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The Journal of Neuroscience, June 15, 1999, 19(12):5026-5033
Attenuation of Ischemia-Induced Cellular and Behavioral Deficits
by X Chromosome-Linked Inhibitor of Apoptosis Protein Overexpression in
the Rat Hippocampus
Daigen
Xu1, 2,
Yves
Bureau4,
Dan C.
McIntyre4,
Donald W.
Nicholson5,
Peter
Liston2, 3,
Yanxia
Zhu1, 2,
Wei Gin
Fong3,
Stephen J.
Crocker1,
Robert G.
Korneluk2, 3, and
George S.
Robertson1, 6
1 Department of Cellular and Molecular Medicine,
Faculty of Medicine, University of Ottawa, Ottawa, Ontario K1H 8M5,
Canada, 2 Apoptogen and 3 Genetics Research
Laboratory, Children's Hospital of Eastern Ontario, Ottawa, Ontario
K1H 8L1, Canada, 4 Department of Psychology, Carleton
University, Ottawa, Ontario K1S 5B6, Canada, and
5 Apoptosis Research Program, Department of Biochemistry
and Molecular Biology and 6 Department of Pharmacology,
Merck Frosst Centre for Therapeutic Research, Kirkland, Quebec H9H 3L1,
Canada
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ABSTRACT |
Transient forebrain ischemia produced by four-vessel occlusion
(4-VO) triggers the delayed death of CA1 neurons in the hippocampus, resulting in behavioral deficits of spatial learning performance. We
demonstrate that CA1 neuronal loss induced by 4-VO (12 min) is preceded
by a selective and marked elevation of catalytically active caspase-3
in these neurons, indicative of apoptosis. Virally mediated
overexpression of the anti-apoptotic gene X chromosome-linked inhibitor
of apoptosis protein (XIAP) prevented both the production of
catalytically active caspase-3 and degeneration of CA1 neurons after
transient forebrain ischemia. CA1 neurons protected in this manner
appeared to function normally, as assessed by immunohistochemical detection of the neuronal activity marker nerve growth factor inducible-A and by spatial learning performance in the Morris water maze. These findings indicate that caspase-3 activation is a key
event in ischemic neuronal death and that blockade of this event by
XIAP overexpression permits CA1 neurons to survive and operate properly
after an ischemic insult.
Key words:
XIAP; caspase-3; DNA fragmentation; water maze; apoptosis; NGFI-A
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INTRODUCTION |
Accumulating evidence indicates that
apoptosis contributes to neuronal cell death in a variety of
neurodegenerative contexts (Choi, 1996 ; Rinkenberger and Korsmeyer,
1997). Recently, a provocative link between developmental apoptosis and
neurodegenerative disease has been shown for acute spinal muscular
atrophy (SMA) by the identification of a candidate gene encoding
neuronal apoptosis inhibitor protein (NAIP) (Roy et al., 1995).
Disruption of NAIP function is thought to precipitate SMA by permitting
unrestrained developmental apoptosis in a variety of sensory and motor
systems. In support of this hypothesis, we have observed a striking
overlap between neuronal populations lost in SMA and the distribution of NAIP immunoreactivity in the rodent CNS (Xu et al., 1997b). We have also demonstrated that treatments that elevate NAIP expression prevent CA1 neuron loss after transient forebrain ischemia (Xu et al.,
1997a).
The molecular mechanisms by which NAIP blocks apoptosis are at least
partly dependent on three conserved domains, ~45-60 amino acids in
length, known as baculoviral-inhibitor of apoptosis repeats (BIRs),
which are shared by all four members of the inhibitor of apoptosis
protein (IAP) family [NAIP, X chromosome-linked IAP (XIAP), human
IAP-1 (HIAP-1), and HIAP-2] (Liston et al., 1996). Overexpression of
each member of the IAP family or just the BIR domains found in these
proteins is sufficient to block apoptosis in a variety of cell lines
produced by several different triggers (Liston et al., 1996; Deveraux
et al., 1997; Roy et al., 1997). Recent evidence suggests that the IAPs
may prevent apoptosis by inhibiting caspase-3 activity (Deveraux et
al., 1997; Roy et al., 1997).
Proteolytic activation of the cysteine protease caspase-3 appears to be
a key event in the execution of programmed cell death (apoptosis) in
the CNS (Johnson et al., 1996; Alnemri, 1997; Nicholson and Thornberry,
1997). The lack of programmed cell death in caspase-3 knock-out mice
suggests that this protease may participate in neuronal apoptosis
during CNS development (Kuida et al., 1996). Recent evidence suggests
caspase-3 may participate in ischemic-excitotoxic brain damage
(Yakovlev et al., 1997; Chen et al., 1998; Endres et al., 1998; Namura
et al., 1998). In the present study, we examined the production of
conformationally active caspase-3 and DNA fragmentation in degenerating
CA1 neurons after transient cerebral ischemia to study the relationship
between ischemia-induced caspase-3 activation and apoptosis. Given that
XIAP has been shown to be a potent inhibitor of both caspase-3
activation and apoptosis, we examined the effects of adenovirally
mediated XIAP overexpression on the these events in CA1 neurons after a
brief episode of forebrain ischemia. The functional status of CA1
neurons 7-14 d after transient global ischemia was assessed at both
the cellular and behavioral levels by immunohistochemical detection of
the neuronal activity marker nerve growth factor inducible-A
(NGFI-A) in CA1 neurons and measurement of spatial learning
performance in the Morris water maze, respectively. Previous studies
have shown that a brief period of global forebrain ischemia severely
suppresses both of these measures of normal CA1 synaptic activity (Auer
et al., 1989; Kiessling et al., 1993; Dragunow et al., 1994; Olsen et
al., 1994; McGahan et al., 1998).
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MATERIALS AND METHODS |
Transient forebrain ischemia model. All animal
procedures conformed to the Guide for the Care and Use of
Experimental Animals (Olfert et al., 1993) endorsed by the Medical
Research Council of Canada. Transient forebrain ischemia was produced
using published modifications of the four-vessel occlusion (4-VO)
method (Pulsinelli and Buchan, 1988). Adult male Wistar rats (250-275
gm; Charles River, Montréal, Canada) were anesthetized using
1.5% halothane 1 d before ischemia. The alar foraminae of the
first cervical vertebra were then approached through a dorsal midline
neck incision. The vertebral arteries were permanently occluded by
electrocauterization. The carotid arteries were isolated through a
ventral midline neck incision, and silk ligatures were placed loosely
around them. The incisions were then closed with topical xylocaine
jelly. The next day, rats were randomly divided into two groups and
subjected to either 12 min of forebrain ischemia (experimental) or
manipulation without carotid occlusion (sham). Forebrain ischemia was
achieved by lifting the common carotid arteries using the silk
ligatures placed around them and occluding the vessels with
microaneurysm clips (#160-863; George Tiemann & Co., Plainview, NY).
Brain temperature was measured indirectly via a thermocouple probe
placed in the temporalis muscle. Body temperature was maintained
between 36 and 37°C by external warming. Ischemia was terminated by
removal of the microaneurysm clips. Animals that convulsed after
reperfusion were omitted from the study, along with those that did not
develop fully dilated pupils or failed to lose righting reflex during ischemia. In the case of sham-treated animals, the carotids were exposed but not occluded.
Stereotaxic injection of adenoviral constructs. Viral
constructs have been described previously by Liston et al. (1996).
Adenoviral vectors (3 µl injected over 10 min; 1 × 106/µl particles) were injected into the dorsal
hippocampus using a 28 gauge needle at the following coordinates (in
mm): anteroposterior,  3.6; mediolateral, ±2.2 (left and right
sides); and dorsoventral, 3.2 from bregma (Paxinos and Watson,
1986).
Antibodies, immunohistochemistry, cell counting, and in
situ end labeling. The antibody for catalytically
active caspase-3 was raised in rabbits against the
(p17/p12)2 conformer corresponding to the catalytically
competent enzyme. The specificity of this antisera has been confirmed
by immunoprecipitation of active caspase from multiple cell lines
induced to undergo apoptosis by a variety of triggers (Rasper et al.,
1998). Fresh frozen sections (12-µm-thick) were incubated with PBS
containing 0.1% Triton X-100. Next, the sections were incubated with
primary antibody (1:2000) for 48 hr at 4°C. After three washes with
PBS (5 min each), the sections were incubated with
CY3-labeled donkey anti-rabbit IgG (1:500; Amersham,
Buckinghamshire, UK) for 2 hr at room temperature. For double
immunofluorescence labeling, biotinylated
acetyl-aspartyl-glutamyl-valyl-aspart-1-aldehyde (DEVD-CHO)
(1:1000; BIOMOL">Biomol, Plymouth Meeting, PA) and FITC-labeled streptavidin
(1:50; Amersham) were coincubated with the primary and secondary
antibodies, respectively. Immunohistochemical detection of XIAP
(1:1000),  -galactosidase (1:1000), neuronal nuclei (NeuN) (1:200), or NGFI-A (1:15000) were performed on free-floating sections (12-µm-thick) cut from brain tissue that had been fixed with
paraformaldehyde as described previously (Xu et al., 1997a). The XIAP
antibody is an affinity-purified rabbit polyclonal antisera raised
against recombinant human XIAP. The specificity of this antibody has
been confirmed by Western blotting (Li et al., 1998). A monoclonal antibody for -galactosidase was obtained from Promega (Madison, WI).
NeuN was detected using a monoclonal antibody (also referred to as A60;
generously provided by Dr. R. J. Mullen, University of Utah
School of Medicine, Salt Lake City, UT) (Mullen et al., 1992). NGFI-A
was detected using a polyclonal rabbit antibody (generously provided by
Dr. R. Bravo, Bristol-Myers Squibb Pharmaceutical Research Institute,
Princeton, NJ). Ischemia-induced DNA fragmentation was assessed
using in situ end labeling (ISEL) as described by Xu
et al. (1997a). Cell counts were performed manually on randomly selected sections by an observer who was unaware of the treatment conditions. Cells that displayed fragmented DNA or immunoreactivity for
NeuN, NGFI-A, or active caspase-3 were counted in the medial, intermediate, and lateral regions of CA1 (400 µm each in length) of
the hippocampus using computer-assisted image analysis. Sections cut at
levels 2.8, 3.6, and 4.0 mm posterior to bregma (two sections per
level) were analyzed for each animal. Neuronal density (cells/100 µm
of CA1 layer) was calculated by averaging these values.
Morris water maze. The task for all of the animals in each
trial consisted of finding a hidden clear plastic platform (~18 cm in
diameter) that was placed 50 cm away from the wall of the water maze
(170 cm in diameter, 90 cm in depth) and 3 cm below the water. The
platform remained in the same position for all sessions and trials. The
starting quadrant was randomized every day, with all animals using the
same order. The animals were faced toward the pool wall before being
released. The time for them to reach the hidden platform was recorded.
The animals were allowed to rest 60 sec on the platform between trials.
If an animal failed to reach the platform in 120 sec, it was manually
guided to the platform. Before surgery, all animals were pretrained.
Each animal received four trials per day for four consecutive days.
These animals then underwent stereotaxic injections of virus, followed by 4-VO surgery a week later. One week after 4-VO surgery, all the
animals were examined as in the pretraining, except that on the last
day they were tested with the platform raised 2 cm above the surface of
the water and wrapped in black. This raised platform session was
performed to determine whether factors other than learning and memory
were responsible for deficits in spatial navigation.
Statistical analysis. A one-way ANOVA was performed
on the cell count and behavioral data. If the analysis was significant, multiple comparisons were performed using Tukey's test.
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RESULTS |
Transient cerebral ischemia selectively activates caspase-3 in
CA1 neurons
To investigate the role of caspase-3 in ischemia-induced CA1
neuronal degeneration, an antibody that selectively recognizes conformationally mature caspase-3 [(p17/p12)2 tetramer]
(Rasper et al., 1998) was used to detect catalytically active
caspase-3 in hippocampal tissue sections from animals subjected to a
brief episode of global ischemia (12 min) produced by 4-VO.
Catalytically active caspase-3 was not present in CA1 neurons of
animals subjected to sham 4-VO surgery (Fig.
1A) or killed 3 hr
after 4-VO (data not shown). Significant levels of
(p17/p12)2 immunoreactivity were apparent in CA1 pyramidal
neurons 24 hr after transient forebrain ischemia. At this time point,
(p17/p12)2 immunoreactivity was confined to the cytoplasm
of most CA1 neurons; however, a small number of CA1 neurons situated
close to midline displayed intense nuclear labeling (Fig.
1B). Nuclear labeling appeared to increase in medial
portions of the CA1 region at 48 hr (Fig. 1C). In contrast, (p17/p12)2 immunoreactivity in lateral aspects of the CA1
region remained moderate and was primarily confined to the cytoplasm of
pyramidal neurons (Fig. 1D). By 72 hr, intense
nuclear labeling was frequently observed in pyramidal neurons located
throughout the entire CA1 subfield (Fig.
1E,F). However, few
(p17/p12)2-positive CA1 neurons were seen 120 hr (5 d)
after transient forebrain ischemia (data not shown). At no time point
was caspase-3 activation detected in CA2 or CA3 sectors of the dorsal
hippocampus. Similar results were obtained in adjacent sections using
biotinylated DEVD-CHO to label conformationally mature caspase-3.
Moreover, double staining demonstrated that the same CA1 neurons were
labeled with the (p17/p12)2-specific antibody (Fig.
1G) and biotinylated DEVD-CHO (Fig. 1H),
suggesting that both methods specifically label catalytically active
caspase-3. Together, these findings indicate that caspase-3 activation
precedes the delayed death of CA1 neurons after an episode of transient forebrain ischemia.
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Figure 1.
Cerebral ischemia activates caspase-3 in CA1
neurons. Brain sections from animals exposed to 12 min of 4-VO were
processed for immunohistochemical detection of conformationally active
caspase-3. Catalytically active caspase-3 was not detected in the
hippocampal CA1 subfield of animals exposed to sham 4-VO
(A). At 24 hr after 4-VO, caspase-3 activation
was seen in CA1 pyramidal neurons. The majority of CA1 neurons
displayed moderate labeling, whereas a small population of neurons
situated close to midline exhibited intense nuclear staining
(B). At 48 hr, a larger number of neurons in the
medial CA1 were intensely labeled (C). Neurons in
the lateral CA1 remained moderately labeled (D,
arrowhead, CA2). By 72 hr, intense nuclear labeling was
frequently observed throughout the entire CA1 subfield
(E, medial CA1; F, lateral CA1;
F, arrowhead, CA2).
Immunoreactivity for conformationally active caspase-3 was restricted
to the CA1 subfield (D, F,
arrowheads, CA2). The (p17/p12)2
tetramer-specific antibody (G) and biotinylated
DEVD (H) labeled the same neurons. Note
moderate cytoplasmic (arrowhead) versus intense nuclear
labeling (arrow) (G). Scale bar:
A-F, 200 µm; G, H, 50 µm.
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Activation of caspase-3 precedes DNA fragmentation
The relationship between caspase-3 activation and apoptosis in CA1
neurons after transient forebrain ischemia was assessed by examining
the appearance of fragmented DNA in CA1 neurons by in situ
end labeling in tissue sections adjacent to those processed for
catalytically active caspase-3 (Fig. 2).
ISEL staining was first observed in medial CA1 neurons 48 hr after
transient cerebral ischemia (Fig. 2C). ISEL was not
seen in lateral aspects of the CA1 region until 72 hr after 4-VO (Fig.
2E,F). At 120 hr, intense ISEL was detected in the majority of CA1 neurons (Fig.
2G,H). Although the time course for DNA
fragmentation clearly occurs later than that for conformationally
mature caspase-3, the two events compliment each other both temporally
and spatially.
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Figure 2.
Cerebral ischemia causes DNA fragmentation in CA1
neurons. Ischemia-induced DNA fragmentation was assessed using
in situ end labeling as described by Xu et al. (1997a).
Labeling was not seen in the CA1 subfield of animals exposed to sham
4-VO (A) or those examined 24 hr after 4-VO
(B). At 48 hr after 4-VO, ISEL was detected in a
small group of CA1 pyramidal neurons located proximal to midline
(C). No labeling was seen in lateral CA1
(D). By 72 hr, ISEL was frequently observed in
neurons of both medial (E) and lateral aspects of
CA1 (F, arrowhead, CA2). At 5 d, the
majority of neurons in the CA1 region displayed intense labeling
(G, medial; H, lateral; H,
arrowhead, CA2). ISEL was restricted to the CA1 region
(H). Scale bar, 200 µm.
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XIAP overexpression reduces ischemia-induced caspase-3 activation
and apoptosis in CA1 neurons
XIAP blocks apoptosis induced by a variety of triggers in several
different cell lines (Liston et al., 1996). At least part of this
anti-apoptotic activity appears to be mediated by the inhibition of
conformationally mature caspase-3 (Deveraux et al., 1997). We have
recently shown that NAIP, another member of the human IAP family,
attenuates the loss of CA1 neurons after a brief period of cerebral
ischemia (Xu et al., 1997a). These observations suggest that IAPs may
reduce ischemic damage by blocking caspase-3 activation in CA1 neurons.
To test this hypothesis, we used an adenoviral construct capable of
overexpressing XIAP in CA1 neurons. This adenoviral construct was
injected unilaterally into the dorsal hippocampus. To control for
effects of the adenoviral construct that were unrelated to XIAP
overexpression, an adenoviral construct encoding bacterial
-galactosidase (lacZ) was injected into the contralateral
hippocampus. Virally mediated overexpression of lacZ and XIAP
was confirmed by immunohistochemical detection of -galactosidase and
human XIAP, respectively (Fig.
3A,B).
All of the animals were subjected to 4-VO (12 min) 7 d after
stereotaxic surgery. In the dorsal hippocampus, which received the lacZ
construct, a large number of (p17/p12)2-positive CA1
neurons (17 ± 3/100 µm) were detected 48 hr after the ischemia
insult (Fig. 3C). In contrast, very few
(p17/p12)2-immunoreactive neurons (2 ± 1/100 µm)
were present in the contralateral hippocampus, which had been injected
with the XIAP construct (Fig. 3D). These results suggest that XIAP overexpression attenuates ischemia-induced caspase-3 activation.
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Figure 3.
Attenuation of ischemia-induced caspase-3
activation and apoptotic death in CA1 neurons by XIAP overexpression.
Animals received intrahippocampal injections of adenoviral constructs
containing either lacZ (left side) or XIAP (right side) and were
subjected to 4-VO or sham surgery 5 d later. Virally mediated
overexpression of lacZ and XIAP was confirmed by immunohistochemical
detection of -galactosidase and human XIAP, respectively. The
majority of immunoreactive cells in the CA1 layer appeared to be
pyramidal neurons. However, a small number of glial cells were also
infected with the viral constructs. Scale bar (in B):
A, B, 75 µm. In animals that received
intrahippocampal injections of the adenoviral constructs followed by
sham 4-VO (n = 4), approximately the same number of
CA1 neurons were immunoreactive for -galactosidase (9 ± 2) and
human XIAP (10 ± 3). Levels of catalytically active caspase-3
were assessed in CA1 neurons by immunohistochemical detection of the
(p17/p12)2 tetramer 48 hr after 4-VO (n = 4) (C, D). In the hippocampus injected
with the lacZ construct, a large number of neurons were stained in the
medial aspect of CA1 (C). In contrast, very few
immunoreactive neurons were present in the contralateral hippocampus
that had been injected with the XIAP construct
(D). DNA fragmentation was examined 5 d
after 4-VO in animals injected with these viral constructs. A larger
number of ISEL-positive CA1 neurons were observed in the lacZ-injected
side (E). In contrast, considerably fewer
ISEL-positive neurons were detected in the XIAP-injected side
(F). Scale bar (in F):
C-F, 75 µm. Immunohistochemistry for NeuN was
performed to stain surviving neurons 7 d after 4-VO (12 min). A
small number of neurons was observed in the CA1 subfield injected with
the lacZ construct (G), whereas the majority of
neurons in the XIAP-injected side remained NeuN-positive
(H). Scale bar (in
H): G, H, 400 µm.
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We next determined whether XIAP overexpression also reduces
ischemia-induced DNA fragmentation in CA1 neurons. Six animals that had
received intrahippocampal injections of the lacZ (left side) and XIAP
(right side) constructs were subjected to 4-VO (12 min) and examined
for ISEL 5 d later. A larger number of ISEL-positive CA1 neurons
(16 ± 2/100 µm) were observed in the lacZ-injected side
compared with the XIAP-injected side (3 ± 1/100 µm) (Fig. 3E,F). Consequently, the
ability of XIAP to block caspase-3 activation was associated with a
reduction of DNA fragmentation in CA1 neurons.
Sections adjacent to those used for ISEL labeling were processed for
immunohistochemical detection of NeuN, a neuron-specific vital marker
(Mullen et al., 1992). In agreement with the inhibitory effects of XIAP
overexpression on caspase-3 activation and DNA fragmentation, there was
a dramatic reduction in the loss of CA1 neurons after a brief episode
of transient forebrain ischemia (Fig.
3G,H). Cell counts revealed that
considerably more CA1 neurons remained in the XIAP-injected side
compared with the contralateral side, which had received the lacZ
construct. Only 4 ± 1 neurons/100 µm length of the CA1 region
were NeuN-positive in the lacZ-injected side. A consistent loss of CA1
neurons was observed throughout the medial to lateral extent of the
dorsal hippocampus. In comparison, 16 ± 2 neurons were detected
in the CA1 region (100 µm length) of the XIAP-injected side. These
findings indicate that XIAP overexpression produced a fourfold
elevation of neuronal survival after global ischemia.
XIAP overexpression attenuates ischemia-induced behavioral and
cellular deficits
To assess the functional status of CA1 neurons that survived the
ischemic episode, we examined spatial learning performance using the
Morris water maze. In agreement with previous reports (Olsen et al.,
1994), we observed that exposure to a brief period of global forebrain
ischemia significantly impaired spatial learning in animals that
received bilateral intrahippocampal injections of the lacZ construct
(Fig. 4A). In contrast,
animals injected bilaterally with the XIAP construct and subjected to
transient forebrain ischemia (XIAP-4-VO) exhibited the same short
latencies as sham-operated animals (lacZ-sham). Accordingly, the
performance of XIAP-4-VO animals was superior to that of the
lacZ-4-VO group. Tissue sections from the dorsal hippocampus of each
animal were processed for immunohistochemical detection of NeuN after
the water maze task. Consistent with the behavioral results,
adenovirally mediated XIAP overexpression significantly reduced the
loss of CA1 neurons 14 d after an episode of transient global
ischemia (Fig. 4B). This finding suggests that CA1
neurons protected from ischemic injury by XIAP overexpression are
functionally normal.
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Figure 4.
XIAP overexpression prevents deficits in spatial
learning and neuronal activity after 4-VO. Spatial learning performance
in the Morris water maze (A). Three groups,
composed of nine animals each, were pretrained to find a submerged
platform over the course of 4 d (4 trials/d). All three groups of
animals displayed similar latencies to reach the platform
(pretraining). Next, two of the three groups
received bilateral intrahippocampal injections of either the lacZ
(LacZ-4VO) or XIAP (XIAP-4VO) adenoviral
constructs and were exposed to transient global ischemia (12 min)
7 d later. The third group, which received bilateral
intrahippocampal injections of the lacZ virus and were subjected to
sham 4-VO, served as controls (LacZ-sham). Compared with
the lacZ-sham group, animals in the lacZ-4-VO group took considerably
longer to find the submerged platform over the course of 3 d
(testing) (p < 0.01). In
contrast, XIAP-4-VO animals exhibited the same short latencies as the
lacZ-sham group (p > 0.05). Furthermore,
the performance of XIAP-4-VO animals was superior to that of the
lacZ-4-VO group (p < 0.01). All three
groups performed equally on the raised platform test
(cued) (p > 0.05).
Quantification of NeuN- and NGFI-A-immunoreactive CA1 neurons
(B, C). Cell counts of
NeuN-immunoreactive neurons revealed that the average neuronal density
for the lacZ-sham, lacZ-4-VO, and XIAP-4-VO groups was 24 ± 1, 2.6 ± 0.7, and 14 ± 2 (cells/100 µm), respectively
(B). Although fewer in number, similar results
were obtained for NGFI-A-immunoreactive neurons
(C). The average density of NGFI-A-immunoreactive
neurons for the lacZ-sham, lacZ-4-VO, and XIAP-4-VO groups was
16 ± 1.3, 1 ± 0.5, and 11 ± 2, respectively.
Statistical analysis revealed that, for both NeuN and NGFI-A
immunoreactivity, significantly more labeled neurons were present in
the XIAP-4-VO than lacZ-4-VO group (p < 0.01).  p < 0.01 relative to lacZ-sham and
XIAP-4-VO; *p < 0.01 relative to lacZ-sham and
lacZ-4-VO. Immunohistochemical detection of NGFI-A
(D). Top, LacZ-sham;
middle, lacZ-4-VO; bottom, XIAP-4-VO.
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To address the functional status of CA1 neurons at the cellular level,
we examined expression of the neuronal activity marker NGFI-A. We have
demonstrated previously that similar results are obtained with NeuN-,
NGFI-A-, and Nissl-stained sections (Xu et al., 1997a; McGahan
et al., 1998; our unpublished observations). Basal expression of
NGFI-A is driven by natural synaptic activity (Worley et al., 1991) and
is severely depressed in CA1 neurons after an episode of global
forebrain ischemia (Kiessling et al., 1993; Dragunow et al., 1994;
Olsen et al., 1994; McGahan et al., 1998). In keeping with performance
in the Morris water maze, XIAP overexpressing CA1 neurons that survived
the ischemic insult displayed considerably higher levels of NGFI-A than
animals injected with the lacZ construct (Fig.
4C,D). The ability of XIAP overexpression to
maintain high basal levels of NGFI-A expression in CA1 neurons after
the ischemic insult is further evidence that these neurons function properly.
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DISCUSSION |
Caspase-3 mediates ischemic neuronal apoptosis of CA1 neurons
We have demonstrated that catalytically active caspase-3 is
generated in CA1 neurons that are undergoing ischemic cell death. This
finding is in agreement with recent reports demonstrating cleavage of
caspase-3 and increased enzymatic activity of this protease in ischemic
brain tissue after transient cerebral ischemia (Chen et al., 1998;
Namura et al., 1998). Furthermore, our observation that
ischemia-induced caspase-3 activation precedes DNA fragmentation in
vulnerable CA1 neurons suggests that these events may be linked. The
pathway by which caspase-3 activation leads to DNA fragmentation in
neurons is unclear. However, a caspase-3-activated deoxyribonuclease, termed CAD, has been identified, which causes the degradation of
chromosomal DNA into nucleosomal units characteristic of apoptosis (Enari et al., 1998). In normal cells, CAD activity is suppressed by
binding to its inhibitor ICAD. Catalytically active caspase-3 cleaves
ICAD, permitting CAD to enter the nucleus and commence DNA
fragmentation (Sakahira et al., 1998). Several nuclear enzymes responsible for genomic integrity, such as poly(ADP-ribose)
polymerase and DNA-dependent protein kinase, are also cleaved by
caspase-3 (Nicholson et al., 1995). The preferential localization of
conformationally mature caspase-3 in the nuclei of degenerating CA1
neurons is therefore compatible with a role for this protease in DNA fragmentation.
XIAP attenuates caspase-3-mediated ischemic apoptosis of
CA1 neurons
X chromosome inhibitor of apoptosis is a member of the human
inhibitor of apoptosis family. XIAP overexpression prevents
apoptosis induced by a variety of triggers in several different cell
lines (Liston et al., 1996). We have shown previously that virally
mediated overexpression of another member of the IAP family, NAIP, in
the rat hippocampus attenuates ischemic neuronal loss in CA1 by
blocking apoptosis (Xu et al., 1997a). The anti-apoptotic property of
the IAPs has been attributed to the ability of these proteins to
selectively inhibit the activity of group II caspases (3 and 7)
(Deveraux et al., 1997; Roy et al., 1997). In keeping with this
proposal, we have demonstrated that attenuation of ischemia-induced CA1 neuronal loss by overexpression of XIAP in the hippocampus is associated with a reduction of caspase-3 activation and DNA
fragmentation. Because XIAP inhibits caspase-3 activity, it is
therefore likely that XIAP overexpression may prevent ischemic neuronal
apoptosis by blocking caspase-3 activation.
In contrast to XIAP, peptide inhibitors such as DEVD-CHO and
benzyloxycarbonyl-Val-Ala-Asp(ome)-fluoromythylketone
discriminate poorly between group I (caspase-1, -4, and -5), II
(caspase-2, -3, and -7), and III (caspase-6, -8, and -9) caspases
(Nicholson and Thornberry, 1997). As a result, it is unclear whether
the neuroprotective properties of these compounds stem from the
inhibition of a single or multiple caspase subtypes. The ability of
XIAP, a selective group II inhibitor, to reduce CA1 injury highlights the importance of caspase-3 as a mediator of ischemic neuronal death
and suggests that small molecules that selectively inhibit this
protease should be neuroprotective.
XIAP overexpression attenuates ischemia-induced cellular and
behavioral deficits
CA1 neurons of the hippocampus play a key role in spatial
learning, as assessed by the Morris water maze. Consequently, ischemic damage to the hippocampus causes deficits in spatial learning (Olsen et
al., 1994). Consistent with this finding, we have observed that the
loss of CA1 neurons produced by a brief episode of transient cerebral
ischemia was associated with impaired performance in the Morris water
maze. XIAP overexpression, which reduced the injurious effects of
cerebral ischemia on CA1 neurons, preserved spatial navigation in the
Morris water maze. Indeed, the performance of these animals was
comparable with that of sham-treated rats. These findings suggest that
neurons protected from ischemic injury by XIAP overexpression retain
normal physiological function.
Neuronal expression of the immediate early gene NGFI-A in CA1 neurons
is driven by natural synaptic activity. This has lead to the suggestion
that NGFI-A expression may be a marker for neuronal activity in the
brain (Worley et al., 1991). Similar to spatial learning performance, a
behavioral measurement of neuronal function, expression of NGFI-A in
CA1 neurons was severely depressed by a brief episode of cerebral
ischemia in CA1 neurons. Overexpression of XIAP in these neurons
attenuated ischemia-induced reduction in NGFI-A expression. This
finding provides evidence that XIAP overexpression maintains cellular
homeostatsis after an experimental stroke.
In summary, the present study provides evidence that CA1 neurons
undergoing delayed cell death after an episode of transient forebrain
ischemia generate catalytically active caspase-3. Caspase-3 activation
preceded DNA fragmentation in CA1 neurons, suggesting that this enzyme
participates in the execution of ischemia-induced neuronal apoptosis.
In support of this proposal, blockade of caspase-3 activation by
overexpression of XIAP, a potent caspase-3 inhibitor, attenuated
ischemic neuronal death in the CA1 region. Moreover, XIAP
overexpression appeared to maintain normal neuronal function after an
episode of cerebral ischemia. These findings suggest that therapeutic
strategies based on XIAP elevation or small molecule inhibitors of
caspase-3 may be useful in the treatment of stroke and possibly other
acute neurodegenerative disorders.
| |
FOOTNOTES |
Received Jan. 6, 1999; revised April 1, 1999; accepted April 6, 1999.
This work was supported by Medical Research Council of Canada Grant
MT-11539 and Heart and Stroke Foundation of Ontario Grant NA-2938.
Correspondence should be addressed to G. S. Robertson, Department
of Pharmacology, Merck Frosst Centre for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada.
| |
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