved
from 69 kDa to 46 kDa during death of PC12 and SCG cells. Taylor et
al5S found that the nuclear enzyme PARP, a caspase substrate, was
degraded from a 116 kDa intact form to an 85 kDa proteolytic fragment
during CGN death. Thus, caspases are activated during staurosporine
death. That this is required for death was suggested by the work of
McCarthy, Rubin and Philpott (submitted for publication) who found
that over-expression of baculovirus p35 in SCG neurones prevented
staurosporine-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 both
staurosporine and growth factor-withdrawal types of death in SCG
neurones. On the other hand, zVAD-fmk was more effective in blocking
NGF death than staurosporine death of these cells (Taylor et al, submitted
for publication). Unexpectedly, in CGNs, zVAD-fmk was very effective in
blocking staurosporine death, but not very good at blocking low K+-death.
Differences in the effectiveness of these inhibitors was also found by Troy
et at14, who compared death of PC12 cells due to either down-regulation
of superoxide dismutase or withdrawal of serum or NGF.
These results suggest that there are several ways of inducing neuronal
apoptosis that involve the same types of morphological and biochemical
pathways. However, it seems that there is variability, both among
different types of neurones and among different initiators of death in an
individual neuronal type, in the particular caspases that cause death. It
will be very important to determine the source of this variability. For
instance, are the very upstream activators of the caspase cascade in the
different types of cell death identical, with differences arising in at least
some of the downstream proteases? Alternatively, do the different
stimulators of death generate different intracellular cascades at their
onset, with perhaps some overlap of downstream caspases?
Bcl-2, bax, and neuronal cell death
Entry into the death cascade in most cell types involves the participation
of bcl-2 and bax-like proteins27. Bcl-2 over-expression appears to block
Bntnh Midical hjlhttn 1997,53 (No 3)
623
Apoptoiis
death, probably by inhibiting entry into the caspase cascade, whereas bax
seems to stimulate the onset of the cascade. It has been clear for some time
that bcl-2 over-expression is anti-apoptotic in neurones. Martinou et at18
injected sympathetic neurones with an expression plasmid for bcl-2 and
found that survival of these cells in the absence of NGF was improved.
Allsopp et al29 obtained similar results for other types of neurotrophindependent neurones, but found, surprisingly, that bcl-2 over-expression
did not block death of neurones dependent on ciliary neurotrophic factor
(CNTF). This group also found that antisense bcl-2 constructs decreased
the ability of neurotrophins to support survival30. However, these
constructs failed to affect the activity of CNTF. This might mean that
CNTF promotes survival by a mechanism dependent on a bcl-2-related
protein. It is clearly the case that other members of the bcl-2 family, such
as bcl-x, support survival31. This is reasonable since many types of adult
neurones have undetectably low levels of bcl-2 with levels of bcl-x being
more substantial. An important role for such related proteins is confirmed
by the observation that there is some neuronal cell death in bcl-2
knockout mice, but not massive malformation of the nervous system.
Bax and related pro-death proteins are important regulators of
neuronal apoptosis. NGF addition to PC12 cells causes a substantial
decrease in their bax levels, suggesting that this is a normal part of
differentiation. This is consistent with the finding that bax is high in
neurones at developmental times when there is significant programmed
cell death, but decreases substantially afterwards59. Interestingly, bax
levels remain high in certain neuronal populations, possibly those that
are particularly susceptible to dying. For instance, bax levels are high in
adult cerebellar Purkinje neurones, which die most readily when the
cerebellum is made ischaemic32. The important role of bax can be
evaluated directly in several ways. One is to engineer its over-expression.
When this is done in sympathetic neurones, they die, even in the presence
of NGF. This death can be blocked by concomitant over-expression of
bcl-x and is also blocked by p35 over-expression, which indicates that
Bax initiates death by activating the caspase cascade59.
Necrosis versus apoptosis in neuronal cell death
One of the most controversial topics amongst investigators focusing on
neuronal cell death occurring under pathological conditions relates to
classification: is it necrosis or is it apoptosis? Everyone agrees that cell
death in development resembles apoptosis. Most investigators now
believe that neurodegenerative disorders, such as Alzheimer's disease,
involve apoptosis to a substantial degree and, until recently, most
624
Bnhsh MadKal Bulletin 1997,53 (No 3)
Neuronal cell death
probably accepted the theory that neuronal death in stroke is
predominantly by necrosis.
An important question is whether or not it is valuable to categorise
neuronal death as being of one type or the other. We will conclude that
there is likely to be some sharing of intracellular pathways in the two
processes. However, although in extreme cases it is not difficult to
distinguish between the two types, one very important issue must centre
on the criteria which can be used in a given situation to distinguish
between the two types of death.
The classical view is that necrotic cells are swollen, due to early
changes in the permeability of their plasma membranes, and are
associated with inflammation in response to leaked cytoplasmic
constituents. Apoptotic cells have intact membranes, distorted organelles, condensed chromatin and are not associated with inflammation
since apoptotic cells are generally engulfed early in the death process.
Ultrastructural analysis should be able to determine the death type, but
this is a time-consuming technique and not useful as a routine
experimental procedure. It is particularly difficult in studies that require
the use of biopsied or post mortem human nervous tissue. As already
mentioned, the rapid disappearance of apoptotic cells presents another
problem in trying to ascertain the type of cell death.
Thus, investigators have sought more convenient procedures, two of
which are now routinely used. The first is to look for labelling with the
TUNEL technique, which is generally considered to be diagnostic for
apoptosis, but can occur during necrosis as well. The second is to isolate
tissue from damaged regions and look for evidence of DNA fragmentation.
DNA from apoptotic cells is cleaved at 180 bp intervals and runs as a
ladder, while that from necrotic cells runs as a smear. However, this
technique is also problematic from two points of view. It is not always easy
to see DNA laddering even in cultured neurones undergoing apoptosis, and
any tissue sample might contain only a small percentage of apoptotic cells,
visible by TUNEL staining or, perhaps, by ultrastructural examination, but
not able to produce enough DNA to make laddering obvious. So, in the
end, many studies simply describe whether or not TUNEL-positive cells or
DNA laddering is seen. The presumption is that if either occurs, cell death
by 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 neurodegenerative diseases
Despite all of these problems, there has been a recent flurry of papers
trying to determine the extent of, especially apoptotic, cell death in
various degenerative diseases. These experiments are difficult, for the
Bnh«/jM«dicolBu/Lhn1997;33(No.3)
625
Apoptosis
reasons just outlined, and compounded by having to use human biopsy
samples, which themselves are often processed slowly, perhaps leading
to more cell death. Nonetheless, reasonable progress has been made.
Using Alzheimer's disease human brain samples, several investigators
have found evidence for TUNEL-positive cells, including cortical and
hippocampal neurones33"35. However, there is some disagreement as to
whether or not these cells have a typically apoptotic morphology.
Further, there is some confusion as to whether there is a direct
correlation between the location of dying cells and the presence of
amyloid plaques and neurofibrillary tangles, the distinguishing pathological features of the disease. In cell culture, the situation is somewhat
clearer. It appears that addition of high concentrations of amyloid-Ppeptide kills cortical neurones by apoptosis, with chromatin condensation, DNA fragmentation and surface blebbing15'36.
Other neurodegenerative diseases have also been studied. In
Huntington's disease samples, TUNEL-positive cells were seen in the
striatum, and there was some indication that these cells were dying by
apoptosis, although it was difficult to find DNA fragmentation
consistently37-39. In status epilepticus in rodents40'41 and scrapie42, there
have been descriptions of apoptosis. Finally, a very interesting case is
that of spinal-muscular atrophy, which is associated with extensive
apoptosis of motor neurones43-44. The gene affected in this disease is
termed NAIP, neuronal apoptosis inhibitor protein, and is homologous
to a baculovirus inhibitor of apoptosis. When over-expressed in different
cell types, not yet including neurones, NAIP inhibits apoptosis, as
expected45.
Cell death in stroke
Of all disorders of the nervous system, stroke has been examined most
carefully for its cell death phenotype. One reason for this (other than the
obvious prevalence and importance of the condition) is the relative
availability of animal models (although the exact correspondence
between the different models and the human disease is frequently
debated). However, another reason is that the neuronal cell death is
fairly extensive and relatively rapid, occurring in hours to days. This is a
tremendous experimental advantage when compared to the slow
degenerative disorders. The common view until recently was that cell
death in stroke was entirely by necrosis, with excess glutamate release
causing Ca2+-overload, cell swelling and death8'9. However, when the
frequency of apoptosis as a death type became clear, investigators were
interested in discovering if it accompanied stroke as well. Reviewing the
626
Bnhih Medical Bulletin 1997J3 (No 3)
Neuronal cell death
now extensive literature would itself require a review of this size, but it is
possible to summarise the essential information fairly succinctly. The
most important point is that there is an apoptotic component to
neuronal death that follows stroke46"50. This has been established by: (i)
the appearance of TUNEL-positive cells; (ii) DNA laddering in tissue
samples; (iii) ultrastructure of dying cells; and (IV) partial inhibition of
death 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 the
hippocampus, in which death is significantly delayed with respect to the
onset of ischaemia51'52. Other significant indications that apoptosis
occurs in stroke include inhibition by agents normally thought of as
being 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 of
how important it is to divide types of death into discrete categories. A
pragmatic position is that the important issue is not categorisation, but
prevention. Of course, it seems logical to expect that knowing the type of
cell death underlying a particular disorder will be very important in that
regard, but this will be difficult for the following reasons. First, some
disorders are associated with dying cells that have some characteristics
normally thought to be associated with apoptosis and some with
necrosis. In some situations, it is very difficult to decide on the dominant
phenotype. Second, the same type of stimulus — hypoxia, glutamate, the
calcium ionophore A23187 — can lead to either necrosis or apoptosis or
both, depending on length of treatment, concentration of drug, etc.
Third, certain agents — neurotrophins, bcl-2, caspase inhibitors —
normally categorised as anti-apoptotic seem to block types of death
often thought of as necrotic.
The situation with A23187 is, in a way, particularly instructive in that
low concentrations kill by apoptosis, high by necrosis56. This presumably means that events underlying apoptosis must begin to occur
when A23187-treated cells are dying by necrosis and would become
obvious if they survived for a long enough time. The same is true of
hypoxia-induced death of cortical cells that, as already mentioned,
undergo delayed apoptosis made apparent if the 'necrotic-type' death is
blocked pharmacologically.
A recent set of experiments carried out by Tsujimoto et al57 is also
extremely important. They placed three different types of cells under
hypoxic conditions. The cell types all underwent a mixture of necrosis
Bnbih Mtdical Bulletin 1997,53 (No 3)
627
Apoptosis
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 suggest
immediately that at least some of the events underlying necrosis and
apoptosis must be similar or even identical.
Conclusion
Apoptosis in neurones, resulting from growth factor withdrawal or
occurring in different neurodegenerative disorders, is fundamentally
similar to that in other cell types. It is normally activated by cytoplasmic
signalling pathways, transmitted via transcription factors and alterations
in gene transcription, and carried out by the appearance or activation of
cytoplasmic effectors. There are cell-specific events, but ICE-like
proteases are key effectors of the death programme, and bcl-2 and
bax-like proteins regulate entry into the pathway. While neuronal cell
death has two extreme phenotypes, apoptosis and necrosis, there may be
many forms of death that are not so simple to distinguish. Nonetheless,
substantial progress has been made in understanding these processes and
in 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. Watson and J. Whitfield) for their hard work and helpful
comments and Ms Helen Blake for help in preparing this article.
References
1 Desphande J, Bergstedt K, Linden T, Kahmo H, Wieloch T. Ultrastructural changes in the
hippocampal CA1 region following transient cerebral ischemia: evidence against programmed
cell death. Exp Brain Res 1992; 88. 91-105
2 Batistatou A, Greene LA. Internucleosomal DNA cleavage and neuronal cell survival/death J
Cell Biol 1993, 122: 523-32
3 D'Mello SR, Galli C, Ciotn T, Callissano P. Induction of apoptosis in cerebellar granule neurons
by low potassium: inhibition of death by insulin-like growth factor I and cAMP. Proc Natl Acad
Set USA 1993; 90. 10989-93
4 Weil M, Jacobson MD, Coles HSR et al. Constitutive expression of the machinery for
programmed cell death. / Cell Biol 1996, 133: 1053-9
5 Philpott KL, McCarthy MJ, Becker D, Gatchalian C, Rubin LL. Morphological and biochemical
changes in neurons: apoptosis versus mitosis. Eur J Neurosa 1996; 8: 1906-15
628
British Modical BulUhn 1997,53 (No. 3)
Neuronal cell death
6 Goldberg MP, Choi DW. Combined oxygen and glucose deprivation in cortical cell culture:
calcium-dependent and calcium-independent mechanisms of neuronal injury. / Neurosa 1993;
13: 3510-24
7 Gwag BJ, Lobner D, Koh JY, Wie MB, Choi DW Blockade of glutamate receptors unmasks
neuronal apoptosis after oxygen-glucose deprivation in vitro Neurosaence 1995; 68: 615-19
8 Choi DW. Calcium: soil center-stage in hypoxic-ischemic neuronal death. Trends Neurosa
1995; 18: 58-60
9 Choi DW, Rothman SM The role of glutamate neurotoxicity m hypoxic-ischemic neuronal
death. Annu Rev Neurosa 1990, 13: 171-82
10 Mattson MP, Lovell MA, Furukawa K, Markesbery WR. Neurotrophic factors attenuate
glutamate-induced accumulation of peroxides, elevation of uitracellular Ca2* concentration,
and neurotoxicity and increase antioxidant enzyme activities m hippocampal neurons. /
Neurochem 1995; 65: 1740-51
11 Nobes CD, Tolkovsky AM. Neutralizmg anti-p21r" Fabs suppress rat sympathetic neuron
survival mduced by NGF, LIF, CNTF and cAMP. Eur J Neurosa 1995, 7: 344-50
12 Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNKp38 MAP kinases on apoptosis. Science 1995, 270: 1326-31
13 Estus S, Zaks WJ, Freeman RS et al. Altered gene expression in neurons during programmed
cell death: identification of c-jun as necessary for neuronal apoptosis. / Cell Biol 1994; 127:
1717-27
14 Ham J, Babi) C, Whitfield J et al. A c-Jun dominant negative mutant protects sympathetic
neurons against programmed cell death. Neuron 1995; 14 927-39
15 Anderson AJ, Pike CJ, Cotman CW Differential induction of immediate early gene proteins in
cultured neurons by (5-amyloid (Af5) association of c-jun with Ap"-mduced apoptosis. /
Neurochem 1995; 65- 1487-98
16 Whitfield JR, Eilers A, Lallemand D, Rubin LL, Ham J. Stress-activated protein kinases, c-jun,
and programmed cell death in the developmg nervous system. Abst Soc Neurosa 1996; In press
17 Freeman RS, Estus S, Hongome K, Johnson Jr EM. Cell death genes in invertebrates and
(maybe) vertebrates. Curr Optn Neurobiol 1993; 3: 25-31
18 Freeman RS, Estus S, Johnson Jr EM. Analysis of cell cycle-related gene expression in
postmitotic neurons: selective induction of cyclin Dl during programmed cell death. Neuron
1994; 12: 343-55
19 Jacobson MD, Raff MC Programmed cell death and Bcl-2 protection in very low oxygen.
Nature 1995, 374: 814-16
20 Shimizu S, Eguchi Y, Kosaka H, Kamnke W, Matsuda H, Tsujimoto Y. Prevention of hypoxiamduced cell death by Bcl-2 and Bcl-xL. Nature 1995; 374: 811-13
21 Gaghardini V, Fernandez PA, Lee RKK et al. Prevention of vertebrate neuronal death by the
crmA gene. Science 1994; 263. 826-8
22 Schulz JB, Weller M, Klockgether T. Potassium deprivation-induced apoptosis of cerebellar
granule neurons: a sequential requirement for new mRNA and protein synthesis, ICE-hke
protease activity, and reactive oxygen species. / Neurosa 1996; 16: 4696-706
23 Martinou I, Fernandez PA, Missotten M et al. Viral proteins E1B19K and p35 protect
sympathetic neurons from cell death induced by NGF deprivation. / Cell Biol 1995; 128: 201-8
24 Troy CM, Stefanis L, Prochiantz A, Greene LA, Shelanski ML. The contrasting roles of ICE
family proteases and lnterleukin-lp" in apoptosis mduced by trophic factor withdrawal and by
copper/zinc superoxide dismutase down-regulation. Proc Natl Acad Set USA 1996; 93: 5635—
40
25 Milhgan CE, Prevette D, Yaginuma H et al. Peptide inhibitors of the ICE protease family arrest
programmed cell death of motoneurons in invo and in vitro. Neuron 1995, 15 385-93
26 Lazebnik YA, Takahashi A, Moir RD et al. Studies of the lamin proteinase reveal multiple
parallel biochemical pathways during apoptotic execution. Proc Natl Acad Set USA 1995; 92:
9042-6
27 White E. Life, death, and the pursuit of apoptosis. Genes Dev 1996; 10: 1-15
28 Garcia I, Martinou I, Tsujimoto Y, Martinou JC. Prevention of programmed cell death of
sympathetic neurons by the bcl-2 proto-oncogene. Science 1992; 258: 302-4
British MtdKal Bulletin 1997,53 (No 3)
629
Apoptosii
29 Allsopp TE, Wyatt S, Paterson HF, Davics AM The proto-oncogene bcl-2 can selectively rescue
neurotrophic factor-dependent neurons from apoptosis. Cell 1993; 73: 295-307
30 Allsopp TE, Kiselev S, Wyatt S, Davies AM. Role of bcl-2 in the brain-derived neurotrophic
factor survival response. Eur ] Neurosa 1995, 7- 1266-72
31 Gonzalez-Garcia M, Garcia I, Ding L et al. Bcl-x is expressed in embryonic and postnatal
neural tissues and functions to prevent neuronal cell death. Proc Natl Acad Set USA 1995; 92
4304-8
32 Kra]ewski S, Mai JK, Krajewska M, Sikorska M, Mossakowski MJ, Reed JC. Upregulation of
bax protein levels in neurons following cerebral ischemia. / Neurosa 1995; 15: 6364-76
33 Smale G, Nichols NR, Brady DR, Finch CE, Horton Jr WE. Evidence for apoptotic cell death in
Alzheimer's disease. Exp Neurol 1995; 133: 225-30
34 Lassmann H, Bancher C, Breitschopf H et al. Cell death in Alzheimer's disease evaluated by
DNA fragmentation in situ. Acla Neuropathol 1995; 89: 35-41
35 Su JH, Anderson AJ, Cummings BJ, Cotman CW. Immunohistocherrucal evidence for apoptosis
in Alzheimer's disease NeuroReport 1994; 5: 2529-33
36 Loo DT, Copani A, Pike CJ et al Apoptosis is induced by fi-amyloid in cultured central nervous
system neurons. Proc Natl Acad Set USA 1993; 90. 7951-5
37 Thomas LB, Gates DJ, Richfield EK, O'Bnen TF, Schweitzer JB, Steindler DA. DNA end
labeling (TUNEL) in Huntington's disease and other neuropathological conditions Exp Neurol
1995, 133: 265-72
38 Dragunow M, Faull RLM, Lawlor P et al In situ evidence for DNA fragmentation in
Huntington's disease striatum and Alzheimer's disease temporal lobes. NeuroReport 1995, 6
1053-7
39 Portera-Cailhau C, Hedreen JC, Price DL, Koliatsos VE. Evidence for apoptotic cell death in
Huntmgton disease and excitotoxic animal models. / Neurosct 1995; 15: 3775-87
40 Dragunow M, Preston K, Dodd J, Young D, Lawlor P, Christie D. Clustenn accumulates m
dying neurons following status epilepticus. Mol Brain Res 1995; 32: 279-90
41 Pollard H, Charriaut-Marlangue C, Cantagrel A et al. Kainate-induced apoptotic cell death in
hippocampal neurons Neurosctence 1994; 63: 7-18
42 Giese A, Groschup MH, Hess B, Kretzschmar HA. Neuronal cell death in scrapie-infected mice
is due to apoptosis. Brain Pathol 1995; 5: 213-21
43 Lefebvre S, Burglen L, Reboullet S et al. Identification and characterization of a spinal muscular
atrophy-determining gene. Cell 1995; 80: 155-65
44 Roy N, Mahadevan MS, McLean M et al. The gene for neuronal apoptosis inhibitory protein is
partially deleted in individuals with spinal muscular atrophy. Cell 1995, 80- 167-78
45 Liston P, Roy N, Tamai K et al. Suppression of apoptosis in mammalian cells by NAIP and a
related family of IAP genes. Nature 1996; 379: 349-53
46 Linnik MD, Zobnst RH, Hatfield MD. Evidence supporting a role for programmed cell death
in focal cerebral ischemia in rats Stroke 1993; 24: 2002-9
47 MacManus JP, Hill IE, Huang ZG, Rasquinha I, Xue D, Buchan AM DNA damage consistent
with apoptosis in transient focal ischemic neocortex. NeuroReport 1994, 5: 493—6
48 Li Y, Sharov VG, Jiang N, Zaloga C, Sabbah HN, Chopp M Ultrastructural and light
microscopic evidence of apoptosis after middle cerebral artery occlusion in the rat. Am ] Pathol
1995; 146 1045-51
49 Charnaut-Marlangue C, Margaill I, Represa A, Popovici T, Plotkine M, Ben-An Y. Apoptosis
and necrosis after reversible focal ischemia: an in situ DNA fragmentation analysis / Cereb
Blood Flow Metab 1996; 16: 186-94
50 Du C, Hu R, Csernansky CA, Hsu CY, Choi DW. Very delayed infarction after mild focal
cerebral ischemia, a role for apoptosis3 J Cereb Blood Flow Metab 1996; 16. 195-201
51 Iwai T, Hara A, Niwa M et al. Temporal profile of nuclear DNA fragmentation in situ in gerbil
hippocampus following transient forebrain ischemia. Brain Res 1995; 671' 305-8
52 Nitatori T, Sato N, Wagun S et al. Delayed neuronal death in the CA1 pyramidal cell layer of
the gerbil hippocampus following transient ischemia is apoptosis. / Neurosa 1995; 15 100111
630
Bnfuh M,dical Bu/I.fm 1997,53 (No 3)
Nouronal cell death
53 Martinou JC, Dubois-Dauphin M, Staple JK et al. Overexpression of BCL-2 in trangenic mice
protects neurons from naturally occurring cell death and experimental ischemia Neuron 1994;
13 1017-30
54 Linnik MD, Zahos P, Geschwind MD, Federoff HJ. Expression of bcl-2 from a defective herpes
simplex virus-1 vector limits neuronal death in focal cerebral ischemia. Stroke 1995; 26: 16705
55 Loddick SA, MacKenzie A, Rothwell NJ. An ICE inhibitor, z-VAD-DCB attenuates ischaemic
brain damage in the rat. NeuroRcport 1996, 7: 1465-8
56 Mah SP, Zhong LT, Liu Y, Roghani A, Edwards RH, Bredesen DE. The protooncogene bcl-2
inhibits apoptosis in PC12 Cells J Neurochem 1993; 60: 1183-6
57 Shimizu S, Eguchi Y, Kamnke W et al. Induction of apoptosis as well as necrosis by hypoxia
and predominant prevention of apoptosis by bcl-2 and bcl-X,. Cancer Res 96; 56: 2161—6
58 Taylor J, Gatchalian CL, Keen G, Rubin LL. Apoptosis in cerebellar granule neurones:
Involvement of interleukjn-lfJ convening enzyme-like proteases. / Neurochem 1997; 68:
1598-605
59 Vekrellis K, McCarthy MJ, Watson A et al. Bax promotes neuronal cell death and is
downregulated daring the development of the nervous system. Development 1997; 124; 1239-49
Bntith Medical Bulletin 1997;53 (No 3)
631