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The Journal of Neuroscience, July 1, 2001, 21(13):4752-4760
Programmed Cell Death of Developing Mammalian Neurons after
Genetic Deletion of Caspases
Ronald W.
Oppenheim1,
Richard A.
Flavell2,
Sharon
Vinsant1,
David
Prevette1,
Chia-Y.
Kuan4, and
Pasko
Rakic3
1 Department of Neurobiology and Anatomy and the
Neuroscience Program, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157, 2 Section of
Immunobiology, Howard Hughes Medical Institute and
3 Section of Neurobiology, Yale University School of
Medicine, New Haven, Connecticut 06510, and 4 Cincinnati
Children's Hospital Research Foundation, Division of Developmental
Biology, Cincinnati, Ohio 45229
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ABSTRACT |
An analysis of programmed cell death of several populations of
developing postmitotic neurons after genetic deletion of two key
members of the caspase family of pro-apoptotic proteases, caspase-3 and
caspase-9, indicates that normal neuronal loss occurs. Although the
amount of cell death is not altered, the death process may be delayed,
and the cells appear to use a nonapoptotic pathway of degeneration. The
neuronal populations examined include spinal interneurons and motor,
sensory, and autonomic neurons. When examined at both the light and
electron microscopic levels, the caspase-deficient neurons exhibit a
nonapoptotic morphology in which nuclear changes such as chromatin
condensation are absent or reduced; in addition, this morphology is
characterized by extensive cytoplasmic vacuolization that is rarely
observed in degenerating control neurons. There is also reduced
terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick
end labeling in dying caspase-deficient neurons. Despite the
altered morphology and apparent temporal delay in cell death, the
number of neurons that are ultimately lost is indistinguishable from
that seen in control animals. In contrast to the striking perturbations
in the morphology of the forebrain of caspase-deficient embryos, the
spinal cord and brainstem appear normal. These results are consistent
with the growing idea that the involvement of specific caspases and the
occurrence of caspase-independent programmed cell death may be
dependent on brain region, cell type, age, and species or may be the
result of specific perturbations or pathology.
Key words:
cell death; neurons; apoptosis; caspases; mouse; embryo; spinal cord
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INTRODUCTION |
Cysteine proteases comprising the
caspase family are considered to be among the most highly conserved
molecules involved in apoptosis and programmed cell death (PCD)
during development, being expressed in animals as diverse as worms and
humans (Cryus and Yuan, 1998 ; Li and Yuan, 1999; Kuan et al., 2000).
They are one of the major classes of pro-apoptotic molecules that are
thought to be essential for many of the degradative events that occur during the cell-death process. By cleaving specific substrates in the
nucleus and cytoplasm, caspases are responsible for many of the
biochemical and cytological changes that define apoptotic PCD.
Previous in vitro studies have shown that various kinds of
stimuli that normally induce apoptosis with chromatin condensation and
DNA degradation fail to do so in neuronal and non-neuronal cells that
are caspase-3-deficient or in which caspase-3 or other caspases are
inhibited (Jänicke et al., 1998a,b; Stefanis et al., 1998, 1999;
Woo et al., 1998; Zheng et al., 1998; Bortner and Cidlowski, 1999;
Cregan et al., 1999; Ferrer, 1999; Keramaris et al., 2000;
Tanabe et al., 1999; Xue et al., 1999; D'Mello et al., 2000; Sperandio
et al., 2000; Williams et al., 2000). Accordingly, caspase-3, and by
extrapolation, caspase-9, which is thought to be upstream of and
required for caspase-3 activation, both appear to be primarily required
for the nuclear changes that occur during apoptosis. In contrast,
in vivo studies of caspase-3- and caspase-9-deficient mice
indicate that these proteases are in fact essential for both the
nuclear changes and the normal PCD of many developing neurons (Kuida et
al., 1996, 1998; Hakem et al., 1998; Roth et al., 1999; Kuan et al.,
2000), whereas many types of PCD outside of the nervous system are
delayed but nonetheless occur in caspase-3- and caspase-9-deleted animals (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998;
Yoshida et al., 1998; Chautan et al., 1999). However, previous studies
of neuronal PCD in caspase-3- and caspase-9-deficient animals focused
almost exclusively on mitotically active or immature neurons in the
forebrain that undergo normal cell death during early developmental
stages before the formation of synaptic connections. The regulation of
neuronal PCD in vitro or in populations in vivo that undergo PCD before the formation of synaptic connections may
differ from the well known target-dependent type of naturally occurring
neuronal death that occurs when synaptic connections are being formed
(Oppenheim, 1999). Therefore, these in vitro and in
vivo studies on the role of caspase-3 and caspase-9 may not
accurately reflect the in vivo role of these proteases in this type of PCD. To examine this, we have focused on several populations of developing neurons that undergo PCD as postmitotic cells
while establishing synaptic connections.
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MATERIALS AND METHODS |
Animals and histology. Heterozygous caspase-3 and
caspase-9 mutant mice were maintained in a mixed C57BL/6 and 129sv
strain background and were interbred to generate homozygous mutants for this study. Sibling animals at indicated embryonic stages were collected and individually genotyped by PCR as described previously (Kuida et al., 1996, 1998). Given the variable severity of caspase-3 and caspase-9 mutant phenotypes, only mutant embryos showing forebrain abnormalities (exencephaly) were included in our analysis. Because most
caspase-9-deficient embryos die in utero after embryonic day
16.5 (E16.5) (Kuida et al., 1998), only E16.5 or younger animals were examined here. Most of the embryos and postnatal animals were
immersion-fixed in either Bouins' or Carnoy's solution; the spinal
cord (with attached DRG) and brain were processed separately, embedded
in paraffin, sectioned serially (10 µm), and stained with either
thionine or hematoxylin eosin (Li et al., 1994). Some animals
were also fixed in 4% paraformaldehyde and 2% glutaraldehyde, post-fixed in 2% osmium tetraoxide, dehydrated in graded alcohol, and
embedded in plastic (Epon); semiserial sections were stained with
toludine blue. A polyclonal antibody specific for the 17 kDa cleaved
subunit of caspase-3 (BD 559565; PharMingen, San Diego, CA) was
used for in situ detection of caspase-3 activation.
FITC-conjugated anti-rabbit IgG secondary antibody (Jackson
ImmunoResearch, West Grove, PA) was used for visualization of the
staining. Spinal cord sections stained with bisbenzimide for cell
nuclei and with FITC-labeled activated caspase-3 were imaged using a
Zeiss LSM510 microscope (Zeiss, Thornwood, NY). Simultaneous
confocal-two-photon excitation using an argon laser (488 nm) for FITC,
and an infrared pulse laser (760 nm) for bisbenzimide was used to
localize cells expressing the active form of caspase-3. For terminal
deoxynucleotidyl transferase (TdT)-mediated biotinylated UTP nick end
labeling (TUNEL) staining, 10 µm paraffin-embedded tissue sections
were first rehydrated and permeabilized with 0.2% Triton X-100 before nick end-labeling with 0.32 U/µl TdT and 2 mM
biotin-16-dUTP (Boehringer Mannheim, Indianapolis, IN). Texas
Red-conjugated streptavidin (PerkinElmer Life Sciences, Emeryville,
CA) was used for visualization of the TUNEL-positive signals.
The number of TUNEL-positive neurons was counted in an equal number of
sections spanning 470 µm in two wild-type (WT) and two
caspase-3 knock-out (KO) lumbar spinal cords on E14.5.
Morphometric analysis. Neurons were identified and counted
according to criteria described by Clarke and Oppenheim (1995), who
indicated that reliable and accurate cell counts can be attained without the use of correction factors or stereological techniques. For
each neuronal population examined, all cells that met these criteria
were counted in every fifth or 10th section, and these totals were
multiplied by 5 or 10 as an estimate of the total number of neurons in
that population. Because neurons dying in the absence of caspase-3 or
caspase-9 exhibit a modified degenerating morphology when examined
using the light microscope (see Results), we have included these cells
as well as typical pyknotic cells in our counts of degenerating
neurons. Counts of surviving and dying neurons in the intermediate gray
matter of the lumbar spinal cord excluded the dorsal and ventral horns,
and cells were counted on only one side (hemisection) in every 10th
section. The degeneration data from caspase-3 and caspase-9 embryos
(control and mutant) were similar and thus have been pooled for further
analysis. Finally, because we have never observed any differences in
neuronal numbers between caspase-3 or caspase-9 homozygotes (+/+) and
heterozygotes (+/ ), these cell counts have been pooled and compared
with caspase-3- and caspase-9-deficient (/) littermates.
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RESULTS |
Gross morphology and cell counts of surviving neurons
In contrast to the severe and obvious forebrain malformations
observed in all of the caspase-3- and caspase-9-deficient animals (Kuida et al., 1996, 1998), the organization and morphology of the
brainstem, spinal cord, and peripheral ganglia of these same animals
appeared completely normal at both embryonic and postnatal stages
(Figs.
1-3).
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Figure 1.
The spinal cord and brainstem of
caspase-3-deficient mice appear morphologically normal. Nissl-stained
transverse sections of postnatal day 10 (P10) mouse spinal cord
(A-H) and brainstem-facial motor nucleus
(I-L) of caspase-3-deficient (A, B, E, F,
I, J) and control animals (C, D, G, H, K,
L) are shown. A-D, Brachial;
E-H, lumbar; I-L, brainstem-facial
nucleus. Scale bars: A, C, E, G, 300 µm (shown in
A); B, D, F, H, 50 µm (shown in
B); I, K, 250 µm (shown in
I); J, L, 120 µm (shown in
J). Dotted lines indicate ventral
horns; an arrow indicates the facial motor
nucleus.
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Figure 2.
Nissl-stained transverse sections of E16.5 lumbar
spinal cord of homozygous littermate control (+/+)
(A) and caspase-9-deficient /
(B) embryos. Dotted lines in
A and B indicate the medial border of the
ventral horn. Scale bar, 100 µm. C, E, G, I,
Degenerating neurons (arrows) from E14.5 caspase-3
(C, E) and caspase-9 (G, I)
control (+/+) embryos. Note fragmented apoptotic bodies in
G and I. Scale bar, 10 µm. D, F,
H, J, Degenerating neurons (arrows) from
caspase-3-deficient (D, F) and
caspase-9-deficient (H, J) (/) embryos. Note
the lack of prominent pyknosis and apoptotic bodies and the increased
cytoplasmic density (compare G and
H) in caspase-deficient dying neurons.
Asterisks indicate normal non-dying cells.
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Figure 3.
Transverse sections through the middle of the
superior cervical ganglion (SCG) of homozygote
(A) and caspase-3 KO (B)
littermates at P8. The overall size and organization of the ganglia
appear normal in both low-magnification (insets) and
high-magnification photomicrographs from KO animals.
Arrows indicate some of the SCG neurons; double
arrows indicate non-neuronal cells. Scale bars: A,
B at full size, 25 µm; insets, 40 µm.
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A quantitative analysis of cell numbers in a variety of different
populations representing spinal interneurons and motor, sensory, and
autonomic neurons showed that cell numbers were comparable with control
values at stages during and after the normal period of naturally
occurring cell death (Tables 1,
2). Because of embryonic lethality,
neuronal numbers in caspase-9-deficient animals were only examined on
E16.5, when the normal cell-death period for spinal motoneurons
(MNs) and DRG cells is almost complete. Although the cell-death period
for cranial MNs and spinal interneurons begins embryonically and
extends into the early postnatal period (Wright et al., 1983; Lawson et
al., 1997; Grieshammer et al., 1998), cell numbers in these populations
in caspase-3-deficient animals were also comparable with control values
when assessed after the cessation of their normal period of cell death
(Table 1). Because the normal PCD of sympathetic neurons occurs
postnatally, we were unable to assess this population in caspase-9 KO
animals. Although not shown in Table 1, MN numbers in the lumbar spinal cord at the beginning of the cell-death period on E13.5 were similar in
control and caspase-3-deficient embryos (controls, 3617 ± 360, n = 4 vs caspase-3-deficient, 4009 ± 479, n = 3).
Assessment of dying neurons
Many neurons undergoing PCD in normal animals exhibit a morphology
featuring a distinct kind of nuclear pyknosis characteristic of an
apoptotic type of degeneration, and the dying cells fragment into
cytoplasmic-nuclear-containing apoptotic bodies (Fig. 2) (Chu-Wang and
Oppenheim, 1978; Oppenheim et al., 1986; Li et al., 1998). When
examined using an electron microscope (Fig.
4), these same cells exhibit the
ultrastructural nuclear and cytoplasmic hallmarks of an apoptotic-like
PCD (Chu-Wang and Oppenheim, 1978; Clarke, 1990; Yaginuma et al., 1996;
Li et al., 1998; Shiraiwa et al., 2001). In contrast, apparently dying
neurons in caspase-3- and caspase-9-deficient embryos exhibit a
somewhat different morphology. When examined with the light microscope,
these cells exhibit cell shrinkage, increased density of the cytoplasm
and nucleus, little if any fragmentation of the cell into apoptotic
bodies, and very little chromatin condensation (Fig. 2). Neurons
exhibiting these characteristics were rarely observed in control
embryos. These same morphological differences were seen even more
clearly when the degenerating neurons were examined at the
ultrastructural level (Fig. 4). Ultrastructural analysis also revealed
the presence of extensive cytoplasmic vacuoles that are seldom observed
in dying neurons of control embryos. Although the degeneration of neurons in the caspase-deficient embryos included swelling and dilation
of mitochondria and rough endoplasmic reticulum (RER) (Fig. 4), similar
changes have also been observed during the normal apoptotic PCD of
avian and mouse neurons (Pilar and Landmesser, 1976; Chu-Wang and
Oppenheim, 1978; Clarke, 1990; Li et al.,1998). For this reason, we
cannot exclude the possibility that these changes may reflect a
sampling bias. A more detailed analysis of the ultrastructure of
caspase-3- and caspase-9-deficient and control neuronal degeneration is
in progress.
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Figure 4.
Photomicrographs of degenerating spinal cord
neurons from E14.5 caspase-3 (Casp3) +/+ (A, C,
E) and caspase-3 (Casp3) KO / (B, D,
F) littermate embryos showing the distinct morphology of
dying neurons in the caspase KO. These cells exhibit reduced chromatin
condensation and nuclear pyknosis, marked cytoplasmic vacuolization,
and dilation of mitochondria and RER compared with neurons from the
caspase-3 +/+ embryos. C, Cytoplasm/soma;
N, nuclei. Asterisks indicate normal
non-dying neurons. The arrow in A
indicates the cytoplasm, the arrows in E
and F indicate mitochondria, and the
arrowheads in D indicate the cell
boundary of this degenerating motoneuron. Note the numerous vacuoles
and abnormal organelles in the cytoplasm of the cells in
D and F. Only part of the cytoplasm of a
dying cell is shown in E. The scale bars in
A, C, and E are the same
for B, D, and F,
respectively.
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Neurons exhibiting similar aberrant morphologies in avian embryos that
have been treated with caspase inhibitors in vivo, when
examined with the electron microscope, are also characterized by
condensation and increased electron density of cytoplasm, dilated mitochondria, cytoplasmic vacuoles, and crystallized ribosomes, but
with only a modest aggregation of nuclear chromatin (Shiraiwa et al.,
2001). This morphology is most consistent with the type 3B cytoplasmic
type of PCD according to the classification of Clarke (1990), whereas
the more typical apoptotic morphology described above is classified as
type 1 by Clarke (1990). Because of the similarity in morphology
between these avian neurons, which we have proven to be degenerating
cells that are phagocytized (Shiraiwa et al., 2001), and the neurons
with aberrant morphology in caspase-3- and caspase-9-deficient embryos
(McCarthy et al., 1997; Hakem et al., 1998; Woo et al., 1998; Tanabe et
al., 1999), these cells have been included in our quantitative
assessment of PCD in control and caspase-deficient embryos (Fig.
5). Few degenerating neurons would have
been counted in caspase-deficient embryos if these profiles had been
excluded. In addition, because we know from the data presented here on
the number of surviving neurons that comparable amounts of cell death
occur in control and caspase-deficient embryos, we consider it highly
likely that, despite their distinct morphology, these cells
represent neurons undergoing PCD. By including them in our quantitative
analysis (Fig. 5), we find that there are similar numbers of
degenerating MNs in caspase-3- and caspase-9-deficient embryos when
compared with the more typical, frankly pyknotic (apoptotic) neurons in
control embryos (Figs. 2, 4). In contrast to MNs, the number of
degenerating spinal interneurons in the caspase-3- and
caspase-9-deficient embryos on E16.5 was increased compared with
control values [caspase-deficient, 10.3 ± 2.5 (mean ± SD)
per section, n = 4 vs wild-type control, 6.1 ± 2.1, n = 4; p < 0.05, t
test]. From these data, we conclude that the PCD of MNs occurs
to the same extent in caspase-3- and caspase-9-deficient embryos as it
does in control embryos, albeit with a distinct morphology, whereas the
increased number of dying interneurons in mutant embryos on E16.5 may
reflect a delayed kinetics of PCD (see Discussion).
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Figure 5.
The number (mean ± SD) of degenerating MNs
in the lumbar spinal cord of control +/+ (CON),
caspase-3 / (Casp3/), and
caspase-9 / (Casp9/) embryos on
E14.5 and E16.5 expressed as the number per 1000 surviving MNs. The
sample size (number of embryos) is indicated in the
bars. In all groups, there was a significant
(p < 0.01) reduction between E14.5 and
E16.5.
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TUNEL
In contrast to our failure to observe a decrease in the number of
degenerating neurons (type 1 and type 3B PCD) between control and
caspase-3- or caspase-9-deficient embryos, the number of cells labeled
by TUNEL was reduced in the spinal cord of caspase-3-deficient embryos
on E14.5 (WT control, mean of 50, n = 2 vs KO, mean of 20, n = 2), consistent with previous reports from
caspase-9 KO embryos (Kuida et al., 1998). Even those neurons that
exhibit TUNEL in KO embryos have less apparent chromatin breakdown and condensation (Fig. 6). Because TUNEL is
primarily a means for detecting DNA fragmentation, these data are
consistent with the suggestion that neuronal PCD in caspase-3- and
caspase-9-deficient embryos occurs without or with reduced DNA
fragmentation, as is also indicated by the distinct morphology of the
apparently dying neurons in Nissl- and bisbenzimide-stained material
from these embryos (Figs. 2, 6). Therefore, TUNEL may not always be a
reliable marker for the PCD of developing neurons, because as shown
here, in some situations neurons can degenerate in vivo
without TUNEL-positive DNA degradation.
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Figure 6.
Caspase-3 immunocytochemistry (A,
B), TUNEL (C, E), and bisbenzimide staining
(D, F) in E14.5 wild-type (A, C,
D) and caspase-3 KO (B, E, F) lumbar
spinal cord. A, Immunocytochemistry shows the staining
of a cleaved (activated) caspase-3 subunit in the spinal motor neuron
column and spinal ganglia in E14.5 wild-type mouse spinal cord
(arrowheads), indicating the activation of
caspase-3. B, In contrast, no staining of activated
caspase-3 subunit is found in E14.5 caspase-3-deficient spinal cord.
However, when the TUNEL method is used to detect cell death (C,
E), it reveals positive staining in both wild-type
(C) and caspase-3-deficient
(E) spinal cord, although the staining patterns
are distinct. The TUNEL-positive profiles in C and
E represent the nucleus of an individual neuron. In
wild-type spinal cord, there are more TUNEL-positive nuclei showing
punctuate nuclear staining (C), which is
correlated with condensed chromatin as revealed by bisbenzimide
staining (D). In contrast, there are fewer
TUNEL-positive cells in caspase-3-deficient spinal cord (see
Results). Moreover, these cells typically show a distinct kind
of TUNEL staining (E) and no clear evidence of
chromatin condensation as indicated by the size of TUNEL-positive
nuclei and reduced bisbenzimide-stained chromatin
(F). Although not obvious in this
photomicrograph, the nucleus indicated by the arrow in
F is weakly labeled with bisbenzimide, indicating
little, if any, chromatin condensation. Scale bars: A,
B, 400 µm; C-F, 20 µm.
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Assays for caspase activation
The normal development of the neuronal populations examined here
in caspase-KO mice raises the question of whether caspase-3 or
caspase-9 is involved in the normal PCD of these cells in control animals. To test this possibility, we examined caspase activity in the
spinal cord of control and caspase KO embryos. Using an antibody
specific for activated caspase-3, numerous positive cells were observed
in control spinal cord and peripheral sensory ganglia (Fig. 6). In
contrast, labeling was never observed in neurons from caspase-3 KO
embryos. These results indicate that caspase-3 is involved in but not
indispensable for normal PCD in these neuronal populations.
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DISCUSSION |
Caspases are considered to be one of the most highly conserved and
ubiquitous families of pro-apoptotic molecules involved in PCD. In the
nematode, CED-3 and CED-4 are required for virtually all
developmental cell deaths (Ellis et al., 1991; Horvitz, 1994), and in
vertebrates, interactions of their counterparts (caspase-3, caspase-9,
and Apaf-1) are also thought to be essential for the PCD of many cell
types, especially neurons. Null mutant mice lacking each of these genes
exhibit a remarkably similar CNS malformation (exencephaly) that has
been attributed in part to a failure of apoptotic PCD of neurons (Kuida
et al., 1996, 1998; Cecconi et al., 1998; Hakem et al., 1998; Yoshida
et al., 1998; Roth et al., 1999). This explanation of the exencephaly
is primarily based on direct evidence for decreased TUNEL-positive and
pyknotic cells and increased numbers of mitotically active cells in the
forebrain (Yoshida et al., 1998). However if TUNEL or a typical
pyknotic morphology is not always a reliable marker for PCD in these
mutants, then it is possible that it is the specific kind of DNA
degradation detected by these methods, but not cell death per se, that
is reduced or absent in the mutants. Accordingly, it seems likely that
other aspects of CNS development may be abnormal (e.g., proliferation) and may contribute to the exencephaly in the forebrain. Although the
cellular mechanisms are not well understood, a failure of neural fold
elevation appears to be involved in virtually all cases of exencephaly
in mice (Harris and Juriloff, 1999).
There is increasing evidence that TUNEL is not a reliable index of PCD
in vitro for either neuronal or non-neuronal cells that lack
caspase-3 (knock-outs) or after reductions in caspase-3 or other
caspases by treatment with peptide inhibitors (Xiang et al., 1996;
Jänicke et al., 1998a,b; Stefanis et al., 1998, 1999; Weil et
al., 1998; Woo et al., 1998; Zheng et al., 1998; Bortner and Cidlowski,
1999; Cregan et al., 1999; Ferrer, 1999; Xue et al., 1999;
D'Mello et al., 2000; Keramaris et al., 2000; Sperandio et al., 2000).
The dying cells in these cases are reported to degenerate in the
absence of the typical nuclear changes that are considered to be
hallmarks of apoptosis, including pyknosis and the DNA degradation
recognized by the TUNEL technique. Although the death of cells in this
situation is reported to be delayed (Cregan et al., 1999; Keramaris et
al., 2000; Williams et al., 2000), they exhibit degradative
changes in the cytoplasm when they do degenerate, and these changes
appear to be distinct from those occurring during classic apoptosis,
including extensive vacuolation (Xiang et al., 1996; Woo et al., 1998;
Sperandio et al., 2000).
Although our results suggest that caspase-3 is normally involved in the
PCD of developing postmitotic neurons in vivo, data from
caspase-3- and caspase-9-deficient animals, together with other
evidence (Shiraiwa et al., 2001), clearly indicates that in the absence
of these caspases the extent of cell death in many neuronal populations
is normal. However, the mode of degeneration includes reduced
TUNEL and nuclear and cytoplasmic changes that are distinct from
classic apoptosis. Because the neuronal populations examined here by us
all undergo PCD as postmitotic cells, it is interesting that we have
not observed the kind of malformations (exencephaly, spina bifida) in
the spinal cord or hindbrain that occur in more rostral, mitotically
active regions of the brain of caspase-3- and caspase-9-deficient mice.
This is consistent with the suggestion that it may be primarily the
delay in PCD and the subsequent increased proliferation of these
rostral forebrain precursor cells that is responsible for the forebrain
abnormality (Kuan et al., 2000). Although additional detailed studies
of the kinetics of neuronal degeneration in caspase-3- and
caspase-9-deficient embryos are needed to determine whether there is in
fact a delay, as occurs in caspase-3-deficient cultured cells (D'Mello
et al., 2000) and in chick embryo neurons in vivo (Shiraiwa
et al., 2001), the evidence presented here for spinal interneurons is
consistent with this possibility. If there is in fact a delay in the
kinetics of cell death in the mutant E16.5 embryos, it is more likely
to be detected in spinal interneurons, which are at the beginning of
their normal period of cell death on E16.5 (Lawson et al., 1997;
Grieshammer et al., 1998), compared with MNs, which are near the end of
their cell-death period at this age (Oppenheim et al., 1986) and
therefore may have had sufficient time to undergo even a delayed cell
death. A delay in the degeneration of caspase-deficient neurons
suggests that caspase-mediated neuronal death is a more efficient means
of PCD than caspase-independent PCD.
We have also observed that the extent of PCD of spinal and cranial
motoneurons and DRG sensory cells is normal in Apaf-1-deficient embryos
(Oppenheim et al., 2001), consistent with the report that the hindbrain
and spinal cord in these mutants appear normal (Cecconi et al., 1998).
This raises the question of what molecular pathways are being used in
place of Apaf-1, caspase-9, and caspase-3 for mediating PCD in these
neuronal populations. One possibility is that these neurons have the
capacity to switch to another, caspase-independent, degradative pathway
of PCD. Interestingly, cultured PC12 cells and peripheral sensory
neurons in which caspase activity is inhibited do in fact switch to a
death-inducing pathway that uses lysosomal proteases (cathepsins)
instead of caspases for PCD, and they exhibit a morphology similar to
that described here for caspase-3- and caspase-9-deficient embryos
(Isahara et al., 1999; Agerman et al., 2000). It is also possible that
neuronal death in these situations involves the recently described
caspase-independent pathway that uses the apoptosis-inducing
factor which activates an apoptotic-like mode of degeneration
(Joza et al., 2001). Alternatively, other caspases may be able to
compensate for the actions of caspase-3 and caspase-9 in mutant mice
(Cryus and Yuan, 1998; Hakem et al., 1998; Stefanis et al., 1998; Woo
et al., 1998; Zheng et al., 1998, 2000). It has been reported recently
that cultured mammalian MNs may use Fas receptors and caspase-8 instead
of caspase-9 during PCD (Raoul et al., 1999). However, because
caspase-3 is thought to be the major downstream caspase in the Fas
pathway, this mode of cell death would not be available for
compensation in the caspase-3 KO animals. Cultured NGF-deprived
sympathetic neurons treated with the pan-caspase inhibitor
bok-asp-fmk (BAF) also continue to undergo cell death, albeit
by an apparent autophagic (Clarke type 2), nonapoptotic pathway (Xue et
al., 1999). The dying neurons in the chick embryo after caspase
inhibition by BAF (Shiraiwa et al., 2001) and those observed here in
caspase-deleted mice do not appear to be undergoing an autophagic cell
death because the cytoplasmic vacuoles in these cases appear to be
empty of lysosomal material. Rather, we suggest that cell death in this situation more closely fits the type 3B "cytoplasmic" mode of embryonic neuronal death reported by Clarke (1990) during normal development. The cytoplasmic vacuoles observed in type 3B PCD appear to
reflect the extreme dilation and swelling of cytoplasmic organelles,
including mitochondria (Pilar and Landmesser, 1976; Chu-Wang and
Oppenheim, 1978). In some respects, dying cells in caspase-deficient
animals resemble necrosis (Hirsch et al., 1997; Lemaire et al., 1998).
However, consistent with several other studies, we consider this form
of degeneration to be a nonapoptotic form of normal PCD (Pilar and
Landmesser, 1976; Chu-Wang and Oppenheim, 1978; Clarke, 1990; Sperandio
et al., 2000). In a recent study of thymocyte apoptosis, it was shown
that during phagocytosis, macrophage-derived lysosomal proteases can
induce cell death and DNA fragmentation in thymocytes by a
caspase-independent, cell-nonautonomous pathway (McIlroy et al., 2000).
Studies are in progress to more fully characterize the mechanisms
involved and the morphological changes that occur in degenerating avian
and mouse neurons after the loss of caspases.
We find it rather remarkable that in the absence of two of the major
pro-apoptotic caspases thought to be required for neuronal cell death,
not only do neurons in the diverse populations examined here continue
to undergo PCD, but they do so in a highly regulated manner such that
the same proportion of cells undergo PCD as do cells in the presence of
caspase-3 or caspase-9. Although we assume that all of these
populations undergo normal PCD in caspase-9-deficient animals, because
we have not been able to examine embryos older than E16.5, we cannot
entirely exclude the possibility that later stages of PCD may not be
normal. Because this seems unlikely, however, our results indicate that
the control of cell number in these neuronal populations by cell death
is regulated at a point upstream of the specific machinery used for
actually killing the cell. Although we have not examined the release of
cytochrome c during the degeneration of caspase-deficient
neurons, several studies have shown that caspase deficiency does not
affect cytochrome c release in dying sympathetic, cortical,
and motor neurons in vitro (Desmukh et al., 2000; Keramaris
et al., 2000; Li et al., 2001).
In summary, many populations of developing postmitotic mammalian
neurons are able to exhibit normal amounts of PCD in the absence of
either caspase-3 or caspase-9. However, the morphology of these
degenerating neurons differs from the more typical apoptotic type of
cell death and the kinetics of their degeneration may be delayed. These
data, together with several other lines of evidence reviewed here,
demonstrate that the use of nuclear changes alone for assessing PCD can
often be misleading. For example, the apparent discrepancy between the
report by Miller et al. (1997) and that by Eldadah et al. (2000)
regarding the role of caspase-3 in the death of cultured cerebellar
granule neurons may be explained by a primary reliance on nuclear
changes alone on the one hand (Eldadah et al., 2000) versus the use of
nuclear changes as well as other markers on the other hand (Miller et
al., 1997; D'Mello et al., 2000) for assessing the death of neurons.
Similarly, whereas the use of TUNEL indicates a significant reduction
in the PCD of DRG cells in E12.5 caspase-9-deficient embryos (Zaidi et
al., 2001), we find no differences in the number of surviving DRG cells in these mutants on E16.5. Together, these and other recent reports (see the introductory remarks) as well as our own in vivo
observations here and elsewhere (Shiraiwa et al., 2001) support the
growing idea that distinct caspases as well as other
caspase-independent pathways may mediate neuronal death in specific
brain regions and cell types, at specific ages, in different species,
and after particular kinds of perturbations (Agerman et al., 2000; Kuan et al., 2000; Zaidi et al., 2001). Finally, these data suggest a note
of caution in attempts to use caspase inhibition as a therapeutic strategy for preventing cell death in neurodegenerative diseases (Kermer et al., 1999; Schulz et al., 1999), because compensatory mechanisms for cell death may be activated when caspase-dependent pathways are blocked.
| |
FOOTNOTES |
Received Jan. 9, 2001; revised March 29, 2001; accepted April 18, 2001.
This work was supported by National Institutes of Health (NIH) Grant
NS20402 (R.W.O.), by the Howard Hughes Medical Institute (R.A.F.), and
by NIH Grant NS14841 (P.R.). We thank Carol FloresDevalgaz, J. Bao, and
J. Musco for technical assistance and T. F. Haydar for
confocal microscopy.
Correspondence should be addressed to Dr. Ronald W. Oppenheim,
Department of Neurobiology and Anatomy, Wake Forest University School
of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1010. E-mail: roppenhm{at}wfubmc.edu.
| |
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Am J Physiol Renal Physiol,
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[Abstract]
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W. Sun, T. W. Gould, S. Vinsant, D. Prevette, and R. W. Oppenheim
Neuromuscular Development after the Prevention of Naturally Occurring Neuronal Death by Bax Deletion
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[Abstract]
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L. K. Chang, R. E. Schmidt, and E. M. Johnson Jr.
Alternating metabolic pathways in NGF-deprived sympathetic neurons affect caspase-independent death
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L. M. Frago, S. Canon, E. J. de la Rosa, Y. Leon, and I. Varela-Nieto
Programmed cell death in the developing inner ear is balanced by nerve growth factor and insulin-like growth factor I
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C. Mbebi, V. See, L. Mercken, L. Pradier, U. Muller, and J.-P. Loeffler
Amyloid Precursor Protein Family-induced Neuronal Death Is Mediated by Impairment of the Neuroprotective Calcium/Calmodulin Protein Kinase IV-dependent Signaling Pathway
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L. K. Chang and E. M. Johnson Jr.
Cyclosporin A inhibits caspase-independent death of NGF-deprived sympathetic neurons: a potential role for mitochondrial permeability transition
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O. Tarabal, J. Caldero, J. Llado, R. W. Oppenheim, and J. E. Esquerda
Long-Lasting Aberrant Tubulovesicular Membrane Inclusions Accumulate in Developing Motoneurons after a Sublethal Excitotoxic Insult: A Possible Model for Neuronal Pathology in Neurodegenerative Disease
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D. Schubert and D. Piasecki
Oxidative Glutamate Toxicity Can Be a Component of the Excitotoxicity Cascade
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TOP
ABSTRACT
INTRODUCTION
