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INTRODUCTION |
Poly(ADP-ribose) polymerase-1
(PARP-11; EC 2.4.2.30), the
most abundant poly(ADP-ribosyltransferase) in mammalian cells, plays an
essential role in excitotoxic neuronal death both in vitro
and in vivo (1-4). The presumptive mechanism for this
neurotoxic effect involves, sequentially, increases in
[Ca2+]i via glutamate receptors, activation of
nitric-oxide synthase, generation of the free radical
peroxynitrite (ONOO
), activation of PARP-1 in response to
genomic DNA damage, consumption of NAD+ during the
formation of poly(ADP-ribose) polymers, and death via energy failure
(5). However, the capacity for PARP-1 activation within the nucleus to
deplete total cellular energy stores, particularly compartmentalized
within mitochondria, remains to be established (4, 6). Because in
addition to being abundant in cell nuclei, PARP-1 and other
ADP-ribosyltransferases are also prevalent in mitochondria (7-9),
where similar to nuclear PARP-1, they facilitate DNA repair in response
to oxidative damage (10, 11), we hypothesized that inhibition of
mitochondrial poly(ADP-ribosylation) may play a pivotal role in
neuronal cell survival under conditions of oxidative stress and excitotoxicity.
Here we show that inhibition of mitochondrial poly(ADP-ribosylation)
preserves mitochondrial transmembrane potential (
m) and
NAD+ content, maintains cellular respiration, and reduces
neuronal cell death triggered by oxidative stress or excitotoxicity.
Treatment with liposome-encapsulated NAD+ or ATP also
preserved 
m and cellular respiration, suggesting that
cells can also be rescued by energy repletion after oxidative stress.
Our findings suggest that NAD+ depletion and energy failure
convert poly(ADP-ribosylation) compartmentalized within mitochondria
from a homeostatic process to a mechanism of neuronal death, providing
a unifying mechanism by which PARP-1 can regulate cell death under
conditions of mitochondrial dysfunction, and identifying multiple
intracellular targets for inhibitors of poly(ADP-ribosylation). These
findings have relevance to both acute and chronic central nervous
system diseases where oxidative stress is a contributing factor,
including stroke, traumatic brain injury, seizures, and Parkinson's
disease and other neurondegenerative diseases (12, 13).
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EXPERIMENTAL PROCEDURES |
PARP Activity
PARP enzyme activity was measured using a commercial kit
(Trevigen, Gaithersburg, MD). Briefly, 1 µg of mitochondrial or
nuclear protein extracts were incubated in reaction buffer containing histones and [32P]NAD ± exogenous DNA with strand
breaks. After incubating at room temperature for 10 min, reactions were
terminated and proteins were precipitated with 20% trichloroacetic
acid. Incorporation of 32P was determined by scintillation counting.
Cell Cultures and Pharmacological Studies
Primary cortical neuron-enriched cultures were prepared from 16 to 17-day-old Sprague-Dawley rat embryos as described (14). Dissociated
cell suspensions were placed in 96-well plates (5 × 104 cells/well) or in plastic dishes coated with
poly-D-lysine (1.3 × 107 cells/well).
Experiments were performed between 7 and 12 days in
vitro.
ONOO
--
Neuron-enriched cultures were exposed to
100-250 µM ONOO
(Cayman Chemical, Ann
Arbor, MI) in buffer for 30 min, whereupon fresh media was replaced.
SIN-1
2 mM SIN-1 (Cayman Chemical) or 20%
Me2SO vehicle in culture media was added to cells in
96-well plates.
Glutamate--
Cells were exposed to varying concentrations of
L-glutamate with 5 µM glycine (Sigma) in
culture media for 10 min. Cultures were pre- or post- treated with
either the PARP inhibitor 5-iodo-6-amino-1,2-benzyopyrone (INH2BP) (6,
15-17) or 20% Me2SO vehicle, the metalloporphyrin-based peroxynitrite decomposition catalyst FP15 (14) or PBS vehicle, or
liposomally encapsulated NAD+ or ATP or vehicles (empty
liposomes or buffer). Liposomal NAD+ and ATP were prepared
using a modification of the thin-film method (18).
Fibroblasts from PARP-1
/
and corresponding
PARP+/+ mouse embryos (17) were cultured in 96-well plates
or in plastic dishes to confluence. For cytotoxicity experiments cells
were exposed to 250 µM ONOO
with or without
100 µM INH2BP.
Immunocytochemistry and Confocal Microscopy
Neurons grown on poly-D-lysine-coated glass
coverslips were fixed for 30 min in 2% paraformaldehyde in PBS (pH
7.4). Cells were incubated with 5% normal donkey serum and 2% bovine
serum albumin in PBS containing 0.2% Triton X-100 for 1 h to
permeabilize cell membranes. The cells were then incubated in a 1:100
dilution of rabbit polyclonal anti-HSP60 (StressGen, Victoria, British Columbia, Canada) and a 1:200 dilution of poly(ADP-ribose) (Biomol, Reading, PA) for 1 h at 37 °C, followed by incubation in the
appropriate secondary antibody. Cells were examined using a Leica TCS
NT confocal tri-laser scanning inverted microscope (Wetzlar, Germany)
as previously described (14).
Western Blot Analysis
Cellular proteins were separated into mitochondrial, nuclear,
and cytosolic fractions as described (14), and Western blotting was
performed using primary antibodies against: poly(ADP-ribose) polymers
(BioMol, Reading, PA), PARP (Cell Signaling, Beverly, MA), the carboxyl
terminus of apoptosis-inducing factor (AIF) (Santa Cruz, Santa Cruz,
CA), cytochrome c (BD Pharmingen), and cytochrome
c oxidase (BD Pharmingen) at optimized dilutions.
Assessment of 
m

m was determined using
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine
iodide (JC-1), a cationic dye that accumulates in mitochondria in a
membrane potential-dependent manner (19). Data are
presented as a green (488/580 nm)/red (535/610 nm) fluorescence
intensity ratio, with an increased ratio representing mitochondrial
depolarization. Cells grown on collagen-coated coverslips were
incubated in 1 µg/ml JC-1 (Molecular Probes, Eugene, OR) for 20 min
at 37 °C. Cells were pretreated with INH2BP, FP-15, or vehicle. The
media was replaced with buffer containing ONOO
for 5 min,
whereupon the ONOO
was removed and media was replaced.
Green/red fluorescent ratios were measured in five predefined fields
containing 2-5 cells/field using an Olympus IX70 microscope with a
×60 oil immersion 1.4 numerical aperature optic and an
automated stage. Image acquisition and analysis were performed using
Simple PCI (Compix, Inc., Cranberry, PA). A minimum of 2 coverslips
were imaged per condition.
Determination of NAD+
Cellular NAD+ levels were measured by the enzymatic
cycling method using alcohol dehydrogenase (20) with modifications.
Cells were homogenized in 0.05 M K phosphate buffer
containing 0.1 M nicotinamide (pH 6.0), frozen rapidly,
placed in a boiling water bath for 5 min, then cooled in an ice bath
for 5 min. Samples were centrifuged for 10 min at 1,000 rpm at 4 °C,
then added to a reaction mixture containing 0.065 M
glycylglycine, 0.1 M nicotinamide, 0.5 M
ethanol, alcohol dehydrogenase,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and
phenazine methosulfate at pH 7.4. NAD+ in the sample
generates NADH, which reduces MTT through the intermediation of
phenazine methosulfate to formazan. The reaction was allowed to run for
5 min and the absorbance determined at A556. A
standard curve was generated using known concentrations of
NAD+ and NAD+ levels were calculated.
Pulsed Field Gel Electrophoresis
Pulsed field gel electrophoresis was performed as described
(14). Briefly, chromosomal DNA samples were prepared in agarose plugs
using a CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad). Fragments were
separated on a 1.2% agarose gel at 14 °C for 20 h. Field strengths were 180 V forward and 120 V reverse, initial and final switching time was set at 5-60 s with a linear ramp. The gel was stained with ethidium bromide and visualized under UV light.
Isolated Brain Mitochondria
Adult Sprague-Dawley rat brain mitochondria were isolated by
differential centrifugation as described (21). Mitochondrial viability
was verified by measuring oxygen consumption. Mitochondria were
suspended in a reaction buffer to a final concentration of 1 mg of
mitochondrial protein/ml and placed in reaction tubes. Drug or vehicle
was added and the mitochondria were placed in a 37 °C water bath for
15 min, then 750 µM ONOO
or pH-adjusted
vehicle were added. This concentration of ONOO
was found
to completely depolarize mitochondria as determined by Safranin-O.
Aliquots of the isolated mitochondrial suspensions were removed at
baseline (
15 min), time 0 and 5 min, centrifuged at 14,000 rpm for 10 min at 4 °C, and mitochondrial pellets and supernatants were frozen
and stored for batch analysis. For assessment of mitochondrial protein
release 20 µl of mitochondria supernatants were dialyzed against PBS
in a mini-dialysis device (Pierce). Mitochondrial pellets were
homogenized in lysis buffer and handled as described above.
Flow Cytometric Analysis of Cell Death
Following cytotoxicity and drug treatments, cells were harvested
using trypsin-EDTA, washed once in ice-cold PBS, and resuspended in 1 ml of Annexin V binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). 1 × 105 cells were stained with 5 µl of Annexin V-fluorescein
isothiocyanate and 5 µg/ml propridium iodide (PI) in 100 µl of
Annexin V binding buffer at 4 °C. After 20 min, 400 µl of binding
buffer was added to each tube and samples were analyzed using a
tri-laser FACS Calibur flow cytometer.
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RESULTS |
Mitochondrial Poly(ADP-ribosylation) in Fibroblasts and
Neurons--
We verified that poly(ADP-ribosylation) could be
stimulated in mitochondria. PARP-1 was detected in both nuclear and
mitochondrial subcellular fractions in fibroblasts from
PARP-1+/+ but not PARP-1
/
mice (Fig.
1a). Endogenous PARP activity,
measured via incorporation of [32P]NAD in protein lysates
incubated in the absence of nicked-DNA, showed poly(ADP-ribosylation)
in both nuclear and mitochondrial cell fractions (Fig. 1b).
A lesser degree of baseline PARP activity was detected in nuclear
fractions from PARP
/
fibroblasts, with activity ~20%
of that seen in PARP+/+ fibroblasts. Poly(ADP-ribosylation)
in control cells suggests that baseline oxidative DNA damage is
occurring within both mitochondria and cell nuclei, and is consistent
with a previous study (22) showing baseline PARP-1 activity in neurons.
Baseline PARP activity was also detected in mitochondrial fractions
from PARP
/
fibroblasts, similarly, activity was ~20%
of that seen in PARP+/+ fibroblasts. PARP enzyme activity
relative to total protein was greater in mitochondrial compared with
nuclear cell fractions (Fig. 1b).

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Fig. 1.
PARP-1 and poly(ADP-ribosylation) in
mitochondria. a, PARP-1 was detected in both
nuclear and mitochondrial fractions from PARP-1+/+ but not
PARP-1 / cells (representative of 5 wells/group).
b, PARP activity in nuclear and mitochondrial protein
lysates obtained from PARP-1+/+ fibroblasts. A lesser
degree of PARP activity was detected in nuclear and mitochondrial
protein lysates obtained from PARP-1 / fibroblasts
(n = 3/group; cpm, counts per min).
c, PARP activity in nuclear and mitochondrial protein
lysates obtained from naive adult rat brain is inhibited by INH2BP in a
dose-dependent manner ( DNA, incubated without
exogenous nicked DNA; mean ± S.D.; n = 4 samples/group; *, p < 0.05 versus no
INH2BP, one-way analysis of variance with Tukey post-hoc
test. d, confocal dual-label immunohistochemical images with
differential interference contrast using antibodies against
poly(ADP-ribose) polymers (red) and the mitochondrial marker
Hsp60 (green) detected poly(ADP-ribosylation) in
mitochondria under baseline conditions (yellow;
arrows).
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Similar to fibroblasts from wild-type mice, PARP activity was seen in
mitochondrial fractions from normal adult rat brain. Mitochondrial and
nuclear protein lysates were incubated with and without nicked DNA to
activate PARP in the presence or absence of the potent PARP inhibitor
INH2BP (6, 15-17). PARP enzyme activity that was inhibited by INH2BP
in a dose-dependent manner was detected in both
mitochondrial and nuclear fractions (Fig. 1c). A portion of
total PARP was previously activated, as protein samples incubated
without nicked DNA demonstrated incorporation of [32P]ADP
to levels that were ~40% of total PARP activity. Similar to
fibroblasts, PARP activity/µg of protein was greater in mitochondrial compared with nuclear protein lysates. Consistent with this finding, poly(ADP-ribosylation), a surrogate marker of PARP activation, was
detected to a greater degree in mitochondria versus nuclei in neurons labeled with antibodies against poly(ADP-ribose) polymers and the mitochondrial marker heat shock protein 60 (hsp60) examined using laser confocal microscopy (Fig. 1d).
Mitochondrial Poly(ADP-ribosylation) Is Inhibited by
5-Iodo-6-amino-1,2-benzyopyrone--
Consistent with the direct
measurement of PARP activity using [32P]NAD+,
poly(ADP-ribose) polymer formation was more abundant in the mitochondrial versus nuclear protein fraction in control
cells (Fig. 2). Thirty min to 2 h
after exposure to ONOO
, there was a non-significant
increase in nuclear poly(ADP-ribosylation) compared with control
samples that were not affected by INH2BP. This non-significant increase
in nuclear PARP activity in neurons using this dose of
ONOO
is similar to reports in fibroblasts, where
500 µM ONOO
is required to stimulate
PARP activity (5). In contrast to nuclear poly(ADP-ribosylation),
mitochondrial poly(ADP-ribosylation) was unchanged 30 min to 2 h
after exposure to ONOO
, however, was significantly
reduced by pretreatment with INH2BP versus vehicle
(p < 0.001). Multiple poly(ADP-ribosylated) proteins were seen in fractions from each cellular compartment, however, patterns differed with primarily lower molecular weight proteins seen in mitochondrial fractions, compared with ~110-140- and 30-kDa bands seen in nuclear fractions. Thus, in neurons using this
experimental paradigm, INH2BP reduces basal PARP activity in
mitochondria.

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Fig. 2.
Inhibition of mitochondrial
poly(ADP-ribosylation) in neurons. Poly(ADP-ribosylation) detected
in nuclear (a) and mitochondrial (b) protein
lysates from untreated neurons and in neurons after exposure to
ONOO . Pretreatment with 100 µM INH2BP
reduced poly(ADP-ribosylation) in mitochondrial but not nuclear
fractions compared with vehicle. Graphs show mean relative
optical densities for poly(ADP-ribosylated) proteins at 30 min to
2 h, obtained from 4 gels and represented as the change
versus control (mean ± S.E., *, p < 0.001 versus vehicle, t test).
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Inhibition of Mitochondrial Poly(ADP-ribosylation) Prevents
Oxidative Stress-induced Mitochondrial Dysfunction and AIF Release and
Neuronal Cell Death--
To determine whether 250 µM
ONOO
produces mitochondrial dysfunction directly,

m, NAD levels, and cellular respiration and were
evaluated. Fig. 3a shows that

m, determined using the fluorescent dye JC-1 (19), is
rapidly lost in neurons exposed to ONOO
, and that PARP
inhibition attenuates loss of 
m. Direct quenching of
ONOO
using the peroxynitrite decomposition catalyst FP15
(14) also attenuates loss of 
m (Fig. 3b). PARP
inhibition also preserved cellular respiration, as determined by
conversion of MTT to formazan (14), in neurons treated with
ONOO
or the nitric oxide donor SIN-1, in a
dose-dependent manner (Fig. 3c). This appears to
be because of maintenance of cellular energy stores, as INH2BP prevents
reductions in NAD+ seen early after exposure to
ONOO
(Fig. 3d). In support of the role for
energy substrate depletion as a mechanism for mitochondrial
dysfunction, post-treatment with both liposome-encapsulated
NAD+ and ATP (18), but not empty liposomes or buffer,
partially preserved cellular respiration (MTT) in neurons 22 h
after exposure to ONOO
(Fig. 3e). Pretreatment
with liposome-encapsulated NAD+ also preserved

m versus empty liposomes (Fig.
3f).

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Fig. 3.
Inhibition of mitochondrial
poly(ADP-ribosylation) or exogenous NAD or ATP preserves cellular
energetics in neurons after oxidative stress. a and
b, pretreatment with INH2BP or FP15 preserves
 m versus vehicle (vehicle, red;
drug, blue; INH2BP alone, green; mean ± S.D.). Immunofluorescent images from representative cells for each
condition from three independent experiments are shown in the panels.
c, treatment with INH2BP preserves cellular respiration at
2 h after ONOO or SIN-1 (n = 12/group for ONOO and 3/group for SIN-1; *,
p < 0.05 versus vehicle). d,
treatment with 100 µM INH2BP preserves cellular
NAD+ content versus vehicle (n = 3-6/group; *, p < 0.05). e, treatment with liposome encapsulated
NAD+ (200 µM) or ATP (200 µM),
but not empty liposomes or buffer, preserves cellular respiration at
22 h (Lipo, liposomes; n = 3/group; *,
p < 0.05 versus vehicle). f,
pretreatment with 200 µM liposomal NAD+
preserves  m versus empty liposomes (empty
liposomes, red; liposomal NAD+,
green; mean ± S.D.). Immunofluorescent images with
differential interference contrast from representative cells for each
condition are shown in the panels. For c, d,
and e, mean ± S.D.; one- or two-way analysis of
variance with Tukey post-hoc test.
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We have previously shown that in neurons ONOO
induces
nuclear translocation of AIF and large-scale DNA fragmentation, the signature event in AIF-mediated cell death, and that these events can
be inhibited by treatment with the ONOO
decomposition
catalyst FP15 (14). Similar to FP15, INH2BP completely blocks nuclear
translocation of AIF and large-scale DNA fragmentation (Fig.
4, a and b),
supporting a role for poly(ADP-ribosylation) in mitochondrial release
of AIF and consistent with the report by Yu and colleagues (4, 6).
Inhibition or poly(ADP-ribosylation) also prevents egress of cytochrome
c from mitochondria to the cytosol after ONOO
exposure. Neuronal cell death induced by ONOO
, assessed
by flow cytometry using PI and Annexin V labeling, is reduced by
pretreatment with INH2BP (Fig. 4c). Compared with vehicle
treatment, PARP inhibition reduced both PI+/Annexin V+ (14 versus 24%, INH2BP versus vehicle, respectively)
and PI
/Annexin V+ (46 versus 27%,
INH2BP versus vehicle, respectively) cell profiles. In
isolated brain mitochondria (21), a dose of ONOO
(750 µM) that completely depolarizes the mitochondrial
membrane initiates rapid release of AIF and cytochrome c
(Fig. 5). This dose of ONOO
is relatively high, although it is likely that some proportion of
ONOO
was quenched by albumin in the mitochondrial
reaction buffer. AIF release, but not cytochrome c release,
was inhibited by pretreating isolated viable brain mitochondria with
INH2BP. Collectively, these data suggest that inhibition of
mitochondrial poly(ADP-ribosylation) preserves 
m and
reduces programmed cell death mediated by AIF.

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Fig. 4.
Inhibition of mitochondrial
poly(ADP-ribosylation) reduces apoptotic cell death in neurons after
oxidative stress. a, INH2BP inhibits nuclear
localization of AIF and appearance of cytosolic cytochrome
c. b, INH2BP prevents large-scale DNA
fragmentation detected by pulsed-field gel electrophoresis at 22 h. c, INH2BP reduced PI+/Annexin V+ and
PI /Annexin V+ cells 24 h after 250 µM ONOO versus vehicle (1 × 105 cells/condition). Representative of three
independent experiments.
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Fig. 5.
Inhibition of poly(ADP-ribosylation)
attenuates release of AIF from isolated brain mitochondria after
depolarization by ONOO . a, complete
depolarization was seen using 750 µM ONOO
and was confirmed by treating mitochondria with carbonyl cyanide
p-(trifluoromethoxy)phenyl hydrazone (FCCP).
INH2BP did not effect membrane potential in isolated mitochondria after
ONOO (data not shown). b, AIF release was
prevented by treating depolarized mitochondria with INH2BP.
Representative of four independent experiments.
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Inhibition of Poly(ADP-ribosylation) Prevents Excitatory Amino
Acid-induced Mitochondrial Dysfunction and AIF Release and Neuronal
Cell Death--
Because PARP plays a key role in nitric oxide- and
glutamate-mediated neuronal death (1, 4), and both mechanisms are felt
to involve ONOO
, the cytoprotective effect of INH2BP in
neurons treated with glutamate was tested. PARP inhibition preserved
cellular respiration after glutamate/glycine exposure (Fig.
6a) and prevented nuclear translocation of AIF (Fig. 6b) compared with vehicle. PARP
inhibition reduced both necrotic (1 versus 12%, INH2BP
versus vehicle, respectively) and apoptotic (4 versus 14%, INH2BP versus vehicle, respectively) cell death profiles induced by exogenous glutamate/glycine (Fig. 6b). Of note, PARP inhibition led to an increase in Annexin
V+/PI
cell profiles (26 versus
1%, INH2BP versus vehicle, respectively), suggesting the
potential of delayed cell death, or a shift to an earlier stage of
apoptosis in this paradigm.

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Fig. 6.
Inhibition of poly(ADP-ribosylation)
preserves cellular respiration and attenuates apoptotic and necrotic
cell death in neurons after glutamate excitotoxicity.
a, increasing concentrations of glutamate + 5 µM glycine reduces cellular respiration and is inhibited
by pretreatment with 100 µM INH2BP (n = 3/group; mean ± S.D.; *, p < 0.05 versus vehicle; #, p < 0.05 versus control; two-way analysis of variance with Tukey
post-hoc test). b, pretreatment with 100 µM INH2BP prevents nuclear localization of AIF at 22 h after 5 µM glutamate, 5 µM glycine.
c, pretreatment with 100 µM INH2BP reduced
PI+/Annexin V+ and PI+/Annexin V cells, but
increased PI /Annexin V+ cells, 24 h
after 5 µM glutamate, 5 µM glycine
versus vehicle (1 × 105 cells/condition;
representative of three independent experiments).
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Mitochondrial PARP-1 and Poly(ADP-ribosylation) in
Fibroblasts--
Similar to a previous report (17),
PARP-1
/
fibroblasts were less sensitive to
ONOO
-induced cytoxicity. The typical reductions in
cellular respiration (MTT), increases in lactate dehydrogenase
release, and cell death seen after ONOO
exposure were
seen in PARP+/+ but not PARP
/
cells (Fig.
7, a-c). These
events were prevented in the case of MTT and lactate dehydrogenase
release was determined 2 h after exposure to ONOO
,
and inhibited in the case of PI extrusion determined 22 h after exposure to ONOO
, by treatment with INH2BP. These data
are consistent with previous reports demonstrating that PARP inhibition
reduces necrotic cell death.

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Fig. 7.
PARP-1 inhibition protects fibroblasts from
oxidative/nitrosative stress. a, cellular respiration
(MTT) is reduced 2 h after exposure to 250 µM
ONOO in PARP-1+/+ cells, but not
PARP-1 / cells or PARP-1+/+ cells pretreated
with 100 µM INH2BP (n = 6/ group;
mean ± S.D.; *, p < 0.05 versus
control, t test). b, lactate dehydrogenase
(LDH) release is increased 2 h after exposure to 250 µM ONOO in PARP-1+/+ cells, but
not PARP-1 / cells or PARP-1+/+ cells
pretreated with 100 µM INH2BP (n = 6/group; mean ± S.D.; *, p < 0.05 versus control, t test). c,
PI+ and Annexin V+ cell profiles are increased
22 h after exposure to 250 µM ONOO in
PARP-1+/+ (WT, wild-type) cells, but not
PARP-1 / (KO, knockout) or
PARP-1+/+ cells pretreated with 100 µM INH2BP
(1 × 105 cells/condition).
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PARP-1 Expression and Proteolysis in Neurons and Fibroblasts
Exposed to Peroxynitrite--
Because PARP-1 cleavage is a feature of
caspase-mediated apoptosis (23), PARP-1 expression and proteolysis were
examined in nuclear and mitochondrial protein fractions. In
fibroblasts, changes in PARP-1 expression were not seen after exposure
to 250 µM ONOO
in either the nuclear or
mitochondrial compartments (Fig.
8a). As expected, PARP-1 was
not detected in PARP-1
/
cells. PARP-1 proteolysis was
not detected after exposure to 250 µM ONOO
,
consistent with necrotic, caspase-independent cell death (Fig. 7). In
primary cortical neurons, baseline PARP-1 proteolysis was detected in
both the mitochondrial and nuclear compartment, consistent with either
apoptosis in dead glial elements typical of neuron-enriched cultures,
or some degree of baseline apoptotic neuronal death (Fig.
8b). After exposure to 250 µM
ONOO
, PARP-1 cleavage did not appear to be altered, with
the exception of the 24-h time point, where a reduction in both PARP-1
and cleaved PARP-1 was seen in both nuclear and mitochondrial
fractions, reflecting ONOO
-induced cell death. Treatment
with INH2BP reduced PARP-1 proteolysis induced by ONOO
in
the mitochondrial, and to a lesser degree the nuclear compartment. These data suggest that INH2BP reduces caspase-mediated proteolysis of
PARP-1 in both compartments, and are consistent with some degree of
caspase-dependent apoptotic cell death in this paradigm
(Fig. 4).

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Fig. 8.
Effect of ONOO on PARP-1
expression and proteolysis in fibroblasts and primary cortical
neurons. a, in fibroblasts, changes in PARP-1
expression were not seen after exposure to 250 µM
ONOO in either the nuclear or mitochondrial compartments.
PARP-1 was not detected in PARP-1 / cells. b,
in neurons, baseline PARP-1 proteolysis was detected in both the
mitochondrial and nuclear compartment. After exposure to 250 µM ONOO , PARP-1 cleavage was not altered.
Treatment with 100 µM INH2BP reduced PARP-1 proteolysis
induced by ONOO in the mitochondrial, and to a lesser
degree the nuclear compartment. The reduction in PARP-1 and cleaved
PARP-1 at 24 h in vehicle-treated cells exposed to
ONOO likely represents cell death.
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DISCUSSION |
Whereas poly(ADP-ribosylation) contributes to cell homeostasis
under basal conditions, pharmacological or genetic inhibition of PARP
during conditions of cellular stress, e.g. energy failure or
oxidative stress, is beneficial (2, 3, 6). Under these circumstances,
it has been proposed that ONOO
produces genomic DNA
damage activating nuclear PARP-1, with subsequent cellular
NAD+ depletion, followed by secondary mitochondrial injury,
AIF release and nuclear translocation, and cell death (4). However, our data suggest that oxidative stress produces mitochondrial dysfunction directly by promoting rapid loss of 
m. Intramitochondrial PARP activity during conditions of limited mitochondrial
NAD+ stores related to impaired NAD recycling would
exacerbate energy failure by consuming mitochondrial NAD+
directly. In support of this, inhibition of basal mitochondrial poly(ADP-ribosylation) preserves 
m, NAD+
levels, and cellular respiration, prevents mitochondrial release of
AIF, and attenuates large-scale DNA fragmentation and cell death after
oxidative stress. Furthermore, replenishment of NAD+ also
preserves 
m and cellular respiration. These data indicate
that inhibition of mitochondrial poly(ADP-ribosylation) and energy
repletion represent effective strategies to protect cells in the face
of insults producing energy failure, and are consistent with the
presumption that energy depletion contributes to PARP-related
cytotoxicity. PARP inhibition has also been shown to directly protect
electron transport chain complexes from inactivation induced by
oxidative stress (24). Similarly, NO inhibits mitochondrial metabolism (25).
Compartmentalization of active PARP, NAD+, and DNA damage
within mitochondria (7, 8, 11, 26-28) explain the rapid beneficial effects of PARP inhibition after exposure to extramitochondrial ONOO
. In addition, compartmentalization of nitric-oxide
synthase and oxygen radicals within mitochondria are a potential source
of internally generated ONOO
(29, 30). Because
mitochondrial nitric-oxide synthase is calcium-dependent,
elevations in [Ca2+]i occurring under conditions
of excitotoxicity could result in both activation of cytosolic and
mitochondrial nitric-oxide synthase providing two sources of
ONOO
(31, 32). The results of this study do not discount
the importance of nuclear PARP-1 activation in terms of cellular
NAD+ consumption; however, it is apparent that
mitochondrial poly(ADP-ribosylation) contributes at least in part to
the determination of cellular fate under circumstances of
NAD+ depletion and energy failure.
Traditionally, PARP-mediated cell death has been felt to be primarily
necrotic in nature (1-3, 5, 6). Recently, however, PARP-regulated
programmed cell death mediated by AIF has been identified (4).
AIF-mediated programmed cell death has many phenotypic features of
developmental apoptosis such as DNA fragmentation, phosphatidylserine
exposure, and regulation by bcl-2 family proteins; however, important
differences also exist, such as large-scale versus
oligonucleosomal DNA fragmentation and cell death that progresses
despite caspase inhibition (33-36). Both necrotic and AIF-mediated
programmed cell death are felt to be caspase-independent, and we have
previously reported that the caspase inhibitors
N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (VAD) and boc-Asp(OMe)-fluromethylketone (BAF) do not protect neurons
from AIF-mediated cell death induced by ONOO
(14). In
contrast, the present data show that the PARP inhibitor INH2BP protects
cells from AIF-mediated cell death induced by ONOO
or
glutamate. Whereas INH2BP reduced apoptotic cell death after ONOO
(PI extrusion and Annexin V binding; Fig.
4c), after glutamate exposure cell death was reduced (PI
extrusion; Fig. 6c) but a pre-apoptotic phenotype was
expressed (PI
/Annexin V binding) in neurons. The concept
that preventing apoptosis may switch the mode of cell death to necrosis
is not novel (37); however, these data suggest the possibility that
preventing necrosis may switch the mode of cell death to apoptosis.
This concept, particularly in paradigms that conserve cellular energy
stores (38, 39), warrants further study. PARP inhibition also reduced necrosis in fibroblasts exposed to ONOO
, demonstrating
differences not only related to the cytotoxicity paradigm chosen, but
also in the cell type used. Differences in response to
ONOO
between fibroblasts and neurons may be related to
dividing versus non-dividing cells, immortalized
versus primary cell cultures, mitochondrial density, or
other factors. Nonetheless, taken together these data are consistent
with a role for mitochondrial poly(ADP-ribosylation) in
caspase-independent cell death.
Whereas activation of PARP during times where cellular energy stores
are limited is detrimental (2, 3, 6), it is important to remember that
poly(ADP-ribosylation) serves many homeostatic functions as well. In
addition to facilitating both genomic and mitochondrial DNA repair (11,
40, 41), poly(ADP-ribosylation) also serves a regulatory role for many
transcription factors including nuclear factor-
B, AP-1, and Stat-1
(42), and also may regulate transcription itself via loosening
chromatin (43). Baseline poly(ADP-ribosylation) within both nuclear and
mitochondrial compartments as demonstrated in this study, support such
homeostatic functions in cells. In neurons, poly(ADP-ribosylation) may
also participate in long-term potentiation (44, 45) and memory
formation (46). In dividing cells such as fibroblasts,
poly(ADP-ribosylation) via PARP-2 regulates mitosis (47). Collectively
these data suggest that poly(ADP-ribosylation) compartmentalized to
both mitochondria and nuclei can be converted from a homeostatic
process to a mechanism of cell death when oxidative stress is
accompanied by energy depletion.
A direct role for mitochondrial poly(ADP-ribosylation) in cell death
associated with loss of 
m, mitochondrial dysfunction, and
release of AIF is implicated. These data do not refute previous hypotheses suggesting that overactivation of nuclear PARP-1 consumes total cellular energy stores contributing to mitochondrial dysfunction and cell death. However, these data do represent a paradigm shift, where poly(ADP-ribosylation) compartmentalized within the mitochondria contributes to AIF release and cell death in the face of cellular energy failure.