preconditioning. Poly(ADP-ribose) polymerase-1 (PARP-1) normally functions in DNA repair, but extensive PARP-1 activation is a
major cause of ischemic cell death. Because PARP-1 can be cleaved and inactivated by caspases, we investigated the possibility that
combined with 0.5 mM 2-deoxyglucose and 5 mM azide (“chemical ischemia”) to model the reversible energy failure that occurs during
longer periods of chemical ischemia. These cultures were also more resistant to the PARP-1 activating agent, N-methyl-N?-nitro-N-
nitrosoguanidine, suggesting reduced capacity for PARP-1 activation after preconditioning. Immunostaining for the 89 kDa PARP-1
cleavage fragment and for poly(ADP-ribose) formation confirmed that PARP-1 was cleaved and PARP-1 activity was attenuated in the
in the cortical cultures. A cause–effect relationship between caspase activation, PARP-1 cleavage, and ischemic preconditioning was
supported by studies using the caspase inhibitor Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO). Cultures treated with DEVD-CHO after
preconditioning showed reduced PARP-1 cleavage and reduced resistance to subsequent ischemia. These findings suggest a novel
interaction between the caspase- and PARP-1-mediated cell death pathways in which sublethal caspase activation leads to PARP-1
A brief period of sublethal ischemia induces resistance to a sub-
emic preconditioning (Nandagopal et al., 2001; Kirino, 2002;
ischemic preconditioning, and the relative importance of these
1997; Ishida et al., 1997; Nandagopal et al., 2001; Kirino, 2002).
Studies in brain suggest involvement of post-translational
changes such as Ras activation (Gonzalez-Zulueta et al., 2000),
altered phosphorylation state of ERK (extracellular signal-
regulated kinase) and NMDA receptor subunits (Shamloo and
Wieloch, 1999; Shamloo et al., 1999), and altered NMDA recep-
tor function (Aizenman et al., 2000). Other studies suggest im-
portant roles for gene transcription and increased expression of
proteins such as hsp70 (Chen et al., 1996; Currie et al., 2000),
anti-oxidant enzymes (Hoshida et al., 2002), or anti-apoptosis
ing and increased resistance to subsequent stress.
Activation of poly(ADP-ribose) polymerase-1 (PARP-1) is a
major cause of neuronal death after brain ischemia (Szabo and
transfer the ADP-ribose groups from NAD?to form branched
strand breaks or kinks. Several PARP family members are now
recognized, with PARP-1 accounting for ?80% of nuclear
poly(ADP-ribose) polymerase activity (Heller et al., 1995;
D’Amours et al., 1999). Although PARP-1 normally functions to
facilitate DNA repair, extensive PARP-1 activation promotes cell
death through processes involving energy depletion and the re-
lease of apoptosis-inducing factor (Ha and Snyder, 1999; Ying et
al., 2002; Yu et al., 2002). Genetic or pharmacological inhibition
of PARP-1 activity reduces infarct size by up to 80% in brains
subjected to transient or permanent ischemia (Eliasson et al.,
1997; Endres et al., 1997; Tokime et al., 1998; Takahashi et al.,
1999). Similarly, inhibition of PARP-1 in cultured neurons sub-
PARP-1 can be cleaved and inactivated by caspase-3,
caspase-7, caspase-8, and calpains (D’Amours et al., 1998; Ger-
activated by cerebral ischemia (Lipton, 1999; Chen et al., 2002),
This work was supported by the Department of Veterans Affairs and by National Institutes of Health Grants
TheJournalofNeuroscience,September3,2003 • 23(22):7967–7973 • 7967
we hypothesized that cleavage and inactivation of PARP-1 could
be an effector mechanism of ischemic preconditioning.
Caspase-3 can be activated by low levels of oxidative stress or
tion has recently been shown to be important for ischemic pre-
conditioning in brain (McLaughlin et al., 2003). Here we show
that sublethal activation of caspase-3 in cultured neurons by a
emia, that would otherwise induce PARP-1-mediated cell death.
Materials. Chemicals were purchased from Sigma-Aldrich (St. Louis,
MO), except where noted otherwise.
the Animal Studies Committee of the San Francisco Veterans Affairs
Medical Center. Cortical astrocyte–neuron cocultures were prepared by
seeding neurons onto a preexisting astrocyte layer, as described previ-
ously (Ying et al., 1999). Wild-type (WT) neurons were prepared from
fetal Swiss–Webster mice (Simonsen, Gilroy, CA). PARP-1 gene defi-
PARP-1?/?strain developed by Wang et al. (1995) on a mixed 129/Sv x
C57BL/6 background. Cocultures were maintained in glial-conditioned
medium prepared by placing MEM with 2 mM glutamine, 50 ?g/ml
and 200 nM ?-tocopherol (Leist et al., 1996) into a flask of confluent
were 14–15 d in vitro.
Experimental procedures. Experiments were performed using a bal-
anced salt solution (BSS) composed of (in mM): 3.1 KCl, 134 NaCl, 1.2
CaCl2, 1.2 MgSO4, 0.25 KH2PO4, 15.7 NaHCO3, as described previously
(Ying et al., 2001). The pH was adjusted to 7.2 while the solution was
equilibrated with 5% CO2at 37°C. Osmolarity was verified at 280–310
mOsm with a Wescor vapor pressure osmometer (Logan, UT). Concen-
trated drug stocks were prepared in BSS and likewise adjusted to pH 7.2
1996) and terminated by washing in BSS and replacement of glial-
Assessment of neuronal injury. Neurons were distinguished from the
underlying astrocyte layer by their phase-bright, process-bearing mor-
after chemical ischemia exposures by propidium iodide (PI) fluores-
cence. PI was added at 0.04 mg/ml to each well, and both the PI-
in four randomly selected optical fields using a Nikon fluorescence mi-
croscope. At least 300 neurons were counted in each well, and results
from each well were expressed as “% neuronal death.”
Immunostaining. Immunostaining for poly(ADP-ribose) (PAR) was
performed according to the method of Burkle et al. (1993) with modifi-
cations. After one wash in cold PBS, the cultures were fixed in 10%
70, 90, and 100% ethanol, and air dried. The fixed cultures were prein-
cubated with blocking buffer (PBS), 10% goat serum, 0.1% Triton
X-100) for 60 min at room temperature, and anti-PAR monoclonal an-
tibody (Trevigen, Gaithersburg, MD) was added at a 1:2000 dilution for
incubation overnight at 4°C. After washing with PBS containing 0.1%
Triton X-100, the cells were incubated with Alexa-fluor 488-conjugated
goat anti-mouse IgG (Molecular Probes, Eugene, OR) at 1:500 dilution
for 1 hr at room temperature and then washed again to remove excess
temperature. After preincubation in blocking buffer, rabbit polyclonal
anti-cleaved PARP-1 antibody (#9544, Cell Signaling Technology, Bev-
erly, MA) was added at a 1:500 dilution and incubated overnight at 4°C.
Antibody visualization was achieved with Alexa-fluor 488-conjugated
goat anti-mouse IgG.
Western blots. Cells were lysed at 4°C in buffer containing 1% SDS, 1
mM Na-vanadate, 0.1 mM phenylmethylsulfonylfluoride, 2.5 ?g/ml pep-
containing 20 ?g of protein were mixed with Laemmli buffer, boiled for
5 min, and electrophoresed on a SDS-polyacrylamide gel. Proteins were
transferred to a polyvinylidene difluoride membrane (Amersham Bio-
sciences, Piscataway, NJ), incubated overnight at 4°C in Tris-buffered
saline containing 5% nonfat dry milk and 0.1% Tween 20, and then
three washes, the membrane was incubated for 90 min with a 1:10,000
dilution of anti-mouse or anti-rabbit IgG horseradish peroxidase anti-
body (Vector Laboratories, Burlingame, CA). The membrane was
washed three additional times, and the signal was detected with the Am-
ersham ECL system (Amersham Biosciences). After antibody stripping,
membranes were immunostained for ?-actin to confirm consistent pro-
1:1500 for rabbit polyclonal antibody to PARP-1 (#9544, Cell Signaling
Technology); 1:1000 for rabbit polyclonal antibody to cleaved caspase-3
(#9661, Cell Signaling Technology); and 1:10,000 for the mouse mono-
the SCION Image system. The analysis of ?-actin expression showed
negligible variation in protein loading.
Caspase-3 activity. Caspase-3 activity was measured as described by
Sordet et al. (2002), with minor modifications. After lysing, cell samples
were centrifuged at 10,000 ? g for 20 min. The protein concentration of
the resulting supernatant was measured by the bicinchonic acid method
(Smith et al., 1985), and 25 ?g protein aliquots were loaded into 96-well
plates. A reaction mixture containing 100 ?M fluorogenic peptide sub-
strate Ac-Asp-Glu-Val-Asp-7-amino-4-methyl coumarin (Calbiochem,
San Diego, CA), 1 mM EDTA, 0.1% 3-[(3-cholamidopropyl)D:methyl
amino]-1-propane sulfonate, 10% glycerol, 20 mM dithiothreitol, and
100 mM HEPES, final pH 7.0 was added to the wells. After 1 hr at 37°C,
fluorescence was measured using an Fmaxfluorescence plate reader with
Softmax Pro software (Molecular Devices, Sunnyvale, CA) at an excita-
tion wavelength of 355 nm and an emission wavelength of 460 nm.
ized to the increase induced by staurosporine.
Caspase inhibition. Effects of caspase inhibition on PARP-1 cleavage
and ischemic preconditioning were tested by adding 25 ?M of Ac-Asp-
Glu-Val-Asp-aldehyde (DEVD-CHO) (Calbiochem) to the culture me-
dium 2 hr before the preconditioning and during the subsequent 24 hr.
Control experiments were performed in parallel by adding only the ve-
hicle, 0.25% dimethylsulfoxide, to the culture medium.
Statistical analyses. Data are presented as means ? SE. Statistical sig-
nificance was assessed using ANOVA and the Student–Newman–Keuls
post hoc test to compare the indicated experimental groups.
A model of ischemic preconditioning in mouse cortical cultures
was established using inhibitors of energy metabolism (Rajdev
and Reynolds, 1994; Aizenman et al., 2000). Oxidative ATP pro-
duction was blocked with 5 mM azide, and glycolytic ATP pro-
duction was blocked by removing glucose and adding 0.5 mM
2-deoxyglucose to prevent metabolism of retained intracellular
glucose (Swanson and Benington, 1996). A 15 min period of CI
was sublethal, resulting in neuronal death no greater than that
caused by medium exchanges alone. In contrast, CI of ?30 min
killed ?75% of the neurons (Fig. 1A,B). Cell death was assessed
24 hr after CI (Fig. 1B). The neuronal death resulting from 30 or
45 min of CI was substantially reduced in cultures that had been
preconditioned with a sublethal, 15 min interval of CI 24 hr
previously. Assessment of neuronal death at 48 or 72 hr after CI
gave nearly identical results. This model of ischemic precondi-
tioning was used for all subsequent studies in this report.
requires several hours or days to become fully manifest (Kirino,
7968 • J.Neurosci.,September3,2003 • 23(22):7967–7973Garnieretal.•IschemicPreconditioningbyCaspaseCleavageofPoly(ADP-Ribose)Polymerase-1
2002; Dirnagl et al., 2003). Consistent with these reports, we
found that significant resistance to subsequent ischemia in this
model first developed between 3 and 6 hr after the precondition-
ing ischemia and increased through at least 24 hr (Fig. 1C). As
shown in Figure 1D, the preconditioning effect was substantially
reduced in cultures treated with the protein synthesis inhibitor
cycloheximide (CHX). Cycloheximide alone (in the absence of
preconditioning) also had a modest neuroprotective effect, as
reported previously (Lobner and Choi, 1996).
Neuronal death caused by CI was substantially reduced by the
PARP inhibitor 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-
death. These results suggested the possibility that the resistance to
ischemia conferred by ischemic preconditioning might likewise re-
tion and neuronal death induced by N-methyl-N?-nitro-N-
nitrosoguanidine (MNNG), a DNA alkylating agent widely used to
trigger PARP-1 activation and PARP-1-mediated cell death (Virag
DPQ (data not shown). PARP-1 activation
was also assessed by immunostaining for
PARP-1. In the absence of preconditioning,
poly(ADP-ribose) immunoreactivity was
evident in most neurons after 40 min of ei-
ther CI or MNNG. These treatments pro-
duced almost no detectable poly(ADP-
ischemic preconditioning, suggesting a re-
duced capacity for PARP-1 activation in the
itor DEVD-CHO negated this effect of pre-
conditioning, whereas CHX had no appre-
vated by caspase cleavage, we examined
reduced PARP-1 activity and PARP-
mediated cell death observed in precondi-
tioned cultures. Cultures were harvested
24 hr after ischemic preconditioning and
tochemistry for the 89 kDa PARP-1 frag-
ment produced by caspase cleavage (La-
zebnik et al., 1994; Le et al., 2002). As
shown in Figure 4A, ischemic precondi-
tioning caused a significant increase in the
89 kDa fragment, and this increase was at-
tenuated in cultures treated with the
caspase inhibitor, DEVD-CHO. Cultures
immunostained with the same antibody
ence of PARP-1 cleavage product in most
neurons (and no astrocytes) after preconditioning (Fig. 4B).
Consistent with the Western blots, appearance of the 89 kDa
PARP-1 cleavage product was significantly reduced in cultures
treated with DEVD-CHO.
Caspase-3 is the major effector of PARP-1 cleavage during
apoptosis (Lazebnik et al., 1994; Le et al., 2002). Procaspase-3
becomes activated by cleavage to a 17 kDa fragment (Cohen,
ern blots were prepared at serial time points after precondition-
ing and probed with an antibody specific to the active 17 kDa
caspase-3 fragment. These studies showed an increase in active
caspase-3 fragment after an interval of 3–6 hr (Fig. 5A). We also
assessed the time course of PARP-1 cleavage. Western blots for
the 89 kDa PARP-1 cleavage product showed a significant in-
crease by 12 hr after preconditioning ischemia (Fig. 5A). Direct
a pattern similar to that observed in the Western blots (Fig. 5B).
hr and maximal by 6 hr after preconditioning ischemia. The
preconditioning-induced caspase-3 activity was blocked by the
caspase inhibitor DEVD-CHO, whereas CHX had no effect. Of
note, the magnitude of caspase-3 activation was modest relative
to that induced by staurosporine, a classic inducer of apoptosis
(data not shown).
clear propidium iodide fluorescence. Scale bar, 100 ?m. B, Effect of preconditioning on neuronal resistance to CI. C, Neuronal
Garnieretal.•IschemicPreconditioningbyCaspaseCleavageofPoly(ADP-Ribose)Polymerase-1J.Neurosci.,September3,2003 • 23(22):7967–7973 • 7969
To further evaluate the role of caspase activation in ischemic
24 hr interval after the preconditioning stimulus. As shown in
Figure 6, cultures treated with 25 ?M DEVD-CHO acquired less
resistance to a second, longer ischemic challenge, whereas the 24
DEVD-CHO reduced the extent of the preconditioning-induced
and by ?100% when tested with a 45 min ischemic challenge.
These differences cannot be compared directly because the de-
gree of preconditioning-induced resistance also differed at the
suggest that the relative contribution of caspase-mediated pro-
cesses to ischemic tolerance may vary with the severity of the
well established (Virag and Szabo, 2002). The increased resis-
tance to ischemic insults resulting from genetic or pharmacolog-
model used here, led us to propose that a block in PARP-1 acti-
vation might similarly contribute to the protective effect of isch-
emic preconditioning. This proposal was supported by the find-
activating agent MNNG. PARP-1 can be irreversibly inactivated
by cleavage of the catalytic site from the DNA binding domain,
through the actions of caspase-3 and other proteolytic enzymes
(Pieper et al., 1999). The present studies provide evidence for
both PARP-1 cleavage and caspase-3 activation in precondi-
tioned neurons. Additionally, treatment with the caspase inhibi-
tor DEVD-CHO blocked PARP-1 cleavage and reduced the de-
velopment of ischemic tolerance. Taken together, these results
suggest that preconditioning provides increased resistance to
ischemia at least in part by inducing caspase-mediated PARP-1
cleavage and thereby blocking the PARP-1-mediated cell death
Ischemic tolerance developed over a period of 6–24 hr after
the preconditioning stimulus, similar to observations made in
vivo and in other cell culture systems (Nandagopal et al., 2001;
Kirino, 2002; Dirnagl et al., 2003). This time course can be com-
pared with the time course of procaspase-3 cleavage, increased
caspase enzymatic activity, and PARP-1 cleavage presented in
maximum values at ?6 hr after ischemic preconditioning, and
PARP-1 cleavage reached a maximum ?12 hr after precondi-
emic tolerance, which first became apparent between 3 and 6 hr
DEVD-CHO. Cultures incubated with DEVD-CHO after ischemic
preconditioning showed no inactivation of PARP-1 activity, as
judged by lack of poly(ADP-ribose) formation in response to CI or
neuronal death after CI. **p ? 0.01; representative of three independent experiments each
7970 • J.Neurosci.,September3,2003 • 23(22):7967–7973Garnieretal.•IschemicPreconditioningbyCaspaseCleavageofPoly(ADP-Ribose)Polymerase-1
The extent of caspase-3 activation induced by ischemic pre-
The relatively modest activation of caspase-3 is consistent with
the observation that preconditioning, unlike staurosporine, did
not lead to neuronal death. Although caspase-3 activation is an
effector mechanism of cell death during apoptosis, caspases can
also be activated in processes that do not lead to cell death. For
example, caspase-3 cleavage of the transcription factor GATA-1
Reversible, sublethal activation of caspase-3 has been demon-
strated previously in neurons (Francois et al., 2001), and
mechanism for synaptic plasticity (Glazner et al., 2000). The
present results suggest that PARP-1 cleavage is an additional
mechanism by which caspase-3 may act outside of its classical
role as an effector of cell death.
of neuroprotection achieved by ischemic preconditioning was
substantial, approaching that achieved with the PARP inhibitor
DPQ and with PARP-1 gene deletion. If the neuroprotective ef-
fect of preconditioning effect is caused primarily by PARP-1
cleavage, a substantial fraction of neuronal PARP-1 must be
cleaved after preconditioning. It is difficult to quantify the frac-
tion of neuronal PARP-1 cleaved after preconditioning because
ies of PARP-1 function showed that preconditioning markedly
treated neurons (Fig. 4B), suggesting that substantial PARP-1
inactivation does occur after ischemic preconditioning.
Modest ischemic insults can trigger apoptosis (Bonfoco et al.,
1995), and it is likely that the sublethal insults that induce pre-
ide production as important upstream components of neuronal
2000; Nandagopal et al., 2001). Although not investigated here,
these upstream events may be coupled to caspase-3 activation in
several ways. One well established sequence involves mitochon-
drial depolarization and cytochrome c release, which triggers the
intrinsic pathway of caspase activation (Lemasters et al., 1999;
Kroemer and Reed, 2000). Evidence also suggests that the Fas
pathway of caspase activation can be induced by transient ATP
depletion (Feldenberg et al., 1999) and that NMDA receptor ac-
tivation and nitric oxide production can trigger caspase activa-
tion through the endoplasmic reticulum stress response (Rao et
It is generally accepted that delayed-onset ischemic tolerance
this, we observed that ischemic tolerance was substantially re-
duced in cultures treated with the protein synthesis inhibitor
CHX. Because caspase-3 is abundant in cells in an inactive form,
it is unlikely that de novo synthesis of caspase-3 is required for
PARP-1 cleavage. Accordingly, we observed no effect of CHX
treatment on the preconditioning-induced caspase-3 activation
or PARP-1 inhibition. This suggests the possibility that precon-
ditioning may induce synthesis of proteins that influence the
PARP-1 cell death pathway at steps downstream of PARP-1 acti-
vation. Alternatively, CHX may have cytoprotective effects
through mechanisms independent of protein synthesis inhibi-
the caspase inhibitor DEVD-CHO (25 ?M). Values above the bands denote the increase ? SE
was almost totally blocked by 25 ?M DEVD-CHO, as observed in the Western blots. Similar
0 hr time point (*p ? 0.05; **p ? 0.01; n ? 3–4). B, Caspase-3 enzymatic activity was
within 3 hr of PC. The increase was blocked in cultures treated with the caspase inhibitor
Garnieretal.•IschemicPreconditioningbyCaspaseCleavageofPoly(ADP-Ribose)Polymerase-1J.Neurosci.,September3,2003 • 23(22):7967–7973 • 7971
tion, as has been suggested by previous studies (Ratan et al.,
During apoptosis, PARP-1 cleavage by caspase-3 and other
enzymes prevents the PARP-1 activation and resultant ATP de-
pletion that would otherwise be triggered during DNA fragmen-
tation (Oliver et al., 1998; Boulares et al., 1999; Le et al., 2002).
Support for caspase-3 as a mediator of PARP-1 cleavage after
ischemic preconditioning was provided in the present study by
biochemical measurements showing increased caspase-3 activity
ment in preconditioned cultures, and by the block of PARP-1
cleavage produced by 25 ?M DEVD-CHO, a concentration at
which DEVD-CHO is a relatively specific caspase-3 inhibitor
(Margolin et al., 1997). These findings are in agreement with a
study published during the preparation of this manuscript that
reports widespread caspase-3 activation in a rat brain model of
ischemic preconditioning (McLaughlin et al., 2003). It is possi-
ble, however, that other proteolytic enzymes may also be in-
volved. PARP-1 can also be cleaved by caspase-7, caspase-8, and
calpains (Germain et al., 1999; Wang, 2000; Le et al., 2002), and
because DEVD-CHO is not entirely specific for caspase-3, the
results obtained with DEVD-CHO do not exclude the possibility
that other caspases or calpains may contribute to PARP-1 inacti-
vation in ischemic preconditioning. Conversely, the possibility
that the observed effect of DEVD-CHO on ischemic precondi-
tioning may underestimate the true contribution of PARP-1
cleavage to this process.
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