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The Journal of Neuroscience, May 15, 2000, 20(10):3687-3694
Neurodegeneration in Lurcher Mice Occurs via
Multiple Cell Death Pathways
Martin L.
Doughty1,
Philip L.
De Jager1,
Stanley J.
Korsmeyer3, and
Nathaniel
Heintz1, 2
1 Laboratory of Molecular Biology and
2 Howard Hughes Medical Institute, The Rockefeller
University, New York, New York 10021, and 3 Harvard
Medical School, Department of Cancer Immunology and AIDS, Dana-Farber
Cancer Institute, Boston, Massachusetts 02115
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ABSTRACT |
Lurcher (Lc) is a gain-of-function
mutation in the  2 glutamate receptor (GRID2) that results in the
cell-autonomous death of cerebellar Purkinje cells in heterozygous
lurcher (+/Lc) mice. This in turn
triggers the massive loss of afferent granule cells during the first
few postnatal weeks. Evidence suggests that the death of Purkinje cells
as a direct consequence of GRID2Lc activation and
the secondary death of granule cells because of target
deprivation occur by apoptosis. We have used mice carrying null mutations of both the Bax and
p53 genes to examine the roles of these genes in cell
loss in lurcher animals. The absence of Bax delayed Purkinje cell death in response to the
GRID2Lc mutation and permanently rescued
the secondary death of granule cells. In contrast, the
p53 deletion had no effect on either cell death pathway.
Our results demonstrate that target deprivation induces a
Bax-dependent, p53-independent cell death
response in cerebellar granule cells in vivo. In
contrast, Bax plays a minor role in
GRID2Lc-mediated Purkinje cell death.
Key words:
lurcher; cerebellum; Bax; p53; caspase-3; apoptosis
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INTRODUCTION |
Evidence supporting a role for
apoptosis in mammalian neurodegenerative diseases has arisen from cell
culture models of neuronal cell death [for review, see Lee et
al. (1999) ] and from descriptive studies documenting the activation of
developmental cell death genes in neurological disorders [for review,
see Heintz and Zoghbi (2000)]. Several issues have arisen from these
studies that require direct investigation in well characterized models
of neurodegeneration in vivo. For example, studies of cell death
in a variety of in vitro culture systems (Choi, 1994;
Deshmukh and Johnson, 1998; Lee et al., 1999), as well as the
investigation of developmental cell death in the nervous system
in vivo (Pettmann and Henderson, 1998), have established
both that different molecules can participate in apoptosis in distinct
cell types and that disparate stimuli can activate distinct cell death
pathways in a single cell type. Descriptive studies of both human and
mouse neurodegeneration in vivo reflect the complex picture
that has arisen from these investigations and support the general idea
that a wide variety of abnormal stimuli can result in the activation of
the effector phase of apoptosis in neurons (Green, 1998; Los et al.,
1999; Vaux and Korsmeyer, 1999). These studies have also
demonstrated a direct role for specific proteins in neuronal cell death
in vitro or in vivo. Thus, analysis of cell death
in Bax null mutant mice has established a direct role in
neuronal cell death in response to trophic factor withdrawal (Deckwerth
et al., 1996) and during the normal development of a variety of brain
structures (White et al., 1998). A role for p53 in both
radiation-induced neuronal cell death in vivo (Woods and
Youle, 1995) and excitotoxic cell death in vitro (Xiang et
al., 1998) has also been demonstrated. These studies provide a
foundation for the investigation of inherited neurodegenerative disease.
Lurcher (Lc) is a semidominant mouse
mutation that results in ataxia because of massive loss of cerebellar
neurons during the first 4 postnatal weeks (Caddy and Biscoe, 1979).
The lurcher mutation results in the activation of the 2
glutamate receptor (GRID2) in cerebellar Purkinje cells, leading to a
constitutive, inward current (Zuo et al., 1997). Death of Purkinje
cells in lurcher animals is cell autonomous (Wetts and
Herrup, 1982a,b) and arises as a direct consequence of the increased
activity of the receptor, because null mutations of
GRID2 do not result in Purkinje cell death (Kashiwabuchi et
al., 1995). Descriptive studies of cerebellar degeneration in
lurcher mice suggest that Purkinje cell death occurs by
apoptosis (Norman et al., 1995; Wullner et al., 1995). This is
consistent with the elevated expression of Bax,
Bcl-X (De Jager, 1998; Wullner et al., 1998), and
procaspase-3 (Selimi et al., 2000) in postnatal
lurcher Purkinje cells and with the increased levels of
active caspase-3 (Heintz and De Jager, 1999) and DNA nicking (Norman et
al., 1995) in the small percentage of these cells that have entered the
effector phase of the cell death pathway. The death of granule cells
and inferior olivary neurons in lurcher animals is secondary
to the death of Purkinje cells and is presumably caused by the loss of
Purkinje cells as targets for these two neuronal populations.
Because of the elevated level of Bax expression observed in
lurcher animals (Wullner et al., 1995; De Jager, 1998) and
the evidence supporting a role for p53 in
Bax-mediated neuronal cell death in response to both
genotoxic and excitotoxic activity in neurons (Xiang et al., 1998), we
have examined the roles of these genes in neurodegeneration in the
lurcher cerebellum. We report here that Purkinje cell death
in +/Lc:Bax / mice is delayed
relative to that in Bax-expressing +/Lc
littermates and that granule cell death is permanently rescued in these
animals. Examination of
+/Lc:p53/ mice revealed no
alteration in cell death. These results demonstrate that granule cell
death as a result of target deprivation in vivo occurs via a
Bax-dependent, p53-independent pathway and that
Bax plays a minor role in cell death induced by the
constitutive activation of the 2 glutamate receptor in cerebellar
Purkinje cells. Finally, in contrast to Bax-expressing
+/Lc littermates, we observed an absence of active caspase-3
in the Purkinje cells of
+/Lc:Bax/, indicating the
activation of a Bax-independent, caspase-3-independent death
pathway in these cells.
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MATERIALS AND METHODS |
Breeding and genotyping of mice. Mice (C57BL/6)
heterozygous for Bax (Knudson et al., 1995) were mated with
Balb/C lurcherJ
(+/LcJ) mice to obtain F1
+/Lc:Bax+/
offspring.
+/Lc:Bax+/ mice
were crossed to generate +/Lc and wild-type (+/+) mice that were either Bax/,
Bax+/, or
Bax+/+. The mice were genotyped for
Bax by single PCR from the wild-type Bax
allele and from the neo cassette using DNA
prepared from tails (Qiagen, Hilden, Germany). A region of the
wild-type Bax allele was amplified using an exon 2 forward
primer (5'-CTTGGGAGAAGAACAACACTGC-3') and an intron 3 reverse primer
(5'-CTGAACAGATCATGAAGACAGG-3'). A region of the neo cassette
was amplified using the forward 5'-GATTGCACGCAGGTTCTCCG-3' and reverse
5'-CCTGGCGAACAGTTCGGCTGG-3' primers. The mice were identified as
+/Lc by their ataxia. The +/Lc genotype was
sometimes confirmed by single PCR from the Lc allele of
GRID2 (GRID2Lc) using an intron
2 forward primer (5'-GCACTGAATGTGTATGACTTCAG-3') and an exon B reverse
primer (5'-GGTGATAGTGAGGAAAGT-3').
Mice (129S3) heterozygous for p53
(Trp53tm1Tyj; The Jackson Laboratory, Bar
Harbor, ME) were mated with Balb/C +/LcJ
mice to obtain F1
+/Lc:p53+/
offspring.
+/Lc:p53+/ mice
were crossed to generate +/Lc and +/+ mice that were either p53/,
p53+/, or
p53+/+. The mice were genotyped for
p53 by single PCR from the wild-type p53 allele
and from the neo cassette using DNA prepared from tails (Qiagen). A region of the wild-type p53 allele was amplified
using an exon 6 forward primer (5'-CCCGAGTATCTGGAAGACAG-3') and an exon 7 reverse primer (5'-ATAGGTCGCCGGTTCAT-3'). The neo cassette
was amplified using the same neo primers used for the
Bax genotyping. The mice were identified as +/Lc
as described above.
Histology and immunocytochemistry. Mice killed for cresyl
violet staining and calbindin immunocytochemistry were deeply
anesthetized with sodium pentobarbital and perfused intracardially with
0.9% NaCl followed by 95% ethanol. The brains were post-fixed in 75% ethanol and 25% acetic acid, dehydrated, and embedded in paraffin. Midsagittal cerebellar sections 10 µm thick were cut, mounted on
slides, and stained with cresyl violet or incubated overnight at 4°C
in a 1:400 dilution of mouse monoclonal anti-calbindin antibodies
(Sigma, St. Louis, MO). The immunolabeling was revealed using the
Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and DAB (Sigma).
Mice killed for anti-active caspase-3 immunocytochemistry were deeply
anesthetized with sodium pentobarbital and decapitated, and the brain
was frozen in ornithine carbamyl transferase compound (Sakura) in an
acetone bath at  20°C. The brains were stored at 80°C until use.
Sagittal cerebellar sections 10 µm thick were then cut, mounted on
slides, fixed in acetone at 20°C, washed in 0.03%
H2O2 to block endogenous
peroxidases, and incubated overnight at 4°C in a 1:500 dilution of
polyclonal anti-active caspase-3 antibodies (PharMingen, San Diego,
CA). Immunolabeling was revealed as described above.
Cell counts. The number of granule cells per midsagittal
cerebellar section was estimated from the total area and granule cell
density of the internal granule cell layer (IGL). The area of the IGL
was measured from an image captured on a video graphics card using NIH
Image software and a CCD video camera attached to a Nikon microscope at
20× magnification. The IGL of the captured image was outlined
freehand, and the area enclosed was measured using NIH Image software.
The IGL cell density was estimated from the number of granule cells
enclosed in a 3600 µm2 area defined by
an ocular graticule at 1000× magnification. Granule cell counts were
taken from the posterior, mid, and anterior cerebellum and used to
calculate an average density.
Purkinje cell counts were performed on calbindin-immunostained
midsagittal cerebellar sections. The total number of immunostained cell
bodies in the Purkinje cell layer (PCL) and molecular layer (ML) of the
section was counted at 200× magnification using Nomarski optics.
All percentages of adult wild-type cell numbers quoted in the text are
calculated from the
+/+:Bax+/ value.
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RESULTS |
Bax deletion reduces lurcher
cerebellar atrophy
The increased expression of the proapoptotic Bcl-2
family member Bax in the dying cerebellar Purkinje cells of
lurcher (+/Lc) mice (Wullner et al., 1995; De
Jager, 1998) strongly favors a role for this protein in the putative
excitotoxic death of the cell. To determine whether Bax
expression is required for +/Lc Purkinje cell death, we
generated +/Lc mice deficient for Bax (+/Lc:Bax/) by
crossing +/Lc heterozygous Bax mice
(+/Lc:Bax+/).
+/Lc:Bax+/
crosses resulted in the death of on average one in four of the neonates, consistent with the neonatal lethality noted in homozygous lurcher pups (Cheng and Heintz, 1997). The remaining pups
developed normally into the third postnatal week when a majority of the litter began to exhibit ataxia characteristic of the +/Lc
mutation (a swaying of the body with a tendency to fall from side to
side). Genotyping revealed that these ataxic mice included
+/Lc:Bax/ as
well as
+/Lc:Bax+/ and
+/Lc:Bax+/+
offspring. No difference was observed in the timing of the onset of
ataxia between the three +/Lc:Bax
genotypes. All these mice remained ataxic into adulthood and were
killed for morphological analysis at postnatal day 30 (P30;
n = 5 mice). After dissection it was evident that the
cerebella of
+/Lc:Bax/ mice
were noticeably larger with deeper, more-pronounced fissures than that
of their
+/Lc:Bax+/ and
+/Lc:Bax+/+
littermates (Fig. 1). This observation
indicated a reduction of the cerebellar atrophy associated with the
+/Lc mutation in mice deficient for Bax. A
similar effect was observed in
+/Lc:Bax/ mice
analyzed at later ages (P60, n = 2 mice, and P300,
n = 3 mice).
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Figure 1.
Bax deletion reduces
+/Lc cerebellar atrophy. Posterior view of the whole
cerebellum lightly stained with cresyl violet. Note the larger lobules
and more pronounced fissure of the
+/Lc:Bax/ compared
with the
+/Lc:Bax+/
cerebellum. Scale bar, 1 mm.
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Bax deletion rescues granule cells and delays Purkinje
cell death in lurcher mice
Granule cells make up the bulk of the cerebellar mass, and cresyl
violet staining of cerebella sections from
+/Lc:Bax/ mice
revealed the presence of a large, cell-dense IGL. The numerous granule cells and normal lobular pattern of the cerebellar cortex in
+/Lc:Bax/ mice
were in stark contrast to the granule cell-sparse, atrophic lobules of
the +/Lc:Bax+/+
and +/Lc:Bax+/
littermates (Fig. 2). Despite the
abundant presence of granule cells, the examination of
+/Lc:Bax/
sections under high-powered objectives revealed the presence of few
Purkinje cells at the interface of the IGL and ML. The large-scale
absence of Purkinje cells in
+/Lc:Bax/ mice
was confirmed by immunolabeling P30 cerebellar sections with antibodies
to calbindin (Fig. 3). Calbindin
immunolabeling also revealed abnormal Purkinje cell morphology in
+/Lc:Bax/
mice: the Purkinje cells were not aligned in a monolayer and had
stunted, poorly developed dendritic trees that failed to reach the pial
surface (Fig. 3b), a morphology that is characteristic of
+/Lc mice.
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Figure 2.
Bax deletion rescues granule cells
from secondary cell death in +/Lc mice. Midsagittal
cerebellar sections of postnatal day 30 mice from
+/Lc:Bax+/
crosses stained with cresyl violet. Note the large numbers of granule
cells in the well lobulated cerebellum of
+/Lc:Bax/
mice compared with the cell-sparse, atrophic lobules of
+/Lc mice containing either one or both alleles of the
Bax gene. Scale bar, 1 mm.
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Figure 3.
Bax deletion partially rescues
+/Lc Purkinje cells from cell-autonomous death.
Calbindin-immunolabeled midsagittal cerebellar sections of postnatal
day 30 mice from
+/Lc:Bax+/
crosses. a, Whole cerebellum view. Note the increased
numbers of immunolabeled Purkinje cells in the posterior cerebellum of
+/Lc:Bax/compared
with the
+/Lc:Bax+/
mice. b, Higher-powered view of the posterior cerebellum
showing the stunted Purkinje cell morphology characteristic of the
Lc mutation in the
+/Lc:Bax+/and
+/Lc:Bax/
mice. Scale bars: a, 1 mm; b, 100 µm.
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We estimated the number of granule cells per midsagittal cerebellar
section at P30 and P300 using morphometric analysis and cell counts.
The data revealed the presence of 60% of the wild-type (+/+:Bax+/) number of
granule cells in
+/Lc:Bax/ mice
at both ages (~32,000 granule cells per section), compared with a
value of 10% or less in the
+/Lc:Bax+/+ and
+/Lc:Bax+/ mice
at P30 (Fig. 4a). The
persistence of 60% of the wild-type number of granule cells in the
+/Lc:Bax/ mice
indicates that the deletion of Bax permanently rescues these cells from target-related cell death. In spite of the evidence of
widespread Purkinje cell death in the calbindin-immunolabeled sections
of +/Lc:Bax/ mice,
Purkinje cell counts revealed significantly increased numbers of
Purkinje cells in the Bax-deficient +/Lc mice at
P30. At this age there was 15% of the wild-type
(+/+:Bax+/) number of
Purkinje cells in
+/Lc:Bax/ mice
compared with 6% or less in +/Lc mice with either one or both Bax alleles (Fig. 4b). However, the
examination
+/Lc:Bax/ mice
at P300 demonstrated that +/Lc Purkinje cell death was
delayed but not prevented by the deletion of Bax, because
the number of Purkinje cells was reduced to 1% of the wild-type value
in these older mice (Fig. 4b). The delay but eventual death
of Purkinje cells in
+/Lc:Bax/ mice
indicates the existence of a Bax-independent cell death pathway in Purkinje cells.
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Figure 4.
Quantification of granule cell and Purkinje cell
survival in postnatal day 30 and postnatal day 300 (Aged) mice from
+/Lc:Bax+/
crosses. a, Bax deletion permanently
rescues granule cells from target-related cell death in
+/Lc mice. The number of granule cells per midsagittal
cerebellar section was estimated from the area and cell density of the
internal granule cell layer. b, Bax
deletion delays but does not prevent cell-autonomous Purkinje cell
death in +/Lc mice. The number of Purkinje cells per
midsagittal cerebellar section was counted from sections processed for
calbindin immunocytochemistry. The data are presented as the mean
value ± SEM (n = 3-5 mice).
Asterisks denote statistically significant differences:
a, *p < 0.000001; b,
*p < 0.05 (unpaired two-tailed Student's
t test).
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We next examined +/Lc mice deficient for Bax at
the younger age of P15, the approximate midpoint in the granule cell
death response in lurcher mice (see Doughty et al., 1999).
By examining these younger mice, we hoped to establish whether
(1) the presence of 60% of the wild-type number of granule
cells at P30 and older was the result of the partial rescue of the cell
population or the prevention of all target-related granule cell death
in the mutant, (2) Purkinje cell death was delayed in all or only a
regional subset of cells, and (3) +/Lc Purkinje cell
development was ameliorated in the absence of Bax
expression. We conducted the same analysis as before using cresyl
violet staining and calbindin immunocytochemistry of midsagittal
cerebellar sections (n = 5 mice). At P15 there was
already a clear increase in the size and density of the IGL of
+/Lc:Bax/
compared with
+/Lc:Bax+/ littermates
(Fig. 5a). Cell counts and
morphometric analysis of the IGL confirmed a significant increase in
granule cell numbers in the
+/Lc:Bax/ mice
(Fig. 5c). The estimated number of granule cells per section at P15 was very similar to the value obtained for
+/Lc:Bax/ mice
at P30 and P300 (33,000 cells per section at P15 compared with on
average 32,000 cells per section in the older animals). The presence of
equal numbers of granule cells at P15, P30, and P300 in
+/Lc:Bax/
mice, in addition to the absence of any pyknotic profiles in the IGL at
P15 (in contrast to the numerous profiles observed in
+/Lc:Bax+/
littermates), suggests that target-related granule cell death in
+/Lc mice is prevented by the deletion of Bax. If
granule cell death is completely rescued by Bax deletion,
then the decrease in total granule cell numbers in
+/Lc:Bax/ mice
is presumably attributable to reduced production of granule cells in
response to local signals from Purkinje cells (Smeyne et al., 1995;
Wechsler-Reya and Scott, 1999). Cell counts revealed greater numbers of
Purkinje cells in the
+/Lc:Bax/ at
P15 (62% of the adult wild-type value) compared with P30 (15% of the
adult wild-type value) (Fig. 5d). Calbindin immunolabeling of P15 cerebellar sections revealed a similar pattern of Purkinje cell
distribution in
+/Lc:Bax/ and
+/Lc:Bax+/
littermates (Fig. 5b), although all of the remaining cells
had poorly developed dendrites. The facts that the number of surviving Purkinje cells in early postnatal
+/Lc:Bax/ animals is
directly proportional to the number of granule cells present in the
adult, that Purkinje cell loss in these animals is eventually complete,
that no further granule cell loss is noted after P15 in
+/Lc:Bax/
mice, and that granule cell production is known to be controlled locally by Purkinje cells (Smeyne et al., 1995; Wechsler-Reya and
Scott, 1999) are all consistent with the complete rescue of secondary
granule cell death in response to loss of Bax expression. However, we cannot exclude the possibility of a
Bax-independent cell death pathway as the cause for the
lower number of granule cells in
+/Lc:Bax/
mice.
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Figure 5.
The effect of Bax deletion on
granule cell and Purkinje cell development in +/Lc mice.
a, Midsagittal cerebellar sections of postnatal day 15 mice from
+/Lc:Bax+/
crosses stained with cresyl violet. Note the already increased
cerebellar size and greater area and cell density of the internal
granule cell layer in the
+/Lc:Bax/
mice. b, Calbindin-immunolabeled midsagittal cerebellar
sections of postnatal day 15 mice from
+/Lc:Bax+/
crosses. Gaps in the Purkinje cell layer reveal cell death in the
developing
+/Lc:Bax+/
and
+/Lc:Bax/
mice. Scale bar: a, b, 1 mm. c,
Quantification of granule cell survival in postnatal day 15 mice from
+/Lc:Bax+/
crosses. The data are the mean estimated number of granule cells per
midsagittal cerebellar section ± SEM (n = 3-5 mice). The asterisk indicates a statistically
significant difference (p < 0.00001;
unpaired two-tailed Student's t test).
d, Quantification of Purkinje cell survival in postnatal
day 15 mice from
+/Lc:Bax+/
crosses. The data are the mean number of calbindin-immunolabeled
Purkinje cells per midsagittal cerebellar section ± SEM
(n = 3-5 mice).
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Bax-mediated neuronal death in lurcher mice
is p53 independent
The expression of the tumor suppressor gene p53 has
been shown to be required for many Bax-mediated cell death
pathways (Aloyz et al., 1998; Xiang et al., 1998; Cregan et al., 1999).
To examine the possible requirement of p53 expression for
the Bax-mediated neuronal cell death in +/Lc
mice, we generated +/Lc mice deficient for p53
(+/Lc:p53/) by
crossing +/Lc mice heterozygous for p53
(+/Lc:p53+/).
We hypothesized that a rescue of cerebellar cell death in
+/Lc:p53/ mice
comparable with that observed in
+/Lc:Bax/ mice
would indicate the need for p53 expression to activate the Bax cell death pathway in the +/Lc cerebellum. As
is the case for Bax, the deletion of p53 failed
to prevent the development of ataxia in the third postnatal week in
+/Lc:p53/
mice. However in contrast to the
+/Lc:Bax/
mice,
+/Lc:p53/ mice
showed no sign of amelioration in the cerebellar atrophy associated
with the +/Lc mutation when killed at P30 (n = 4 mice). Histological analysis confirmed that the cerebella of
+/Lc:p53/ mice
were indistinguishable from that of their
+/Lc:p53+/
littermates (data not shown). The meager presence of Purkinje cells and
granule cells in the cerebella of
+/Lc:p53/ mice
demonstrates that the Bax-mediated death of these cells is
independent of p53 expression.
Caspase-3 activation in lurcher mice is
Bax dependent
Caspase-3, a widespread effector protease in apoptotic cell death,
has been shown to be activated in the dying Purkinje cells and granule
cells of +/Lc mice (Selimi et al., 2000). Interestingly, the
activation of caspase-3 in granule cell cultures switched from medium
containing high to low concentrations of potassium requires the
expression of Bax (Miller et al., 1997). We therefore examined the expression of active caspase-3 in
+/Lc:Bax/ and
+/Lc:Bax+/ mice
during the period of Purkinje cell death (P15 -P20) using an antibody
that only recognizes the active subunit of the protease. The
immunocytochemical labeling of cerebellar sections from
+/Lc:Bax+/ mice
confirmed the expression of active casapse-3 in dying Purkinje cells
and granule cells (data not shown). Consistent with the results of
Selimi et al. (2000), few Purkinje cells were immunolabeled with the
antiserum (on average, three per sagittal section of the cerebellar
vermis; n = 2 mice from 2 litters, 5 sections per animal), indicating the rapid death of the cell after cleavage and
activation of the caspase. In contrast, the simultaneous
labeling of cerebellar sections from
+/Lc:Bax/ mice failed
to detect any active caspase-3 expression in Purkinje cells or granule
cells (n = 3 mice from 3 litters, 5 sections per
animal). These contrasting observations suggest that caspase-3 is
not activated in the dying Purkinje cells of
+/Lc:Bax/ mice and
suggest the existence of a caspase-3-independent,
Bax-independent cell death pathway in the cell.
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DISCUSSION |
Previous studies of +/Lc mice suggest an
apoptotic mechanism of Purkinje cell death involving Bax
expression and induction of caspase-3 activity (Norman et al., 1995; De
Jager, 1998; Wullner et al., 1998; Selimi et al., 2000). By applying a
genetic approach, we have demonstrated the involvement of
Bax in both the primary, cell-autonomous death of Purkinje
cells and the secondary loss of granule cells in +/Lc mice.
The deletion of Bax expression in +/Lc mice
permanently rescued granule cells from target-related cell death and
delayed but did not prevent Purkinje cell death. From these
loss-of-function experiments, we conclude there is an absolute
requirement for Bax expression in target-related granule cell death, despite the presence of only 60% of the +/+ number in
adult and aged
+/Lc:Bax/
mice. This conclusion is based on the strong evidence of a positive mitogenic role of Purkinje cells in granule cell production (Smeyne et
al., 1995; Wechsler-Reya and Scott, 1999). Thus the retarded development of Purkinje cells in
+/Lc:Bax/ mice
is likely to reduce granule cell production in these mice. The delay
but failure to prevent +/Lc Purkinje cell death by the deletion of Bax expression indicates the presence of both
Bax-dependent and Bax-independent death pathways
in these cells. Next, we examined the role of p53, a
molecule widely implicated in the regulation of Bax-mediated
neuronal cell death (Aloyz et al., 1998; Xiang et al., 1998; Cregan et
al., 1999). Our analysis showed that the deletion of p53
expression in +/Lc mice did not prevent target-related granule cell death, demonstrating that Bax is expressed
independently of p53 in these cells. Finally, we suggest
that the activation of caspase-3 in +/Lc cerebella (Selimi
et al., 2000) requires the expression of Bax. Because
Purkinje cells continue to die in
+/Lc:Bax/
mice, this observation suggests the activation of a
Bax-independent, capase-3-independent death pathway in the cell.
Bax activity is essential for many programmed cell death
(PCD) events in CNS development (Deckwerth et al., 1996; White et al.,
1998), and the protein has been shown to regulate neuronal sensitivity
to both excitotoxic and genotoxic injury (Xiang et al., 1998; Cregan et
al., 1999). Therefore the observation that Bax expression is
increased in +/Lc Purkinje cells (De Jager, 1998; Wullner et
al., 1998) suggested a role for this protein in the cell-autonomous
death of this cell. Interestingly, increased Bax expression
was not reported in the cerebellar granule cells of +/Lc
mice by these authors, despite the large-scale death of these neurons
after the loss of target Purkinje cells. Contrary to these
observations, our loss-of-function experiments clearly demonstrate that
the target-related death of granule cells is dependent on
Bax expression, whereas Bax deletion does not
prevent +/Lc Purkinje cell death. Nevertheless, the rate of
+/Lc Purkinje cell death was slowed in the absence of
Bax. This indicates the involvement of Bax in
Purkinje cell death, but it also reveals the activation of a
Bax-independent pathway in the cell.
The presence of a Bax-independent cell death pathway in
Purkinje cells is not surprising considering the strong activation of
the 2 glutamate receptor in response to the lurcher
mutation. Thus, induction of both Bax and Bcl-X
has been demonstrated in +/Lc Purkinje cells (De Jager,
1998; Wullner et al., 1998), suggesting a possible role for the
proapoptotic isoform of Bcl-X in a parallel pathway for
activation of the effector phase of apoptotic cell death in these
neurons. This is consistent with the demonstration that granule cells
from Bax/ mice will undergo
excitotoxic death in response to NMDA application in culture (Miller et
al., 1997). Alternatively, the removal of Bax as a
rate-limiting component of the cell death pathway active in
+/Lc Purkinje cells may provoke a less well defined pathway. The observation that active caspase-3 is not detected in
+/Lc:Bax/
Purkinje cells demonstrates that this alternative cell death pathway
does not involve the activation of this protease.
The permanent rescue of secondary granule cell loss in
+/Lc:Bax/ mice is
consistent with previous studies of apoptotic cell death in response to
target deprivation. Thus, a requirement for Bax in cell
death attributable to trophic factor withdrawal for sympathetic and
motor neurons has been demonstrated using
Bax/ mice (Deckwerth et al.,
1996). Furthermore, partial rescue of inferior olivary neurons from
target-related cell death by the overexpression of Bcl-2
in +/Lc mice has been reported (Zanjani et al., 1998) (it
should be noted that Bcl-2 was not overexpressed in granule
cells in these mice). These examples support the activation of a
common, Bcl-2 family-mediated cell death response to the loss of target support. In spite of experiments indicating a role for
p53 in cell death models induced by DNA damage (Woods and Youle, 1995), excitotoxicity (Xiang et al., 1998), PCD, and trophic factor withdrawal (Aloyz et al., 1998) and the expression of
p53 in developing cerebellar granule cells (van Lookeren
Campagne and Gill, 1998), our results failed to reveal a role for
p53 in granule cell death in response to target
deprivation. This is interesting considering the reported
induction of a Bax-dependent cell death pathway in
response to adenovirus-mediated delivery of p53 in granule
cells in vitro (Cregan et al., 1999). We conclude that the
activation of Bax in response to target deprivation
in vivo occurs by an alternative p53-independent
mechanism, indicating the presence of multiple pathways for the
activation of Bax-dependent cell death in cerebellar granule
neurons. Although we have not tested directly the requirement for
caspase-3 in granule cell death in
+/Lc:Bax/
mice, the fact that active caspase-3 was not observed in these cells
strongly supports an important role for this protease.
The data presented here suggest that the constitutive activation of
GRID2Lc induces multiple death pathways in
the Purkinje cell that involve apoptotic (Bax expression and
caspase-3 activation) and unknown, possibly necrotic, mechanisms. The
complete rescue of granule cells in
+/Lc:Bax/
mice, on the other hand, confirms that these cells die by a purely apoptotic mechanism. Recently, a number of studies (Martinou et al.,
1994; Ankarcrona et al., 1995; Krajewski et al., 1995; Chen et al.,
1998; Namura et al., 1998) have indicated that apoptosis is a key
component of ischemic brain injury [for a review of current perspectives, see Lee et al. (1999)]. Thus, the features of
+/Lc cell death in many ways mirror the events of brain
injury caused by transient cerebral ischemia (Heintz and Zoghbi, 2000).
In both cases, the primary insult is the activation of glutamate
receptors, and this leads first to the expression of neuroprotective
Bcl-2 family members that, once overwhelmed, results in the
activation of cell death pathways that include Bax
expression and caspase activation, as well as unknown molecules. In
turn, the primary lesion leads to the secondary loss of afferent
neurons by apoptosis. However, unlike models of cerebral ischemia, the
site of the primary lesion is well characterized in +/Lc
mice, and this advantage should prove invaluable in the use of this
mutant as a model to study cell death mechanisms in ischemic brain injury.
| |
FOOTNOTES |
Received Dec. 15, 1999; revised Feb. 15, 2000; accepted March 2, 2000.
This work was supported by the Howard Hughes Medical Institute (N.H.),
the National Institutes of Health (NIH) National Institute of
Neurological Disorders and Stroke Program Project PHS NS 30532 (N.H. and M.L.D.), and the NIHNational Institute of General Medical Sciences Grant GM 07739 (P.L.D.J.).
Correspondence should be addressed to Dr. Nathaniel Heintz, Laboratory
of Molecular Biology, The Rockefeller University, 1230 York Avenue, New
York, NY 10021. E-mail: heintz{at}rockvax.rockefeller.edu.
Dr. De Jager's present address: Department of Medicine, Beth Israel
Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215.
| |
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TOP
ABSTRACT
INTRODUCTION
