 |
 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 1998, 18(13):4914-4928
Induction of Caspase-3-Like Protease May Mediate Delayed Neuronal
Death in the Hippocampus after Transient Cerebral Ischemia
Jun
Chen1,
Tetsuya
Nagayama1,
Kunlin
Jin1,
R. Anne
Stetler1, 2,
Raymond L.
Zhu1,
Steven H.
Graham1, 2, and
Roger P.
Simon1
1 Department of Neurology, University of Pittsburgh
School of Medicine, Pittsburgh, Pennsylvania 15213, and
2 Neurology Service, Department of Veterans Affairs Medical
Center, Pittsburgh, Pennsylvania 15261
 |
ABSTRACT |
Delayed neuronal death after transient cerebral ischemia may be
mediated, in part, by the induction of apoptosis-regulatory gene
products. Caspase-3 is a newly characterized mammalian cysteine protease that promotes cell death during brain development, in neuronal
cultures, and in other cell types under many different conditions. To
determine whether caspase-3 serves to regulate neuronal death after
cerebral ischemia, we have (1) cloned a cDNA encoding the rat brain
caspase-3; (2) examined caspase-3 mRNA and protein expression in the
brain using in situ hybridization, Northern and Western
blot analyses, and double-labeled immunohistochemistry; (3) determined
caspase-3-like activity in brain cell extracts; and (4) studied the
effect of caspase-3 inhibition on cell survival and DNA fragmentation
in the hippocampus in a rat model of transient global ischemia. At
8-72 hr after ischemia, caspase-3 mRNA and protein were induced in the
hippocampus and caudate-putamen (CPu), accompanied by increased
caspase-3-like protease activity. In the hippocampus, caspase-3 mRNA
and protein were predominantly increased in degenerating CA1 pyramidal
neurons. Proteolytic activation of the caspase-3 precursor was detected
in hippocampus and CPu but not in cortex at 4-72 hr after ischemia.
Double-label experiments detected DNA fragmentation in the majority of
CA1 neurons and selective CPu neurons that overexpressed caspase-3.
Furthermore, ventricular infusion of Z-DEVD-FMK, a caspase-3 inhibitor,
decreased caspase-3 activity in the hippocampus and significantly
reduced cell death and DNA fragmentation in the CA1 sector up to 7 d after ischemia. These data strongly suggest that caspase-3 activity contributes to delayed neuronal death after transient ischemia.
Key words:
cerebral ischemia; caspase-3; apoptosis; cysteine
protease; neuronal death; neuron
| |
INTRODUCTION |
Transient global ischemia results in
delayed neuronal death in selectively vulnerable brain regions such as
the hippocampal CA1 sector and caudate-putamen (Pulsinelli et al.,
1982 ). A number of recent studies suggest that cell death in this
setting involves apoptosis-an active and genetically controlled cell
suicide process. Histological and biochemical characteristics of
apoptosis are present in dying neurons after ischemia (MacManus et al.,
1993; Kihara et al., 1994; Nitatori et al., 1995), and inhibition of new protein synthesis protects CA1 neurons after ischemia (Goto et al.,
1990; Papas et al., 1992). Several apoptosis-regulatory genes are found
to be induced in ischemic cells. Bax, a bcl-2 homolog that effects
apoptosis, is upregulated in neurons destined to die after global
ischemia (Krajewski et al., 1995; Chen et al., 1996), whereas the
apoptosis-suppressor gene bcl-2 is expressed in neurons that survive
ischemia (Shimazaki et al., 1994; Chen et al., 1997a). Furthermore,
bcl-2 overexpression in rodent brain reduces ischemic injury (Martinou
et al., 1994; Linnik et al., 1995; Lawrence et al., 1996). Taken
together, these observations suggest that endogenously induced
apoptosis-regulatory genes may play a role in determining the fate of
ischemic neurons.
The interleukin-1 -converting enzyme (ICE) family of cysteine
proteases, now referred to as caspases, is another group of apoptosis-regulatory genes that may play a role in ischemic brain injury (Bredesen, 1995). The ICE family, consisting of at least 11 members (caspases-1-11), represents mammalian homologs of ced-3, an
essential cell death gene in Caenorhabditis elegans (Yuan et al., 1993; Fernandes-Alnemri et al., 1994; Xue et al., 1996). When
overexpressed, the caspases trigger apoptosis in cultured cells (Miura
et al., 1993). Among the identified caspases, caspase-3 (also termed
cpp32, Yama, or Apopain) exhibits the highest sequence homology to
ced-3 (Tewari et al., 1995). Caspase-3 is a potent effector of
apoptosis triggered via several different pathways in a variety of
mammalian cell types (for review, see Alnemri et al., 1996). Caspase-3
promotes neuronal death during brain development (Kuida et al., 1996).
In neuronal cultures, induction of caspase-3-like protease promotes
apoptosis induced by withdrawal of trophic support,
K+ deprivation, or glutamate excitotoxicity
(Deshmukh et al., 1996; Schulz et al., 1996; Du et al., 1997; Keane et
al., 1997; Ni et al., 1997). Moreover, inhibition of ICE- or
caspase-3-like protease provides protection in rodent brains subjected
to focal ischemia or direct excitotoxic insults (Hara et al., 1997) or
to traumatic brain injury (Yakovlev et al., 1997). Thus, caspase-3 and
related caspases could be important neuronal death effectors in the
brain under certain pathological conditions.
We, therefore, characterized the regional and temporal profiles of
caspase-3 gene expression in the rat brain at both mRNA and protein
levels after transient global ischemia. Then we determined whether
caspase-3-like protease activity is altered in the brain after ischemia
and how the altered enzymatic activity correlates with regional
vulnerability to ischemia. Lastly, by inhibiting caspase-3-like
activity in the hippocampus in vivo, we investigated the
role of induction of this important cell death gene in determining the
fate of ischemic neurons.
| |
MATERIALS AND METHODS |
Animal model of transient global ischemia
Experiments were performed on a total of 238 male Sprague Dawley
rats weighing 300-350 gm (Hilltop Sprague Dawley, Scottdale, PA).
Transient global ischemia (15 min) was induced in
isoflurane-anesthetized rats using a previously described method
(Pulsinelli et al., 1982), with modifications (Chen et al., 1996).
Blood pressure, blood gases, and blood glucose concentration were
monitored and maintained in the normal range throughout the
experiments. Rectal temperature was continuously monitored and kept at
37-37.5°C using a heating pad and a temperature-regulated heating
lamp. Brain temperature was monitored by a 29 ga thermocouple implanted
in the left striatum and kept at 36.4 ± 0.2°C during ischemia
and at 37-37.5°C thereafter. An electroencephalogram (EEG) was
monitored in all animals to ensure isoelectricity within 10 sec after
carotid artery occlusion. A sham operation was performed in additional
animals using the same anesthesia and surgical exposure procedures
except that the arteries were not occluded; these brains were used as
nonischemic controls.
cDNA cloning
Construction of a cDNA library. Hippocampi were
dissected from rat brains subjected to 15 min of ischemia followed by
8, 24, or 72 hr of reperfusion (three brains per time point).
Polyadenylated mRNAs were isolated from the hippocampi using a fast
track mRNA isolation kit (Invitrogen, San Diego, CA) and were used as
templates for cDNA synthesis. An ischemic brain cDNA library was
constructed using a SuperScript plasmid system for cDNA synthesis and a
plasmid cloning kit according to the manufacturer's instructions
(Gibco BRL, Gaithersburg, MD). In brief, first-strand cDNA was
synthesized using the oligo-dT12-NotI primer
adapter and SuperScript II reverse transcriptase (RT); second-strand
synthesis was catalyzed by Escherichia coli DNA polymerase I
in combination with E. coli RNase H and E. coli
DNA ligase. Double-stranded DNA was restricted with NotI and
SalI, selected on a cDNA size fraction column to include
molecules of >500 bp, ligated into plasmid pSPORT 1, and transformed
into E. coli DH5a. The constructed cDNA library was examined
by the color-selection method using
isopropyl-1-thio--D-galactoside-X-gal and titer
measurement after amplification. The titer of the amplified library was
1011 pfu/ml.
Isolation of a cDNA encoding caspase-3. In preliminary
studies, a 345 bp cDNA fragment encoding the rat caspase-3 was
generated by RT-PCR using primers designed according to the conserved
sequences in human and mouse caspase-3 (Fernandes-Alnemri et al., 1994; Tewari et al., 1995). This cDNA fragment was labeled with
[32P]dATP using random primers and SuperScript II
RT (Gibco BRL) and then was purified using a NucTrap probe purification
column (Stratagene, La Jolla, CA). To obtain a cDNA containing the open reading frame encoding the caspase-3 protein, we used the labeled cDNA
probe to screen the ischemic cDNA library. Colonies of the cDNA library
were plated onto 137 mm filters, denatured, and neutralized. The
filters were hybridized in 50% formamide-containing 5× saline-sodium phosphate-EDTA buffer, 1% SDS, 2× Denhardt's solution, and 5% dextran sulfate with the 32P-labeled caspase-3 cDNA probe
(5 × 106 cpm/ml) at 42°C for 18 hr. The
resulting positive clones were sequenced on both strands using the
dideoxy chain termination technique (Sequenase II; United States
Biochemicals, Cleveland, OH). Sequence analysis was performed using
MacVector software (International Biotechnologies, New Haven, CT) and
aligned using the BLAST program (www.ncbi.nlm.nih.gov).
In vitro transcription and translation. To confirm that
the cDNA contains the full open reading frame, we performed in
vitro transcription and translation to detect its protein product.
Transcription was performed using an RNA transcription kit according to
the manufacturer's instructions (Stratagene). In brief, 1 µg of
linearized plasmid DNA was incubated at 37°C for 1 hr in a mixture
containing 40 mM Tris-HCl, pH 8.0, 8 mM
MgCl2, 2 mM spermidine, 50 mM NaCl, 40 mM DTT, 2 mM dNTP, and
10 U of T7 RNA polymerase. After DNA digestion using 200 U of
RNase-free DNase (Promega, Madison, WI), RNA was extracted from the
reaction mixture with phenol and chloroform followed by precipitation
with 0.5 volume of ammonium acetate and 2.5 volumes of ethanol.
In vitro translation was then performed at 30°C for 1 hr
in a reaction system containing 100 ng of extracted RNA, 2 µl of
translation reaction mixture (Boehringer Mannheim, Indianapolis, IN),
10 µl of rabbit reticulocyte lysate, 100 mM potassium
acetate, and 1 mM magnesium acetate in the presence of
[35S]methionine (>800 Ci/mmol; NEN, Boston, MA).
Translated protein was electrophoresed in a 12% SDS-polyacrylamide gel
and detected by autoradiography.
Northern blot analysis
Total RNA was isolated from the hippocampi of brains subjected
to 15 min of ischemia followed by 8 and 24 hr of reperfusion (n = 3 per time point) or brains 24 hr after sham
operation (n = 3) as described previously (Chen et al.,
1996). RNA was electrophoresed on a 1.0% agarose-formaldehyde gel,
blotted onto Hybond-N membrane (Amersham, Arlington Heights, IL), and
prehybridized for 2 hr at 42°C. The membranes were subsequently
hybridized with the rat caspase-3 cDNA probe for 24 hr at 42°C. The
32P-labeled cDNA probe was prepared using the random primer
method. cDNA inserts were released from the plasmid with restriction
enzyme digestion and purified using the Gene Clean kit (BIO 101, La
Jolla, CA). Approximately 25 ng of cDNA inserts was dissolved in 20 µl of distilled water in a microcentrifuge tube, heated for 5 min in
a boiling water bath, and then immediately cooled on ice. Two microliters of a reaction mixture containing 0.5 mM each of
dATP, dTTP, and dGTP; 10 µl of random primer buffer (Gibco BRL); 50 µCi of [ -32P]dCTP; and 3 U of the large fragment of
DNA polymerase I were added to the cDNA preparation and incubated at
room temperature for 1 hr. The labeled probe was purified using the
NucTrap probe purification column (Stratagene). Labeling efficiency of
the probe was determined by measuring percentage incorporation of the
radioisotope. After the hybridization and washing procedures,
autoradiography was performed at  80°C overnight with an
intensifying screen. Autoradiograph signals were quantified by a gel
densitometric scanning program using the Microcomputer Imaging Device
(MCID) image analysis system. To control for variation in the amount of
total RNA in different samples, we stripped off the original probe in a
solution containing 0.1× SSC and 0.5% SDS at 100°C for 15 min. The
blot was rehybridized with an oligodeoxynucleotide probe
(5'-ACGGTATCTGATCGTCTTCGAACC-3') corresponding to 18S RNA. All
densitometric values for caspase-3 were normalized to values for 18S
RNA obtained on the same lane.
In situ hybridization
The 35S-labeled single-stranded RNA probe was
prepared from plasmids containing the rat caspase-3 cDNA inserts. For
the preparation of the antisense probe, the plasmid was linearized by
XhoI digestion. A product complementary to the rat caspase-3
mRNA was transcribed by the T3 RNA polymerase in the presence of 125 µCi of 35S-UTP (NEN) using an in vitro
transcription kit according to the manufacturer's instructions
(Stratagene). For the sense probe, the plasmid was linearized by
PstI and subsequently transcribed using the T7 RNA
polymerase (Stratagene). The transcription reaction was performed for
45 min at room temperature, and the cDNA template was then digested for
10 min at 37°C using DNase I (10 U) in the presence of tRNA (20 µg). The RNA probe was extracted from the reaction mixture using
phenol and chloroform followed by precipitation with 0.5 volume of
ammonium acetate and 2.5 volumes of ethanol. After centrifugation, the
pellet was rinsed with cold graded ethanol, air dried, and resuspended
in 50 µl of 10 mM DTT. Labeling efficiency of the probe
was determined by measuring percentage incorporation of the
radioisotope. Frozen sections from ischemic, sham-operated, or naive
control brains were prepared as described previously (Chen et al.,
1996). Coronal sections at the levels of the dorsal hippocampus
(anteroposterior, 3.5 to 4.0 mm from the bregma) were selected and
processed for in situ hybridization. Neuronal degeneration
at these levels after 15 min of global ischemia has been characterized
and described elsewhere (Chen et al., 1996, 1997a, 1998). The sections
were treated under RNase-free conditions with 4% paraformaldehyde in
PBS for 20 min, rinsed twice for 5 min in PBS, and then acetylated
twice for 5 min with acetic anhydride in 0.1 M
triethanolamine-HCl, pH 7.5. After washing in PBS for 5 min and saline
for 5 min, the sections were dehydrated in graded ethanol and
air-dried. The sections were hybridized with the labeled RNA probe
(1 × 107 cpm/ml) in a hybridization cocktail
for 18 hr at 55°C. The slices were then washed twice for 10 min in
2× SSC (300 mM sodium chloride and 30 mM
sodium citrate, pH 7.4), once for 2 hr in 0.1× SSC at 50°C, and then
twice for 10 min in 0.5× SSC at room temperature. They were then
dehydrated, air dried, and autoradiographed onto Kodak SB-5 film for 3 weeks. Control and ischemic brain slides were processed together and
developed on the same film. Relative changes in regional mRNA
expression were semiquantitated using the MCID system (St.
Catharine's, Ontario, Canada) as described previously (Chen et al.,
1998). Cellular localization of the labeled mRNA was evaluated by
coating slides with Kodak NTB-2 emulsion. Sections were exposed at
4°C for 5 weeks, developed in Kodak D-19, and counterstained with
cresyl violet.
Western blot analysis
Animals were killed at 4, 8, 24, or 72 hr after 15 min of
ischemia or 24 hr after sham operation (n = 4 per
experimental condition). The hippocampus, striatum, and cortex were
separately dissected, homogenized, and lysed. The lysates were cleared
by centrifugation at 14,000 × g for 30 min at 4°C.
The protein was denatured in SDS gel-loading buffer (100 mM
Tris-HCl, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol
blue, and 20% glycerol) at 100°C for 6 min and then separated on
12% SDS-polyacrylamide gels (40 µg per sample). Immunoblotting was
performed as described previously (Chen et al., 1996), using a
chemiluminescent detection system (Clontech, Palo Alto, CA). The
antibody used to detect the caspase-3 protein was a custom-made rabbit
polyclonal antibody (Biosynthesis, Dallas, TX) raised against the
deduced C-terminal sequence (NH2-QACRGTELDCGIETD-COOH) of
the rat caspase-3 larger active form p17. The working dilution for
caspase-3 antibody in the present study was 1:2000. The synthesized peptide for immunization was also used in preabsorption experiments to
confirm the specificity of the detected immunoreactivity. This was done
by incubating the peptide (5 µg/ml) with the primary antibody for 30 min at 37°C before the immunoblotting was performed. For
immunoblotting to detect poly(ADP-ribose) polymerase (PARP), a
monoclonal antibody against the C-terminal part of the DNA binding domain of PARP was used at a working dilution of 1:5000, as suggested by the manufacturer (Biomol, Plymouth Meeting, PA). A purified bovine
PARP protein was used in preabsorption experiments to confirm the
specificity of PARP immunoreactivity. Immunoreactivity for caspase-3 or
PARP on each individual lane of the blots was quantified by a gel
densitometric scanning program using the MCID image analysis system.
Immunohistochemistry
Animals were anesthetized with 8% chloral hydrate at 4, 8, 24, or 72 hr after 15 min of ischemia or 24 hr after sham operation (n = 4 per time point). They were then perfused with
200 ml of heparinized 0.9% saline followed by 500 ml of 4%
paraformaldehyde in 0.1 M PBS, pH 7.4. The brains were
removed, immersed in 4% paraformaldehyde for 5 d, and processed
for paraffin embedding and cutting (6 µm thick) on a rotary
microtome. Coronal sections at the levels of dorsal hippocampus and
midcaudate (anteroposterior, +0.2 mm from the bregma) were selected and
processed for immunohistochemical staining. The sections were
deparaffinized in xylene, rehydrated through graded ethanol, and
pretreated with protease K (10 µg/ml for 10 min). After three 10 min
washes in PBS, the sections were microwaved in sodium citrate buffer,
pH 6.0, for 5 min before immunostaining. Immunohistochemical staining
for caspase-3 was performed using the same antibody used for Western
blot analysis. Briefly, after the preblocking step using the rabbit
preimmunizing serum, sections were incubated overnight at room
temperature in the primary antibody diluted 1:1000 in PBS, pH 7.4, containing 2% goat serum, 0.2% Triton X-100, bovine serum albumin (5 mg/ml), and 0.2% glycine. Sections were washed in PBS three times for 10 min each and then incubated for 1 hr at room temperature at a 1:2500
dilution with goat anti-rabbit Cy3.18 immunoconjugate (Jackson
ImmunoResearch, West Grove, PA). Sections were then washed in PBS four
times for 15 min each on a orbital shaker (model 361; Fisher
Scientific, Houston, TX), mounted in gelvatol, and coverslipped. A
Zeiss light microscope equipped for epifluorescent illumination was
used for observation. For the assessment of nonspecific immunostaining, alternating sections from each experimental condition were incubated without the primary antibody or, in some cases, with antibody that was
preabsorbed with the synthetic caspase-3 peptide (8 µg/ml) for 30 min
at room temperature before immunohistochemistry. Immunoreactivity was
compared in sections from animals killed 24 hr after ischemia, with or
without preabsorption of the primary antibody.
Detection of DNA fragmentation
In situ detection of DNA fragmentation in brain cells
after ischemia or after sham operation was performed using terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) as described previously by Gavrieli et al. (1992), with modifications (Chen et al., 1997b). For double-label experiments, paraffin-imbedded sections, at the levels of dorsal hippocampus and
midcaudate, obtained 24 and 72 hr after ischemia and 24 hr after sham
operation (n = 4 per experimental condition) were used. The sections were pretreated as described above and then incubated at
37°C for 90 min in 1× terminal deoxynucleotidyl transferase (TdT)
buffer containing 100 U/ml TdT and 20 nmol/ml biotin-conjugated 16-dUTP
(Boehringer Mannheim). After three washes in PBS (10 min/wash), the
sections were incubated at room temperature for 15 min in fluorescein-avidin D cell sorting (DCS) (cell sorting grade; Vector Laboratories, Burlingame, CA) diluted in PBS at 8 µg/ml. Sections were then processed for caspase-3 immunohistochemistry as described above, and all steps were performed in the dark. Sections were examined
by fluorescence microscopy using excitation/emission wavelengths of
550/565 nm (red) and 495/515 nm (green-yellow) for caspase-3 and
TUNEL, respectively.
Activities of caspase-3- and ICE-like proteases
Measurement of caspase-3- and ICE-like protease activity in
brain cell extracts was performed as originally described (Enari et
al., 1996) with the slight modifications suggested by Clontech. The
animals were anesthetized using 8% chloral hydrate and decapitated. Brains were quickly removed. Tissues were dissected separately from the
hippocampus, striatum, cortex, or cerebellum of brains at 1, 4, 8, 24, and 72 hr after ischemia, at 24 hr after sham operation, or from naive
animals (n = 4-5 per experimental condition). Protein
extracts were prepared on ice by Dounce homogenization of tissues in a
lysis buffer containing 25 mM HEPES, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM DTT, and 10 µg/ml each of pepstatin, leupeptin,
aprotinin, and PMSF (Sigma, St. Louis, MO). Cell lysate was centrifuged
at 15,000 rpm for 10 min, and the supernatant was used for the
enzymatic assay. One hundred micrograms of the extracted proteins were
incubated for 1 hr at 37°C with the reaction buffer (25 mM HEPES, pH 7.5, 10% sucrose, 0.1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate, 5 mM DTT, and 5 mM EDTA) in a total volume of 150 µl containing 25 µM colorimetric peptide substrate
(Biomol). The following two substrates were used for the assays:
acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA) for caspase-3-like protease activity and
acetyl-Tyr-Val-Ala-Asp-p-nitroanilide (Ac-YVAD-pNA) for ICE-like protease activity.
Enzyme-catalyzed release of p-nitroanilide was measured at
405 nm in a microtiter plate reader (Molecular Devices, Palo Alto, CA).
One unit of protease activity corresponds to the caspase-like activity
that cleaves 1 pmol of pNA per minute at 37°C at
saturating substrate concentrations. In certain experiments, the
extracted protein sample was diluted in the reaction buffer and first
incubated with inhibitors for caspase-3 (DEVD-CHO) and ICE (YVAD-CHO)
at room temperature for 30 min (Lazebnik et al., 1994; Nicholson et
al., 1995).
In vivo inhibition of caspase-3-like protease
N-Benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp-(OMe)-fluoro-methylketone
(Z-DEVD-FMK) (Enzyme Systems Products, Livermore, CA), a
peptide methylketone caspase-3 inhibitor (Nicholson et al., 1995;
Rodriguez et al., 1996), was dissolved in DMSO at a concentration of
18.75 mg/ml and further diluted 1:25, 1:75, or 1:225 in mock CSF.
Peptide infusions were performed using a 10 µl Hamilton syringe
(Hamilton, Reno, NV) through a preimplanted 21 ga cannula in the left
ventricle (from the bregma: anteroposterior, 0.8 mm; lateral, 1.5 mm;
depth, 3.5 mm). Each animal received three ventricular infusions of 2 µl each over a 5 min time period 30 min before ischemia and 2 and 24 hr after ischemia, except as indicated otherwise. The resulting peptide
doses for infusion treatment were 0.057 µg × 3, 1.5 µg × 3, 0.5 µg × 3, and 0.167 µg × 3 per animal. Infusions of
diluted DMSO (0.04%) served as vehicle controls. All treatments were
assigned to animals in a randomized and blinded manner. Brain and
rectal temperatures were monitored in all animals before, during, and
up to 2 hr after ischemia. The rectal temperature was also measured at
24, 48, and 72 hr after ischemia.
The doses of Z-DEVD-FMK used in the present study were suggested by the
focal ischemia studies of others (Hara et al., 1997) and determined by
a series of experiments in which the effectiveness of this peptide to
inhibit caspase-3 activity in the hippocampus after ischemia was
evaluated (see Results). For the measurement of caspase-3-like protease
activity in peptide- or vehicle-infused brains, the brains were quickly
removed 48 hr after ischemia and processed for the enzyme assay as
described above.
For histological outcome experiments, rats were killed at 3 or 7 d
after ischemia. Their brains were perfused and paraffin-imbedded, and 6 µm sections were cut. Coronal sections at the level of the dorsal
hippocampus (anteroposterior, 4.0 mm from the bregma) were selected
and stained with cresyl violet. Adjacent brain sections were processed
for TUNEL staining using the method described above, except that the
sections were pretreated with 1% H2O2 for 15 min to quench endogenous peroxidase before incubation in TdT buffer, and the biotin-16-dUTP tailing was detected using the
horseradish-streptavidin-peroxidase method (Chen et al., 1997b).
Sections were examined by two investigators who were blinded to the
experimental conditions. Surviving hippocampal CA1 neurons (showing
normal morphology by cresyl violet staining) and cells containing DNA
fragmentation (TUNEL positive) were quantified with the assistance of a
computerized scanning program (MCID; St. Catharine's).
To determine the potential role of the ICE-like protease in ischemic
cell death, we subjected additional rats to ventricle infusion of the
ICE inhibitor
N-benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-CH2F (Z-YVAD-FMK) (Enzyme Systems Products) before and after ischemia using
the same protocol described for Z-DEVD-FMK. Rats received either
Z-YVAD-FMK at 0.5 µg × 3 or 1.5 µg × 3 or the same
volume of 0.04% DMSO (n = 6 per group). Three days
after ischemia, the brains of these rats were processed for cresyl
violet staining, TUNEL staining, and subsequent cell counting in the
hippocampal CA1 sector.
Data analysis
All data are reported as mean ± SEM. Comparisons of
caspase-3 mRNA expression, caspase-3 protein expression, caspase-3
activity, ICE-like activity, CA1 cell survival, or TUNEL-positive cells at different durations of reperfusion with or without inhibitor treatment versus sham controls were made using ANOVA and post hoc Fisher's PLSD tests. A level of p < 0.05 was
considered statistically significant.
| |
RESULTS |
cDNA cloning of rat brain caspase-3
To characterize the expression pattern of the caspase-3 gene in
the brain after ischemia, we cloned a cDNA containing the entire open
reading frame of caspase-3 from the ischemic brain cDNA library.
Sequence analysis revealed that this cDNA encodes an open reading frame
of 277 amino acids (Fig.
1A). The deduced amino
acid sequence had 84.1 and 92.1% identity to the published sequences
of human and mouse cpp32 (Fernandes-Alnemri et al., 1994; Tewari et
al., 1995), respectively. The positions of several predicted functional
residues were also identical to those of human and mouse cpp32,
including the two aspartic acid residues (28 and 175) predicted to be
the cleavage sites, the three residues required for substrate binding
(Arg64, His108, and Arg207), and the QACRG motif responsible for
binding of the protease to the aspartic acid residues in the substrate
cleavage sites (Nicholson et al., 1995; Juan et al., 1996). After the
completion of this work, two cDNA sequences encoding the rat caspase-3
homologs were published. One clone, obtained from a rat brain cDNA
library and referred to as interleukin-converting enzyme-related
protease (IRP) (Ni et al., 1997), was 99.3% identical to our amino
acid sequence. The other clone (cpp32), cloned from a rat colon cDNA library (Juan et al., 1996), was identical to our sequence at both
nucleotide and amino acid levels. Thus, caspase-3 seems to be highly
conserved across species and, within a species, across different
organs.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 1.
A, Deduced amino acid sequence of
rat brain caspase-3 from the cDNA clone. This sequence shares 84.1%
identity with the human cpp32 (Fernandes-Alnemri et al., 1994) and
99.3% with the rat brain IRP (Ni et al., 1997). The
boxes mark the different amino acids between caspase-3
and the rat brain IRP. Arrowheads indicate the known
proen-zyme cleavage sites for caspase-3 (Asp28-Ser29 and
Asp175-Ser176) that yield the p17 and p12 active forms. The peptide
sequence used to raise the caspase-3 antibody is
underlined. B, SDS-PAGE analysis of
extract of in vitro translation assay product from
caspase-3 cDNA. Lane 1, Negative control. cRNA was
omitted from the assay. Lane 2, Translation product
(arrow) from the caspase-3 cDNA. C,
Northern blot analysis of caspase-3 mRNA in the hippocampus after sham
operation (lane 1) or 8 hr (lane 2) or 24 hr (lane 3) after ischemia. Total RNA was isolated from
the hippocampi (three brains per time point) and electrophoresed
through a 1% agarose-formaldehyde gel (20 µg of RNA per
lane). The only transcription species resulting from
hybridizing with the caspase-3 cDNA probe is ~2.6-2.7 kb
(arrow), consistent with the predicted molecular size of
rat caspase-3 mRNA. Bottom, The same blot hybridized
with the 18S RNA probe as a control for sample loading.
|
|
Using the cloned cDNA as a template, we found that the in
vitro transcription and translation assay produced a protein at ~32 kDa (Fig. 1B), the predicted size for
caspase-3.
Evidence of caspase-3 gene induction after ischemia
Previous studies suggest that expression of caspase-3 mRNA and
protein is retained in most adult tissues (Juan et al., 1996; Krajewska
et al., 1997; Ni et al., 1997). In the present study, we have examined
the expression of caspase-3 at both mRNA and protein levels in normal
and ischemic brains, focusing on regions such as the hippocampus and
caudate-putamen where cells are particularly vulnerable to transient
global ischemia. Using Northern blot analysis, we detected caspase-3
mRNA (~2.6-2.7 kb) in all samples tested (Fig. 1C).
Although caspase-3 mRNA was readily detected in the normal hippocampus,
the levels were increased at 8 hr (1.8-fold) and 24 hr (2.3-fold) after
15 min of ischemia.
As shown in Figure
2A-C, the cellular
distribution of the mRNA was examined further using in situ
hybridization in nonischemic brains and in brains 4, 8, 24, 72, or 96 hr after ischemia (n = 3 per time point). Consistent
with the findings in Northern blots, basal expression of caspase-3 mRNA
was readily detectable in the whole hippocampal formation, including
pyramidal neurons in the CA1-CA3 sectors and granule cells in the
dentate gyrus (Fig. 2A). Low levels of basal
expression were also present in medium-sized neurons in the
caudate-putamen and thalamus and in large- and medium-sized neurons in
the cortex. Eight hours after ischemia, significantly increased
caspase-3 mRNA was first detected in the dentate granule cells (Fig.
2B). It subsided to near control levels in this region
thereafter. In the CA1 sector, a slight increase in caspase-3 mRNA was
first seen at 8 hr. The signals were markedly increased in this region
at 24 and 72 hr after ischemia. Examination of emulsion-coated sections
revealed that many neurons in the thalamus and dorsolateral putamen
also showed increased caspase-3 mRNA signal at 24-72 hr after
ischemia. In the cortex, however, only a few scattered shrunken neurons
in layers III-V showed increased mRNA. In control experiments,
sections hybridized with the sense cDNA probe showed a low-level
background signal that was homogeneous throughout the brain sections
(Fig. 2A,C).
View larger version (81K):
[in this window]
[in a new window]
|
Figure 2.
Caspase-3 in situ hybridization.
A, Representative autoradiograms through the level of
dorsal hippocampus in a sham-operated brain and in brains at 8, 24, 72, and 96 hr after 15 min of global ischemia. Caspase-3 mRNA is slightly
increased in the dentate gyrus (DG) granule cell layer 8 hr after ischemia but markedly increased in the hippocampal
CA1 sector at 24 and 72 hr after ischemia
(arrowheads). A section (24 hr after ischemia;
Sense) hybridized with the sense cDNA probe results in
only background signals. B, Relative caspase-3 mRNA
changes in the hippocampal CA1 sector, CA3 sector, and dentate gyrus at
4, 8, 24, 72, and 96 hr after ischemia versus sham controls
(n = 3 per group), determined by optical density
measurement on autoradiograms. Data are reported as mean ± SEM
and represent fold changes in ischemic brains versus sham controls; *
p < 0.01, and ** p < 0.001 versus sham controls (ANOVA and post hoc Fisher's PLSD
tests). C, Representative emulsion-coated sections
counterstained with cresyl violet from a brain 72 hr after ischemia
(a, c, e) and a sham
control brain (b, d,
f). Caspase-3 mRNA is predominantly increased in
the CA1 sector of ischemic hippocampus (a;
arrows) compared with the control brain
(b). Under a high-power field (400×), increased
amounts of silver grains localize to CA1 pyramidal neurons
(c) and to scattered neurons in the
caudate-putamen (e; arrows) compared with
the controls d and f, respectively. The
open squares in a and b
mark the regions from which the high-power views in c
and d are taken, respectively. g, Sense
control in caudate-putamen from the same ischemic brain. Scale bar, 30 µm.
|
|
Evidence of caspase-3 protein alteration after ischemia
Normally, caspase-3 protease is synthesized in cells as an
inactive precursor (32 kDa). After activation, it is cleaved at the C
terminal of two specific aspartic acid residues to form two mature
subunits, p17 (17 kDa) and p12 (12 kDa) (Nicholson et al., 1995). Using
the antibody against the deduced C-terminal sequence of the larger
subunit (see Materials and Methods), we found that Western blots
recognized both precursor and p17 but not p12 in brain protein extracts
(Fig. 3). Normal nonischemic hippocampus
and caudate-putamen showed fairly high levels of the caspase-3
precursor but none or very low levels of the p17 subunit. After
ischemia, caspase-3 protein was increased in these regions (Fig. 3). In
the hippocampus, the p17 subunit began to increase at 4 hr and markedly
increased at 24-72 hr after ischemia. The levels of the precursor
protein were also increased in this region at 8-72 hr. In the
caudate-putamen, the p17 form was significantly increased and peaked at
8-24 hr after ischemia. The levels of the precursor protein, however,
were unchanged after ischemia. The specificity of the caspase-3
immunoreactivity was confirmed in duplicate Western blots by
preabsorbing the primary antibody with the specific caspase-3
peptide (5 µg/ml), which abolished the signals (data not shown).
Neither the p17 nor the precursor protein was increased in the cortex
4-72 hr after ischemia (Fig. 3).
View larger version (79K):
[in this window]
[in a new window]
|
Figure 3.
Western blot analysis of caspase-3 protein in the
hippocampus (Hi), caudate-putamen (CPu),
and cortex (Ctx) from brains after sham operation
(lane 1) or 4 hr (lane 2), 8 hr
(lane 3), 24 hr (lane 4), or 72 hr
(lane 5) after ischemia. Immunoreactivity of both
caspase-3 precursor protein (32 kDa) and its larger cleavage form (17 kDa) is increased in the hippocampus after ischemia. Immunoreactivity
of the 17 kDa cleavage form is also increased in the caudate-putamen
but not in the cortex. Graphs, Semiquantitative changes
of caspase-3 protein after ischemia, determined by optical density (OD)
measurements on Western blot autoradiograms (OD × area). Data are
mean ± SEM (n = 4 per time point) and
represent fold changes in ischemic brains versus sham controls;
*,#p < 0.05 versus sham controls
(ANOVA and post hoc Fisher's PLSD
tests).
|
|
The cellular distribution of caspase-3 immunoreactivity in the brain
was examined using immunohistochemistry. In contrast to the Western
blot findings in normal nonischemic brains, neurons throughout most
forebrain regions, including hippocampus, caudate-putamen, thalamus,
and cortex, contained very weak or no caspase-3 immunoreactivity. As
shown in Figure 4, basal caspase-3
immunoreactivity was exclusively present in the cell cytoplasm. Only
very few scattered, shrunken neurons in the cortex and thalamus were
highly caspase-3-immunoreactive and exhibited nuclear caspase-3
immunoreactivity. These results are consistent with recent observations
in adult human brain tissues (Krajewska et al., 1997). Immunostaining
in ischemic brains showed increased caspase-3 reactivity in several
regions affected by global ischemia (Fig. 4a-f). In
the hippocampal CA1 (but not CA3) neurons, increased caspase-3
immunoreactivity began to be detectable at 8 hr after ischemia and
became maximal at 72 hr. At the earlier time points (8-24 hr), most
cells in the CA1 sector had normal morphology, and the immunoreactivity
was predominantly present in the cytosol. At 72 hr after ischemia,
however, the majority of cells in this region (>90%) showed pyknotic
changes (shrinkage of the cell body and condensation of the nucleus).
The increased immunoreactivity was distributed throughout the entire
cell, including the nucleus. The hippocampal dentate granule cell
layers also showed mildly increased caspase-3 immunoreactivity at 8 hr
but not at 24 or 72 hr after ischemia. In the dentate, immunoreactivity was diffusely distributed through the cell bodies. The other brain region showing a marked increase in cellular caspase-3 immunoreactivity after ischemia was the caudate-putamen. Many neurons in this region showed increased caspase-3 immunoreactivity 24-72 hr after ischemia. Many cells had pyknotic changes and showed increased immunoreactivity in the nucleus.
View larger version (94K):
[in this window]
[in a new window]
|
Figure 4.
Immunofluorescent images of caspase-3 protein
(a-f, i, j) and TUNEL
labeling (g, h) in the hippocampal
CA1 sector (left) and caudate-putamen
(right) after ischemia. Compared with that in the
control brain (a, b), caspase-3
immunofuorescence is increased in the cytoplasm of CA1 pyramidal
neurons (c) and in scattered neurons in the
caudate-putamen (d; arrowheads) at 24 hr
after ischemia. Caspase-3 immunofluorescence is increased further in
neurons in these regions with both cytosolic and nuclear localization
at 72 hr after ischemia (e, f;
arrowheads mark representative positive cells).
Double-label (TUNEL) in the same sections shows a colocalization of DNA
fragmentation (g, h;
arrowheads mark representative positive cells) and
increased caspase-3 immunofluorescence in most CA1 neurons
(e vs g) and in many caudate neurons
(f vs h) at 72 hr after ischemia.
Note that TUNEL-positive neurons show a condensed and shrunken nucleus.
Consecutive sections of c and d incubated
with the primary antibody preabsorbed with the synthetic caspase-3
peptide show background fluorescence only (i, j).
Insets in a, c, and
e show representative cresyl violet staining in the CA1
sector. In keeping with the delayed manner of cell death in this model,
CA1 neurons show normal morphology in control brain
(a) and in the brain 24 hr after ischemia
(c) but show pyknotic changes in the brain 72 hr
after ischemia (e). Scale bar, 50 µm.
|
|
To address the potential role of caspase-3 protein induction in
ischemic cell death, we performed double staining for caspase-3 protein
and DNA fragmentation in brain sections obtained 24 and 72 hr after
ischemia, when substantial cell death began to occur in the
caudate-putamen and hippocampus, respectively. In the hippocampal CA1
sector, the majority of DNA-fragmented (TUNEL-positive) neurons showed
increased nuclear caspase-3 immunoreactivity at 72 hr (Fig. 4e-h) and vice versa. In the caudate-putamen, especially
the dorsolateral portion, many pyknotic cells showed colocalization of
increased nuclear caspase-3 immunoreactivity and DNA fragmentation at
24 and 72 hr after ischemia (Fig. 4f,h).
Colocalization of increased nuclear caspase-3 immunoreactivity and DNA
fragmentation was also detected in a few scattered neurons in layers
III and V of the cortex. In sham-operated control brains
(n = 4), zero to three cells per section showed DNA
fragmentation. No colocalization of DNA fragmentation and caspase-3
immunoreactivity was found in these brains.
TUNEL staining was also performed in adjacent brain sections using the
horseradish-streptavidin-peroxidase method (Chen et al., 1997b), and
positive versus negative cells in the hippocampus and caudate-putamen
(CPu) were quantified. Twenty-four hours after ischemia, 31.5 ± 8.9% (mean ± SEM; n = 4)
of the total number of cells in the CPu at the midcaudate level showed
TUNEL-positive staining, whereas <0.5% of the CA1 neurons stained
positively. At 72 hr after ischemia, however, 86.5 ± 4.3% of the
neurons in the CA1 and 52.7 ± 11.4% of the cells in the CPu were
TUNEL positive (mean ± SEM; n = 4 at each coronal
level). In both regions, the majority of TUNEL-positive cells exhibited
shrinkage of cytoplasm and nucleus and condensation of chromatin. Many
cells in the CPu also showed formation of two or more dense masses
around the nucleus suggestive of apoptotic bodies, as described by
others (Li et al., 1995; Charriaut-Marlangue et al.,
1996). Meanwhile, only a small portion of
TUNEL-positive cells in the CA1 sector (<10% of the total number of
positive cells), mainly in CA1a, exhibited significant morphological
changes indicative of necrosis, such as diffused cytosol staining or
loss of cellular structures (Charriaut-Marlangue et al., 1996). Thus,
many but not all ischemic neurons in this model show morphological
features of apoptotic cell death. These observations are consistent
with previous findings in similar animal models (Kihara et al., 1994;
Nitatori et al., 1995; Chen et al., 1996).
Alteration of caspase-3- and ICE-like protease activities
after ischemia
To assay for protease activity, we incubated cell lysates with
Ac-DEVD-pNA as a substrate for caspase-3 and
Ac-YVAD-pNA as a substrate for ICE and monitored release of
p-nitroanilide. Lysates from normal nonischemic brains
showed low levels of both ICE- and caspase-3-like peptide cleavage
activities, which were blocked, but not completely, by saturated
concentrations of the ICE inhibitor YVAD-CHO (5 µM) and
the caspase-3 inhibitor DEVD-CHO (5 µM). The levels
measured after inhibitor treatment were considered nonspecific background signals present in the lysates and were subsequently subtracted from all readings, as suggested previously (Enari et al.,
1996). Lysates from the ischemic hippocampus and caudate-putamen exhibited an increase in Ac-DEVD-pNA peptide cleavage
activity that was detectable at 8 hr and maximal at 24-72 hr after
ischemia (Fig. 5, top). The
increased caspase-3-like protease activity was completely inhibited by
DEVD-CHO but was not affected by YVAD-CHO (data not shown). Moreover,
lysates from ischemic hippocampus also exhibited a mild increase in
Ac-YVAD-pNA cleavage activity that was detectable only at 72 hr after ischemia (Fig. 5, bottom). By contrast, there was
no significant induction of ICE-like activity in the caudate-putamen at
any time point tested. Neither caspase-3-like nor ICE-like protease
activity was significantly increased in the cortical or cerebellum
lysates at any time point after ischemia.
View larger version (27K):
[in this window]
[in a new window]
|
Figure 5.
The caspase-3-like (top) and
ICE-like protease activity (bottom) in cell extracts
from the hippocampus (Hi), caudate-putamen
(CPu), cortex (CTx), and cerebellum
(Cereb) after sham operation or 1, 4, 8, 24, or 72 hr
after ischemia. The protease activities are measured by determining the
ability of cell extracts to cleave the colorimetric substrates
Ac-DEVD-pNA (for caspase-3-like activity) and
Ac-YVAD-pNA (for ICE-like activity). One unit of
protease activity corresponds to the caspase-like activity that cleaves
1 pmol of pNA per minute at 37°C at saturating
substrate concentrations. Data are presented as mean ± SEM
(n = 4-5 per time point); *p < 0.05, and **p < 0.01 versus sham controls
(ANOVA and post hoc Fisher's PLSD tests).
|
|
The finding that ischemia markedly induces Ac-DEVD-pNA
cleaving activity in both the hippocampus and caudate-putamen prompted an analysis of poly(ADP-ribose) polymerase (PARP) cleavage and other
potential caspase-3 cleavage substrates. Western blots using the
C-2-10 monoclonal antibody detected increased cleavage of intact PARP
(116 kDa) to the 85 kDa apoptosis-related cleavage fragment in ischemic
brains (Fig. 6). The 85 kDa fragment was present at negligible levels in normal nonischemic brains. However, these levels were significantly increased after ischemia (24-72 hr
after ischemia in the caudate-putamen and 72 hr after ischemia in the
hippocampus). Duplicate Western blots performed using the primary
antibody preabsorbed with the purified bovine PARP protein (10 µg/ml)
showed a complete loss of signal at both 116 and 85 kDa (Fig.
6a), confirming the specificity of the immunoreactivity recognized by the PARP antibody. Moreover, PARP cleavage was not detected in the cortex 4-72 hr after ischemia (data not shown).
View larger version (73K):
[in this window]
[in a new window]
|
Figure 6.
Western blot analysis of PARP in the hippocampus
and caudate-putamen after ischemia. a, Lanes
1-2, Positive controls for intact PARP (116 kDa) and cleaved
PARP (85 kDa) using cell extracts from uninduced human HL60 leukemia
cells and HL60 induced to undergo apoptosis, respectively. Lanes
3-6, Cell extracts from the hippocampus after sham operation
(lane 3) or 8 hr (lane 4), 24 hr
(lane 5), or 72 hr (lane 6) after
ischemia. Note that the 85 kDa form of PARP is increased at 72 hr.
Lane 7, The same cell extract used in lane
6 incubated with the primary antibody preabsorbed with purified
bovine PARP protein (10 µg/ml). Lanes 8-10, Cell
extracts from the caudate-putamen after sham operation (lane
8) or 24 hr (lane 9) or 72 hr (lane
10) after ischemia. Note that the 85 kDa form is increased in
both ischemic samples. b, Semiquantitative analysis of
relative PARP changes in the hippocampus (Hi) and
caudate-putamen (CPu) after ischemia. Data are mean ± SEM (n = 3 per time point);
*p < 0.05 versus sham controls (ANOVA and
post hoc Fisher's PLSD tests).
|
|
Considering that induced caspase-3 protease activity after ischemia may
result in the cleavage of other substrates in the ischemic brain as
well, we also analyzed two other proteins, the DNA-dependent protein
kinase and actin. Both proteins have been found to be cleaved under
certain apoptotic conditions in several cell systems (Casciola-Rosen et
al., 1995; Mashima et al., 1995; Song et al., 1996; McConnell et al.,
1997), however, no specific cleavage products from either of the two
proteins were detected in cell extracts from the hippocampus or
caudate-putamen with or without ischemic insults (data not shown).
Effects of inhibition of caspase-3-like protease activity
after ischemia
To investigate the cell death regulatory role of caspase-3 in
ischemic brain injury further, we infused Z-DEVD-FMK, an inhibitor of
caspase-3, into the ventricles beginning 30 min before the induction of
ischemia. DMSO was used as a vehicle. The doses of Z-DEVD-FMK used in
the histological outcome studies were predetermined by a set of
dose-response experiments, in which the ability of Z-DEVD-FMK to
inhibit caspase-3-like protease activity in ischemic brains was
examined.
Animals that received either no infusion, vehicle infusion, or peptide
infusion all survived the experiments. At the highest dose (1.5 µg × 3), Z-DEVD-FMK did not cause any notable behavioral abnormalities in the animals or microscopic evidence of cell death in
normal nonischemic brains, determined by cresyl violet staining 72 hr
after infusion (n = 4). Brain and rectal temperatures
were not altered by infusion of the peptide before, during, or after ischemia. The EEG reached isoelectricity within 10 sec of the induction
of ischemia in all animals. In the brains of animals subjected to 15 min of ischemia followed by 48 hr of reperfusion, Z-DEVD-FMK infusion
decreased Ac-DEVD-pNA peptide cleavage activity in the
hippocampus in a dose-dependent manner (Fig.
7aE).
View larger version (32K):
[in this window]
[in a new window]
View larger version (18K):
[in this window]
[in a new window]
|
Figure 7.
a, Quantitative analysis of effect
of in vivo caspase-3 inhibitor treatment on hippocampal
CA1 neuron survival (A, B), DNA
fragmentation (C, D), and caspase-3-like
activity after ischemia (E). A,
B, Dose-dependent increase in CA1 neuron survival after
ischemia by the caspase-3 inhibitor Z-DEVD-FMK, either injected before
the induction of ischemia (vehicle or total dose of 0.5, 1.5, or 4.5 µg) or after ischemia (post-is with total dose of 4.5 µg). Cresyl
violet staining and cell counting were performed either 3 or 7 d
after ischemia. C, D, Dose-dependent
decrease in the amount of cells with DNA fragmentation (TUNEL positive)
in CA1 after ischemia by inhibiting caspase-3-like protease activity.
No DNA fragmentation is present in CA1 in naive or sham-operated brains
(data not shown). Sections through the same level of the dorsal
hippocampus (bregma, 4.0 mm) are used for the analysis. Cell counting
includes the entire CA1 sector at this level. E,
Dose-dependent inhibition of caspase-3-like activity in the hippocampus
by Z-DEVD-FMK. Vehicle (n = 4) or the peptide
(total dose of 0.17, 0.5, 1.5, or 4.5 µg) was infused beginning 30 min before ischemia (n = 5 each dose).
Caspase-3-like activity was measured in hippocampal cell extracts 48 hr
after ischemia. All data are reported as mean ± SEM; *
p < 0.01, and ** p < 0.001 (ANOVA and post hoc Fisher's PLSD tests).
b, Quantitative analysis of effect of in
vivo ICE inhibitor Z-YVAD-FMK treatment on hippocampal CA1
neuron survival (A) and DNA fragmentation
(B) 3 d after ischemia. No significant
protection by Z-YVAD-FMK was detected. Injection Side,
The hemisphere receiving inhibitor infusion; contralateral
side, the hemisphere receiving no infusion. The
number in parentheses indicates the
number of animals in that experimental group.
|
|
In histological outcome studies, Z-DEVD-FMK partially but significantly
decreased neuronal death in the hippocampal CA1 sector at 72 hr after
ischemia (Fig. 7aA,aB). The protective effect of Z-DEVD-FMK was detectable at higher doses, and the effect was more
apparent in the hippocampus on the side ipsilateral rather than
contralateral to the infusion. To test the efficacy of Z-DEVD-FMK administrated after ischemia, we infused the peptide into the ventricles beginning 2 hr after ischemia and followed with a second infusion 22 hr later (total dose, 4.5 µg). Post-treatment with Z-DEVD-FMK increased CA1 neuron survival in the hippocampus bilaterally 72 hr after ischemia, with an efficacy similar to that of pretreatment. To determine whether Z-DEVD-FMK promotes CA1 neuron survival after a
longer period of reperfusion, we gave two additional groups of animals
either vehicle or peptide pretreatment (1.5 µg × 3), and brain
sections were obtained for analysis 7 d after ischemia. Z-DEVD-FMK
significantly increased CA1 neuron survival, although to a lesser
extent compared with the outcome at 3 d (Fig. 8). In all treatment
paradigms tested (before or after treatment or a longer period of
reperfusion), Z-DEVD-FMK significantly decreased the number of CA1
neurons that exhibited DNA fragmentation after ischemia (Fig.
7aC,aD). Representative micrographs showing
histological outcome and DNA fragmentation after ischemia with or
without peptide infusion are presented in Figure
8.
View larger version (108K):
[in this window]
[in a new window]
|
Figure 8.
Effect of in vivo caspase-3
inhibition on CA1 neuron survival and DNA fragmentation.
A, Low-power fields (40×) showing representative cresyl
violet staining (a, c, e)
and TUNEL (b, d, f)
in the hippocampus 3 d after sham operation (a,
b), after ischemia plus vehicle infusion
(c, d), or after ischemia plus caspase-3
inhibitor infusion (total dose, 4.5 µg; e, f).
Arrows in c and e mark
cell death in the CA1 sector. Arrows in d
and f mark DNA-fragmented (TUNEL-positive) cells in the
CA1 sector. B, High-power fields (400×) showing
representative cresyl violet staining (a,
c, e, g) and TUNEL
counterstained with cresyl violet (b, d,
f, h) in the CA1 sector. Three days after
sham operation (a, b), no cell death or
DNA fragmentation is present in CA1; 3 d after ischemia plus
vehicle infusion (c, d), the majority of
CA1 neurons show pyknotic changes (c) and TUNEL
labeling (d); 3 d after ischemia plus
caspase-3 inhibitor infusion (e,
f), many neurons show normal morphology
(yellow arrowheads), and decreased amounts of
neurons show pyknotic changes (e) or TUNEL
labeling (red arrowheads; f); and
7 d after ischemia plus inhibitor infusion
(g, h), both survival neurons
(yellow arrowheads) and TUNEL-positive cells
(red arrowheads; h) are present in the
CA1. Scale bar, 50 µm.
|
|
Because Z-DEVD-FMK, like all other commercially available caspase
inhibitors, may act on multiple caspases instead of inhibiting caspase-3 only, another caspase inhibitor, Z-YVAD-FMK, which has a
preferential action on ICE, was also tested in the present study. At
both doses equivalent to the effective doses for Z-DEVD-FMK (0.5 µg × 3 and 1.5 µg × 3), Z-YVAD-FMK failed to show
significant protection in CA1 neurons 3 d after ischemia (Fig.
7b).
| |
DISCUSSION |
A number of gene products may be modulators of neuronal apoptosis
resulting from cerebral ischemia and related brain insults (for review,
see Bredesen, 1995; Koistinaho and Hokfelt, 1997). In the present
study, we demonstrate that caspase-3, a key member of the ICE protease
family, was induced in neurons after transient global ischemia.
Caspase-3 mRNA and protein were increased in the hippocampus and
caudate-putamen, which are selectively vulnerable to ischemic injury.
The caspase-3 precursor was proteolytically activated, and
caspase-3-like protease activity was increased in these vulnerable
brain regions before and coincidental with cell death. The specific
caspase-3 protease substrate PARP was cleaved in these regions when
substantial amounts of cell death occurred. Finally, inhibition of
caspase-3-like protease activity using a tetrapeptide inhibitor
significantly decreased neuronal death and DNA fragmentation in the
hippocampal CA1 sector up to 7 d after ischemia. These results
strongly support the hypothesis that the caspase-3 protease is an
inducible cell death effector in the brain after ischemic injury.
Consistent with previous observations, caspase-3 mRNA and protein are
present at low levels in the adult brain (Krajewska et al., 1997; Ni et
al., 1997). After transient global ischemia, expression of both
caspase-3 mRNA and protein was markedly increased in the brain.
Although caspase-3 gene expression was transiently and modestly
increased in less vulnerable cells such as dentate granule cells 8 hr
after ischemia, a greater and prolonged increase, lasting up to 72 hr
after ischemia, was found in selectively vulnerable CA1 and striatal
neurons destined to die. These data confirm recent findings regarding
cpp32 mRNA expression in a rat model of cardiac arrest (Gillardon et
al., 1997). This pattern of expression is also similar to that of Bax,
another proapoptotic gene studied in similar animal models of global
ischemia (Krajewski et al., 1995; Chen et al., 1996). With rare
exceptions, cells containing fragmented DNA also showed highly
increased caspase-3 immunoreactivity (Fig. 4). Such colocalization of
DNA fragmentation and elevated caspase-3 immunoreactivity was detected
in a majority of neurons in the CA1 sector and many neurons in the CPu
at 72 hr after ischemia. These results are consistent with a potential
role for caspase-3 as a neuronal death effector in ischemic
neurons.
As seen with other ICE family proteases, post-translational activation
of caspase-3 requires proteolytic cleavage of the precursor protein, in
this case into two subunits (p17 and p12), the larger of which contains
the catalytic site (Fernandes-Alnemri et al., 1994). In the present
study, an increase in p17 immunoreactivity began to be detectable in
the hippocampus and CPu 4 hr after ischemia and peaked at 24-72 hr
(Fig. 3). Nearly coincident with the time course of caspase-3
proteolytic cleavage after ischemia, caspase-3-like protease activity
was selectively increased in the same brain regions, suggesting that
proteolytic cleavage of caspase-3 may directly contribute to the
increase in caspase-3 protease activity after ischemia. The increased
caspase-3 protease activity was detected in brain regions in which cell
death was eventually substantial (the hippocampus and CPu) but not in
regions either less vulnerable to ischemia (the cortex) or unaffected
by ischemia in this model (the cerebellum). Thus, we suggest that cell
death after global ischemia is associated with post-translational
activation as well as gene induction of caspase-3, with the proteolytic
cleavage event slightly preceding gene upregulation. The mechanism by
which the caspase-3 gene is induced and the caspase-3 precursor is
cleaved after ischemia is unclear. Ischemia-induced glutamate release could be a trigger for caspase-3 gene induction (Du et al., 1997). A
number of studies suggests that caspase-3 can be either autoactivated or activated by ICE or nedd2, Mch2a/CAP, Mch4, and Mch5 (Kumar et al.,
1994; Tewari et al., 1995; Fernandes-Alnemri et al., 1996; Liu et al.,
1996). It is possible that ischemia activates ICE or other upstream
proteases, which lead to the activation of caspase-3. Subsequently,
active caspase-3 and overexpression of caspase-3 could contribute to
autoactivation of this enzyme. Accordingly, we speculated that ICE
might be induced or activated during earlier reperfusion periods after
ischemia. However, we could not detect increased ICE mRNA expression in
the brain 0.5-72 hr after ischemia (data not shown). Moreover,
ICE-like protease activity was only modestly increased in the
hippocampus and only after 72 hr of reperfusion and was not increased
in the CPu (Fig. 5). The ICE inhibitor Z-YVAD-FMK failed to provide
significant protection in CA1 (Fig. 7b). These data do not
support ICE as the main trigger responsible for the activation of
caspase-3 and subsequent neuronal death after global ischemia.
The regional and temporal profile of caspase-3 induction after ischemia
supports a role of this enzyme in ischemic neuronal death; however, a
causal relationship between these two events cannot be deduced from the
expression data alone. Consequently, we have studied the effect of
inhibiting caspase-3 activity on cell survival after ischemia. The
tetrapeptide inhibitor Z-DEVD-FMK significantly reduced caspase-3-like
protease activity in ischemic hippocampus and significantly decreased
CA1 cell loss, with a similar efficacy whether given before or after
ischemia. The protective effect of Z-DEVD-FMK on CA1 neuron survival is
unlikely to be caused by its effects on brain temperature or ischemia
induction (as judged by EEG measurement). However, we must interpret
these data with great caution. Because the tetrapeptide inhibitor was designed based on the PARP cleavage site (Lazebnik et al., 1994), caspase-3 may not be the sole caspase inhibited by Z-DEVD-FMK. Several
caspase-3-related proteases including Mch2a and Mch3a are also capable
of cleaving PARP or the
N-acetyl-Asp-Glu-Val-Asp-(7-amino-4-methylcoumarin) (DEVD-AMC) substrate reminiscent of the PARP cleavage site
(Fernandes-Alnemri et al., 1995). Ich-2/Tx and Ich-1/nedd2 can cleave
PARP, but this requires very high enzyme-substrate ratios (for review,
see Zhivotovsky et al., 1996). Thus, we cannot exclude the possibility
that the protective effect achieved by the caspase-3 inhibitor is via
the inhibition of other caspase-3-like enzymes. To address this issue, it will be helpful to compare ischemic injury in naive and
caspase-3-deficient (knock-out) animals. Nevertheless, the fact that
the caspase-3 gene is induced and its enzymatic activity is increased
after ischemia provides strong evidence to link ischemic cell death to
the activation of caspase-3.
Caspase-3 may mediate ischemic cell death via several mechanisms.
Mature caspase-3 can cleave specific cellular proteins. Several
proteins have been suggested as potential targets for caspase-3 during
apoptosis. The best-characterized death substrate is PARP, an enzyme
involved in DNA repair and maintenance of genome integrity (Althaus and
Richter, 1987; Satoh and Lindahl, 1992). Under many apoptotic
conditions, PARP is cleaved by caspase-3 to generate the characteristic
85 and 24 kDa fragments (Kaufmann et al., 1993; Fernandes-Alnemri et
al., 1995; Nicholson et al., 1995). Other proteins thought to be
targets for caspase-3 include DNA-dependent protein kinase (DNA-PK)
(Casciola-Rosen et al., 1995; Han et al., 1996), protein kinase C
(Hugunin et al., 1996), the transcription factors SREBPs (Wang et al.,
1996), and actin (Mashima et al., 1997; Song et al., 1997). In the
present study, PARP was partially cleaved to its 85 kDa fragment in
vulnerable brain regions after ischemia. No degradation of DNA-PK or
actin was detected. Thus, PARP may be a specific substrate for
caspase-3 during ischemic cell death, although whether degradation of
PARP is an event leading to ischemic cell death is unclear. First, as
shown in this study, cleavage of PARP is a late event after ischemia.
Second, PARP is a nuclear protein, and although caspase-3 may be
present in the nucleus of degenerated neurons (Fig. 4), there is no
direct evidence that mature caspase-3 is relocated to the nucleus from
the cytosol before cell death. Third, under certain circumstances such
as focal cerebral ischemia and excitotoxicity, gene disruption or
pharmacological inhibition of PARP improves neuronal survival (Eliasson
et al., 1997; Endres et al., 1997). Accordingly, cleavage of PARP may
not be responsible for neuronal death after ischemia. Instead, PARP
degradation may reflect the overall cellular destruction in the final
stages of the apoptotic cascade. Another potential mechanism via which
caspase-3 might effect cell death after ischemia is by activating other
caspases such as Mch2a and Mch6 involved in the apoptotic cascade
(Srinivasula et al., 1996). Finally, caspase-3 may promote cell death
via the activation of caspase-activated deoxyribonuclease (CAD), a key DNA-cleavage enzyme responsible for DNA fragmentation during apoptosis. Enari et al. (1998) recently suggest that caspase-3 activates CAD by
cleaving and releasing the CAD inhibitory protein that normally binds
CAD. Future work identifying specific cellular substrates for caspase-3
at early stages of cell death would greatly enhance our understanding
of the role of this death protease in ischemic brain injury.
In summary, the present study provides evidence that the caspase-3 gene
is induced and its protein product is activated in selectively
vulnerable brain regions after ischemia. Caspase-3 activation precedes
and its regional distribution correlates with delayed cell death. The
caspase-3 inhibitor decreases protease activity in the hippocampus and
increases cell survival in this region after ischemia. The results
strongly support a cell death-effector role for caspase-3-protease in
ischemic brain injury. Future development of highly specific methods to
inhibit caspase-3-like protease activity or its consequences may have
therapeutic significance in the treatment of stroke and related
neurological disorders.
| |
FOOTNOTES |
Received Feb. 5, 1998; revised April 7, 1998; accepted April 10, 1998.
This work was supported by National Institutes of Health Grants NS
35965 to R.P.S., S.H.G., and J.C., NS 24728 to R.P.S., and NS 36736 to
J.C. J.C. was also supported in part by the Competitive Medical
Research Fund of the University of Pittsburgh Medical Center. S.H.G.
was also supported by the Department of Veterans Affairs Merit Review
Program. We thank Dr. David A. Greenberg for critical review and
helpful comments on this paper, Jingyun Luan for technical assistance,
and Pat Strickler for secretarial support.
Correspondence should be addressed to Dr. Jun Chen, Department of
Neurology, S506-Biomedical Science Tower, University of Pittsburgh
School of Medicine, Pittsburgh, PA 15213.
| |
REFERENCES |
-
Alnemri ES,
Livingston DJ,
Nicholson DW,
Salvesen G,
Thronberry NA,
Wong WW
(1996)
Human ICE/CED-3 protease nomenclature.
Cell
87:171[ISI][Medline].
-
Althaus FR,
Richter C
(1987)
ADP-ribosylation of prot
|
Top
Abstract
Introduction
