Poly(ADP-ribose) (PAR) polymer is a death signal

Hurn, Guy G. Poirier, Valina L. Dawson, and Ted M. Dawson Sasaki, Judith A. Klaus, Takashi Otsuka, Zhizheng Zhang, Raymond C. Koehler, Patricia D.

Shaida A. Andrabi, No Soo Kim, Seong-Woon Yu, Hongmin Wang, David W. Koh, Masayuki

doi:10.1073/pnas.0606526103 published online Nov 20, 2006; PNAS

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Poly(ADP-ribose) (PAR) polymer is a death signalShaida A. Andrabi*†, No Soo Kim*†, Seong-Woon Yu*†‡, Hongmin Wang*†§, David W. Koh*†, Masayuki Sasaki*†,Judith A. Klaus¶, Takashi Otsuka¶, Zhizheng Zhang¶�, Raymond C. Koehler¶, Patricia D. Hurn¶�, Guy G. Poirier**,Valina L. Dawson*†,††‡‡§§, and Ted M. Dawson*†,††§§

*Institute for Cell Engineering, Departments of †Neurology, ††Neuroscience, ‡‡Physiology, and ¶Anesthesiology�Critical Care Medicine, Johns HopkinsUniversity School of Medicine, Baltimore, MD 21205; and **Health and Environment Unit, Laval University Medical Research Center, Centre HospitalierUniversitaire de Quebec, Ste-Foy, QC, Canada G1V 4G2

Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved September 28, 2006 (received for reviewJuly 31, 2006)

Excessive activation of the nuclear enzyme, poly(ADP-ribose) poly-merase-1 (PARP-1) plays a prominent role in various of models ofcellular injury. Here, we identify poly(ADP-ribose) (PAR) polymer,a product of PARP-1 activity, as a previously uncharacterized celldeath signal. PAR polymer is directly toxic to neurons, and degra-dation of PAR polymer by poly(ADP-ribose) glycohydrolase (PARG)or phosphodiesterase 1 prevents PAR polymer-induced cell death.PARP-1-dependent, NMDA excitotoxicity of cortical neurons isreduced by neutralizing antibodies to PAR and by overexpressionof PARG. Neuronal cultures with reduced levels of PARG are moresensitive to NMDA excitotoxicity than WT cultures. Transgenic miceoverexpressing PARG have significantly reduced infarct volumesafter focal ischemia. Conversely, mice with reduced levels of PARGhave significantly increased infarct volumes after focal ischemiacompared with WT littermate controls. These results reveal PARpolymer as a signaling molecule that induces cell death andsuggests that interference with PAR polymer signaling may offerinnovative therapeutic approaches for the treatment of cellularinjury.

excitotoxicity � poly(ADP-ribose) glycohydrolase � poly(ADP-ribose)polymerase � stroke

Poly(ADP-ribose) polymerase-1 (PARP-1) is an abundant nu-clear protein that is involved in the DNA base excision repair

system, where it is potently activated by DNA strand nicks andbreaks (1, 2). Using NAD� as a substrate, PARP-1 builds uphomopolymers of ADP ribose units on various nuclear proteinsincluding histones, DNA polymerases, topoisomerases, DNA li-gase-2, transcription factors (3, 4), and PARP-1 itself (5, 6).Although the exact physiologic function of PARP-1 is not com-pletely understood, in some tissues it plays an important role inDNA repair and genomic stability (5, 7, 8). Poly(ADP-ribose)(PAR) catabolism and metabolism is a dynamic process, with PARglycohydrolase (PARG) playing the major role in the degradationof the polymer (9).

Recent studies using pharmacologic inhibition of PARP orgenetic KO of PARP-1 indicate that PARP-1 plays a dramatic andsignificant role in cellular injury after stroke, trauma, ischemia–reperfusion of the heart, spleen, skeletal muscle, and retina, ar-thritis, �-islet cytotoxicity�diabetes mellitus, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of Parkinson’s disease,experimental autoimmune encephalomyelitis (EAE) model of mul-tiple sclerosis, endotoxic shock, multiple-system organ failure, andliver damage (for review, see refs. 1 and 10). PARP-1 activation alsoplays a prominent role in NMDA excitotoxicity, because PARP-1KO mice are remarkably resistant both in vitro and in vivo to theexcitotoxic effects of glutamate and NMDA (11, 12). A cell-suicidehypothesis has been proposed (1, 2, 13, 14) to explain the actionsof PARP-1 in mediating cell death. However, studies in micelacking PARG suggest that PAR polymer formed during theactivation of PARP-1 might play a role in PARP-1-dependent celldeath. PARG KO mice die at embryonic day 3.5 because of thefailure to hydrolyze PAR polymer and its subsequent accumulation(15). Moreover, the loss of PARG in Drosophila melanogaster leads

to lethality in the larval stage at a normal developmental temper-ature (25°C) and progressive neurodegeneration with reducedlocomotor activity and a short lifespan at 29°C because of theaccumulation of PAR polymer (16). Here, we explore the role ofPAR polymer in PARP-1-dependent cell death and report thatPAR polymer is a cell-death signal that plays an important rolein PARP-1-dependent cell death.

ResultsPAR Polymer Is Toxic. An extensive amount of PAR polymer isformed, and it remains significantly elevated for a prolonged periodafter a variety of toxic insults in which PARP-1 activation plays aprominent role, (12, 17–19). To determine whether PAR polymeris sufficient to induce neuronal cell death, viability was monitoredby Hoechst 33342 and propidium iodide (PI) staining by usingquantitative computer-assisted counting of viable and dead neurons(20) after BioPorter-mediated delivery of PAR polymer. BioPorterreagent is a cationic lipid formulation TFA-DODAP:DOPE thatenables delivery of recombinant proteins, peptides, or antibodiesinto viable cells (21). The PAR polymer was synthesized by in vitroautomodification of PARP-1 and has a mean length of 40 ADP-ribose residues, as determined by HPLC methods and gel electro-phoresis (22), and, accordingly, the concentration of PAR is givenas a function of polymer molecules with a mean size of 40ADP-ribose units. The range of size of PAR in this mix is 6- through100-mer ADP-ribose units (22, 23). PAR polymer is effectivelydelivered into cortical neurons by the BioPorter reagent, as re-vealed by using PAR polymer antibody and confocal imaging. PARpolymer is observed in the majority of neurons (Fig. 5, which ispublished as supporting information on the PNAS web site). Underthese conditions, PAR polymer induces �50% cell death in corticalneurons, which is prevented by pretreatment of the PAR polymerwith PARG or phosphodiesterase 1 (PD1), which degrade PAR

Author contributions: S.A.A. and N.S.K. contributed equally to this work; S.A.A., N.S.K.,S.-W.Y., H.W., R.C.K., P.D.H., G.G.P., V.L.D., and T.M.D. designed research; S.A.A., N.S.K.,S.-W.Y., H.W., D.W.K., M.S., J.A.K., T.O., and Z.Z. performed research; G.G.P. contributednew reagents�analytic tools; S.A.A., N.S.K., S.-W.Y., H.W., D.W.K., R.C.K., P.D.H., V.L.D., andT.M.D. analyzed data; and S.A.A., N.S.K., S.-W.Y., V.L.D., and T.M.D. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS direct submission.

Abbreviations: DIV, days in vitro; MCAO, middle cerebral artery occlusion; MNNG,N-methyl-N-nitro-N-nitrosoguanidine; PAR, poly(ADP-ribose); PARG, PAR glycohydrolase;PARP-1, PAR polymerase-1; PD1, phosphodiesterase 1; poly(A), poly (adenine); Tg,transgenic.

‡Present address: Department of Neurology, Pharmacology, and Toxicology, B-436 LifeScience Building, Michigan State University, East Lansing, MI 48824.

§Present address: Medical Biotechnology Center, Room N355, UMBI Building, University ofMaryland, 725 West Lombard Street, Baltimore, MD 21201.

�Present address: Department of Anesthesiology and Perioperative Medicine, OregonHealth & Science University, UHS-2, 3181 Southwest Sam Jackson Park Road, Portland, OR97239.

§§To whom correspondence may be addressed at: Institute for Cell Engineering, JohnsHopkins University School of Medicine, 733 North Broadway, Suite 731, Baltimore, MD21205. E-mail: tdawson@jhmi.edu or vdawson@jhmi.edu.

© 2006 by The National Academy of Sciences of the USA

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polymers [Fig. 1B (24, 25)] (Fig. 1 A and C). Because PAR polymeris a highly negatively charged molecule, the same concentration ofpoly (adenine) [poly(A)], also negatively charged, was applied byBioPorter to cortical neurons. BioPorter-mediated delivery ofpoly(A) fails to elicit neuronal cell death (Fig. 1 A and C). Todetermine whether PAR polymer induces cell death in the absenceof PARP-1, PAR polymer was delivered to PARP-1 KO corticalneurons, and cell death was monitored (Fig. 1D). PAR polymerdelivery induces cell death in PARP-1 KO cortical neurons that isattenuated by pretreatment with PARG (Fig. 1D). These resultstaken together indicate that PAR polymer is toxic and that PARpolymer can act as a death signal in PARP-1 KO cultures.

High-Molecular-Weight PAR Polymers Are Toxic. These latter studieswere performed with a mixture of PAR polymers of different sizeand complexity. To determine the nature of the PAR polymer thatmediates cell death, the ability of isolated PAR polymers to inducecell death was evaluated by using PAR polymers of different sizeand complexity, ranging in length from 16 to �60 ADP-riboseresidues. The DEAENPR fractions, as defined by Kiehlbauch andJacobson (23), were used for these studies. Increasing length andcomplexity of PAR polymers leads to increased cell death, withcomplex polymers �60 ADP-ribose units inducing �80% cell deathat a concentration of 80 nM polymer (Fig. 2A). At an equivalentpolymer concentration, polymers of 16 ADP-ribose residues elicita small amount of cell death, and polymers of 30 ADP-riboseresidues induce modest cell death (Fig. 2A). Thus, consistent withformation of high-molecular-weight complex polymers afterPARP-1-dependent cell death (12, 18, 19), PAR polymers ofincreasing complexity and molecular weight are more toxic. A

dose–response relationship of complex polymers �60 ADP-riboseunits on cortical neuron cell death was performed. Concentrationsas low as 20 nM PAR polymer induce cell death, and cell deathincreases with increasing concentration of PAR polymer (Fig. 2B).These concentrations, length, and complexity of PAR polymers arewithin the range of polymer concentrations and size found in intactcells during NMDA excitotoxicity (12, 18, 19) (see Fig. 4), andPARP-1-dependent [N-methyl-N-nitro-N-nitrosoguanidine(MNNG)] HeLa cell toxicity (data not shown). Thus, it is likely thatcomplex and high-molecular-weight PAR polymers act, in part, toplay a role of PARP-1 in cell death.

PAR Polymer-Induced Cell Death Is Caspase-Independent. PARP-1-dependent cell death is, in part, caspase-independent (18, 19). Todetermine whether PAR polymer-induced cell death is caspase-independent, we monitored cell death of cortical neurons afterBioPorter-mediated delivery of PAR polymer in the presence andabsence of the broad-spectrum caspase inhibitor Z-VAD.fmk.Z-VAD fails to prevent PAR polymer-induced cell death (Fig. 2 Cand D). PAR polymer also fails to directly activate caspases (datanot shown). To ascertain whether these findings extend to nonneu-ronal cells and whether PAR polymer death signaling occursoutside the nervous system, we evaluated PAR toxicity in HeLacells. Similar to neurons, complex polymers, �60 ADP-ribose units,cause HeLa cell death, with 70% cell death at a PAR polymerconcentration of 80 nM (Fig. 6A, which is published as supportinginformation on the PNAS web site). Z-VAD also fails to preventPAR polymer-induced cell death in HeLa cells (Fig. 6B). Takentogether, these results indicate that PAR polymer induces cell deathin a concentration-dependent and caspase-independent manner.

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Fig. 1. PAR polymer induces neuronal cell death. (A) Representative fluo-rescent microscopic images of PI (red) and Hoechst (blue) staining aftertreatment of neurons with BioPorter-mediated delivery of PAR polymer, PARpolymer � PARG, PAR polymer � PD1, and poly(A). (B) Silver staining ofpurified PAR polymer, showing degradation of PAR polymer after incubationwith PARG (PAR�PARG) and PD1 (PAR�PD1). These experiments (A and B)have been replicated in separate experiments at least three times with similarresults. (C) Quantitative analysis of Hoechst- and PI-stained WT neurons afterBioPorter-mediated delivery of PAR polymer, PAR polymer � PARG, PARpolymer � PD1, and poly(A). Purified PAR, cleaved PAR by either PARG or PD1pretreatment, or synthesized poly(A) was diluted with PBS, mixed with Bio-Porter reagent, and added to neuronal cultures at a final concentration of 80nM in serum-free MEM. The neurons were cultured for 3 h to allow thepolymers to be imported into neurons and then subjected to fluorescentmicroscopy and quantitative computer-assisted cell counting after Hoechstand PI double staining 24 h later. Data are the mean � SD, n � 3; *, P � 0.05.(D) PAR polymer can also induce cell death in PARP-1 KO neurons. PAR polymer(80 nM) and cleaved PAR (80 nM PAR polymer � PARG) was delivered intoneurons by BioPorter-mediated delivery, and cell death was analyzed byHoechst and PI staining. Data are the mean � SD, n � 3; *, P � 0.05.

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Fig. 2. PAR polymer toxicity is length- and dose-dependent and caspase-independent. (A) Purified PAR polymer was fractionated into fractions ofvarying polymer length. Different PAR polymer fractions with average poly-mer size (16, 30, and 60 mer) were delivered into the cortical neurons by theBioPorter delivery reagent at a final concentration of 80 nM. The mostabundant fraction containing PAR polymer of 40–100 (average polymer size60 mer) ADP-ribose residues induces robust neuronal cell death. Neurons weretreated with BioPorter alone and serve as a control. Data are the mean � SEM,n � 6; *, P � 0.05. (B) PAR polymer-induced neuronal death is dose-dependent.Different concentrations of PAR polymer (20–160 nM) were administered tocortical neurons by the BioPorter reagent, and cell death was assessed byHoechst and PI staining. Data are the mean � SD, n � 4; *, P � 0.05. (C)Representative pictures of PI- (red) and Hoechst- (blue) stained neurons weretreated with BioPorter alone (Control), purified PAR polymer (PAR), or puri-fied PAR polymer in the presence of z-VAD (z-VAD � PAR), by using BioPorterreagent as a delivery system. PI-positive cells were considered dead cells. (D)Quantification of the PAR polymer-mediated cell death in the presence ofz-VAD. z-VAD is unable to protect against PAR polymer-mediated cell deathin cortical neurons. BioPorter alone serves as a control. Cell death was assessedby Hoechst and PI staining. Data are the mean � SEM, n � 6; *, P � 0.05.

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PAR Polymer Mediates PARP-1-Dependent Cell Death. To determinewhat role PAR polymer formation might play in NMDA excito-toxicity, a pathophysiologically relevant form of PARP-1-dependent cell death, a neutralizing polyclonal antiserum to PARpolymer was used (Fig. 3). Anti-PAR antiserum is capable ofimmunodepleting both purified PAR polymer and PARP-1-activated nuclear supernatant containing endogenous PAR poly-mer [SN(PAR)], as assessed by dot–blot analysis and Western blotanalysis, respectively (Fig. 3A). Anti-PAR antiserum was deliveredinto cortical neurons by the BioPorter reagent before NMDAtreatment. With this protein-delivery system, �95% of the neuronsreceive anti-PAR antiserum, as indicated by the presence of afluorescent marker (data not shown), which was coadministeredwith the antiserum. BioPorter-mediated delivery of anti-PARantiserum reduces NMDA excitotoxicity, whereas BioPorter-mediated delivery of an equivalent concentration of preimmuneserum or normal rabbit serum fails to protect neurons fromNMDA-induced cell death (Fig. 3B). Similar neuroprotectionagainst NMDA toxicity was obtained with a different anti-PARantiserum (data not shown). To further evaluate the role of PARpolymer in NMDA-induced excitotoxicity, a PARG adenovirus wasconstructed by using the exon-1-deleted form of PARG, which isexclusively localized to the cytoplasm (Av PARG WT) (26). Inaddition, we constructed a catalytically inactive exon-1-deletedmutant PARG adenovirus (Av PARG Mut) in which two criticalglutamates at residues 756 and 757 were mutated to alanines (27).Adenoviral-mediated overexpression of WT and mutant PARGwas assessed by Western blot analysis, and we observe a several-foldincrease in the level of both WT and mutant exon-1-deleted PARG(Fig. 3C). Adenoviral-mediated overexpression of WT cytosolicPARG leads to a reduction of cytosolic PAR polymer (Fig. 3D) and

an �50% reduction of NMDA-induced neuronal cell death (Fig.3E), whereas overexpression of the catalytically inactive mutantPARG fails to reduce cytosolic PAR polymer levels (Fig. 3D) andNMDA toxicity (Fig. 3E). To control for nonspecific effects ofadenoviral-mediated gene expression, adenoviral-mediated over-expression of GFP was evaluated and was found to have no effecton NMDA excitotoxicity (Fig. 3E).

To determine whether the anti-PAR antibody and adenoviral-mediated overexpression of WT cytosolic PARG could blockagainst other forms of PARP-1-dependent cell death in nonneu-ronal cells, we assessed the ability of the neutralizing PAR anti-serum and adenoviral-mediated overexpression of WT cytosolicPARG to block PARP-1-dependent cell death induced by MNNGin HeLa cells. HeLa cells die in a PARP-1-dependent�caspaseindependent manner after MNNG treatment, because treatment ofHeLa cells with 50 �M MNNG for 15 min leads to cell death thatis blocked by the PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ) (30 �M), but the pancaspaseinhibitor z-VAD (100 �M) has no effect (Fig. 7A, which is publishedas supporting information on the PNAS web site). BioPorter-mediated delivery of anti-PAR antibody reduces MNNG cell death,whereas a preimmune serum and normal rabbit serum have noeffect and adenoviral-mediated overexpression of WT cytosolicPARG reduces MNNG induced cell death, whereas control virushas no effect (Fig. 7B). The residual toxicity observed with reducingPAR polymer levels in neurons and HeLa cells may be due toalternative pathways of cell death or residual PAR polymer, be-cause our methods of depleting PAR polymer are not complete.Consistent with this notion is our observation that concentrationsof PAR polymer as low as 20 nM are still toxic (see Fig. 2B).

To ascertain whether reducing the level of PARG leads to

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Fig. 3. Interfering with PAR signaling reduces NMDA-induced neuronal cell death. (A) Immunodepletion of purified PAR polymer (Upper) and SN(PAR) (Lower)by anti-PAR antibody. Supernatants after immunoprecipitation have reduced level of PAR polymers. NRS, normal rabbit serum; Pre-Im, preimmune serum; �-Par,anti-Par antibody. (B) Pretreatment with neutralizing PAR antiserum reduces NMDA-induced neuronal cell death. PAR antiserum, normal rabbit serum, orpreimmune serum was delivered into cortical neurons with the BioPorter reagent. At 3 h after delivery, the neurons were treated with NMDA (500 �M for 5 min).Cell death was assessed 18–24 h later by Hoechst and PI staining. Data are the mean � SEM, n � 6–8; *, P � 0.05. (C) Cortical neurons were infected with a cytosolicform (exon-1 deleted) PARG adenoviral construct (Av PARG WT) or catalytically inactive (Av PARG Mut) virus, and, after 48 h, the cells were harvested in lysisbuffer containing protease inhibitors. Because of low basal levels of PARG, nondetectable levels of PARG are seen in adeno-GFP- and PBS-treated cultures,whereas high levels of PARG expression are seen in cultures that are treated with Av PARG WT or Av PARG Mut virus. (D) Overexpression of cytosolic, WT PARGdecreases PAR levels in cortical neurons after NMDA treatment. Neurons [11 days in vitro (DIV)] were infected with an exon 1-deleted, cytosolic WT (Av PARGWT) and mutant (Av PARG mut) PARG adenoviral constructs, or a GFP-adenoviral control construct (Av GFP). After 48 h, the transduced cultures were treatedwith NMDA (500 �M for 5 min), and neurons were harvested at indicated time points and subjected to Western blotting analysis with anti-PAR antibody. Theseexperiments (A, C, and D) have been replicated in separate experiments at least three times with similar results. (E) Overexpression of PARG reducesNMDA-induced neuronal cell death. Neurons (12 DIV) were infected with a Av PARG WT or catalytically inactive Av PARG Mut virus, GFP-adenoviral controlconstruct (Adeno-GFP), or no-adenovirus (PBS). After 48 h, the neurons were treated with NMDA (500 �M for 5 min), and 18–24 h after NMDA administration,cells were stained with Hoechst and PI to assess cell death. Data are the mean � SEM, n � 6; *, P � 0.05.

18310 � www.pnas.org�cgi�doi�10.1073�pnas.0606526103 Andrabi et al.

enhanced PARP-1-dependent cell death, we evaluated NMDAexcitotoxicity in mice lacking one copy of the PARG gene. PARGKO mice die at embryonic day 3.5, but the heterozygotes are viable(15). We first determined whether there is increased PAR polymerformation in PARG�/� cortical neurons compared with WT cor-tical neurons after a toxic dose of NMDA (Fig. 4 A and B).PARG�/� cortical neurons have enhanced PAR polymer forma-tion after NMDA administration, as assessed by Western blotanalysis with an anti-PAR antibody (Fig. 4A). Quantitation of theamount of PAR polymer formed in WT cultures indicates that �80nM of PAR polymer is detected at 60 min after NMDA adminis-tration (Fig. 4B). There is almost a 2-fold increase in PAR polymerin PARG�/� mice cortical neurons compared with WT corticalneurons (Fig. 4B). We next examined neuronal cell death and foundthat there is an �20% increase in cell death after NMDA admin-istration in PARG�/� cortical neurons compared with WT corticalneurons (Fig. 4C). The 20% increase in cell death in PARG�/�

cortical neurons after NMDA toxicity is essentially identical to thepercentage increase in cell death after a comparable increase of theconcentration of BioPorter-mediated delivery of purified PARpolymer (see Fig. 2B).

To determine whether reducing PARG levels in HeLa cellsincreases their susceptibility to PARP-1-dependent cell death,HeLa cells were treated with an siRNA to PARG (Fig. 8A, whichis published as supporting information on the PNAS web site).siRNA treatment of HeLa cells leads to a significant reduction inPARG catalytic activity, as assessed by a PARG activity zymogramactivity assay (Fig. 8A), and protein, as assessed by Western blotanalysis (Fig. 8A). PARG siRNA leads to enhanced PAR polymer

formation after MNNG treatment (Fig. 8B). In mock and scram-bled siRNA-transfected cells, 50 �M MNNG for 15 min inducestransient PAR polymer formation (Fig. 8B). PARG siRNA treat-ment enhances the amount of PAR polymer detected after MNNGtreatment of HeLa cells (Fig. 8B). PAR polymer accumulation issubstantially enhanced after PARG siRNA treatment but eventu-ally is degraded by the residual PARG activity (Fig. 8B). Underconditions in which PARG siRNA reduces PARG activity, weassessed MNNG toxicity in HeLa cells (Fig. 8C). MNNG (50 �M)for 15 min leads to killing of �50% of the HeLa cells, and PARGsiRNA treatment enhances MNNG-induced cell death by 30%(Fig. 8B). Taken together, these data generated from different anddiverse experiments are consistent with PAR polymer acting as asignal that initiates the cell-death cascade after PARP-1-dependentcell death.

PAR Polymer Mediates Neuronal Injury in Vivo. To evaluate whetherPAR polymer formation plays a role in the susceptibility of neuronsto a pathophysiologically important insult in vivo, PARG transgenicmice overexpressing WT PARG under the direction of the mouseprion promoter in which PARG message and protein are overex-pressed (Fig. 4D) and PARG�/� mice, which have reduced levelsof PARG protein (Fig. 4F), were subjected to transient middlecerebral artery occlusion (MCAO) by the intralumenal filamenttechnique as described by Goto et al. (28). The effect of overex-pression of PARG on focal ischemic injury was tested by comparinginfarct volume after 120 min of transient MCAO in PARG trans-genic (Tg) mice and WT littermates. For the PARG transgenicstudies we used aged-matched littermate controls from crosses

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Fig. 4. Enhanced PAR polymer formation and toxic-ity in PARG�/� mice, and PAR polymer mediates PARP-1-dependent cell death in vivo. (A) Western blotsshowing PAR polymer formation in mouse corticalneurons prepared from PARG�/� mice and their WTlittermates. The neurons were subjected to NMDAstimulation for 5 min on DIV 14. Samples were har-vested 1 h after NMDA stimulation and probed forPAR polymer by using anti-PAR antibody. PARG�/�

neurons that express reduced levels of PARG accumu-late more PAR polymer than their WT littermates.PARG�/� neurons also have higher basal PAR polymerlevels. The same blot was stripped and probed for�-tubulin, serving as a loading control. This experi-ment has been replicated in separate experiments atleast three times with similar results. (B) Cortical neu-rons from PARG�/� KO mice and WT littermate cul-tures were treated with 500 �M NMDA for 5 min onDIV 14, and intracellular PAR polymer levels weredetermined at the indicated times points after NMDAadministration by using an ELISA as described in Ma-terials and Methods. Data are the mean � SD, n � 4;

*, P � 0.05. (C) Cortical neurons from PARG�/� KO miceand WT littermate cultures were subjected to NMDA-stimulation (500 �M for 5 min) on DIV 14. PARG�/�

neurons that have reduced expression of PARG aremore sensitive to NMDA toxicity than their WT litter-mates. Control cultures were treated with control saltsolution (CSS) alone. Data are the mean � SD, n � 6; *,P � 0.05. (D) PARG Tg mice overexpress PARG. (a)Expression of the PARG gene is under the control ofthe PrP promoter. (b) Northern blot analysis for PARGin WT vs. PARG Tg mice. �-actin was used as a loadingcontrol. (c) Immunoblotting of mouse PARG in whole-brain and testis homogenates of transgenic mice ex-pressing the PARG and WT control mice. �-tubulin was used as a loading control. (E) Infarct volume (�SEM) in cerebral cortex, striatum, and total hemisphere(expressed as a percent of contralateral structure after correction for swelling) in 10 WT and 11 PARG Tg mice subjected to 120 min of MCAO. *, P � 0.05 betweengroups. (F) Decreased levels of PARG protein in PARG�/� mice. Immunoblot analysis of full-length (110-kDa) PARG protein levels in brain and testis extracts fromPARG�/� and PARG�/� mice. These results have been replicated at least two separate times with similar results. (G) Infarct volume (� SEM) in cerebral cortex,striatum, and total hemisphere (expressed as a percent of contralateral structure after correction for swelling) in 15 WT and 16 PARG�/� mice subjected to 90min of MCAO. *, P � 0.05 between groups.

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between PARG heterozygote Tg and WT mice; thus, all mice usedin these studies should have similar genetic backgrounds. Inductionof MCAO reduced cortical perfusion in the MCA territory mea-sured by laser-Doppler flowmetry (LDF) to an equivalent extent inWT (15 � 7% of baseline; n � 10) and PARG Tg (18 � 5%; n �11) mice. Rectal temperature was similar between groups at 10 minof MCAO (WT 36.9 � 0.5°C vs. PARG Tg 37.0 � 0.6°C) and at theend of MCAO (WT 36.2 � 0.1°C vs. PARG Tg 36.2 � 0.2°C). Theneurologic-deficit score was similar in WT (2.4 � 0.3) and PARGTg (2.8 � 0.3) during MCAO. After transient MCAO, overallinfarct volume in the hemisphere of PARG Tg mice is significantlyreduced, by 62%, compared with WT littermates (Fig. 4E). On aregional basis, infarct volume is reduced by 52% in striatum and by59% in cerebral cortex of PARG Tg mice. The effect of decreasingPARG expression (Fig. 4F) on focal ischemic injury was next testedon PARG�/� mice. For the PARG�/� studies, we used aged-matched littermate controls from crosses between PARG�/� mice;thus, all mice used in these studies should have similar geneticbackgrounds. The reduction in LDF during MCAO in PARG�/�

mice (18 � 7% of baseline; n � 15) was not different from that inWT littermates (16 � 6%; n � 16). Rectal temperature was similarbetween groups at 10 min of MCAO (WT 37.0 � 0.6°C vs.PARG�/� 37.3 � 0.3°C) and at the end of MCAO (WT 36.2 �0.2°C vs. PARG Tg 36.2 � 0.2°C). During MCAO, the neurologic-deficit scores were equivalent (WT 2.9 � 0.6 vs. PARG�/� 2.7 �0.4). After 90 min of transient MCAO, infarct volume is signifi-cantly increased, by 62%, in cerebral cortex, 56% in striatum, and56% in the entire hemisphere of PARG�/� mice compared withWT littermates (Fig. 4G). These results taken together suggest thatPAR polymer is the nuclear signal initiating the cell-death cascadeafter PARP-1-dependent cell death in vivo.

DiscussionThe major finding of this article is that PAR polymer acts as apreviously uncharacterized signaling molecule that induces celldeath after PARP-1 activation. Evidence supporting PAR polymeras a cell-death signaling molecule include the observations thatPAR polymer is directly toxic and that its toxicity is abolished bypretreatment of the PAR polymer with either PD1 or PARG, whichdegrade PAR polymer. Moreover, interfering with PAR polymersignaling through neutralizing PAR antibodies or PARG overex-pression reduces PARP-1-dependent NMDA excitotoxicity andMNNG-induced cell death. In addition, Tg mice overexpressingPARG have markedly reduced infarct volumes after 2 h of transientMCAO. Consistent with the notion that PAR polymer is toxic areour observations that PARG heterozygote KO cortical cultures aremore sensitive to NMDA excitotoxicity, siRNA knockdown ofPARG leads to enhanced MNNG toxicity, and PARG heterozy-gote KO mice have larger infarct volumes after 1.5 h of transientMCAO.

During PARP-1-dependent cell death, an extensive amount ofPAR polymer is formed through consumption of intracellularNAD� stores. Energy depletion was thought to be the primarymechanism by which excessive PARP-1 activation kills cells (10).Although we cannot exclude the possibility that decrements inNAD� contribute to cell death, because sustained energy depletionclearly can kill cells, the ability of neutralizing PAR antibodies andoverexpression of PARG to reduce NMDA excitotoxicity andMNNG toxicity, the reduction in focal ischemia-induced neuronalinjury by PARG overexpression and the direct toxicity of PARpolymer in energy-replete cells supports the idea that NAD�

decrements are not sufficient for PARP-1-dependent cell death.Consistent with this notion are studies showing that energy deple-tion alone may be an insufficient explanation for why PARP-1activation kills cells in vivo (28).

In summary, our results suggest that excessive activation ofPARP-1 leads to a unique intrinsic cell-death program and thatPAR polymer participates in PARP-1-dependent cell death. Inter-

fering with PAR actions may offer innovative therapeutic ap-proaches to treat cellular injury.

MethodsPrimary Cortical Cultures. Primary cortical neuron cultures wereprepared from gestational day 14–15 fetal mice as described (20),and, in mature cultures, neurons represent 80–90% of the totalnumber of cells (29).

PAR Polymer Synthesis and Purification. Purification and synthesisof PAR polymer were performed as described (23, 30)

BioPorter Protein-Delivery System. BioPorter reagent was purchasedfrom Gene Therapy Systems (San Diego, CA) (21). Antibodies,PAR polymer, or cleaved PAR polymer with either PD1 (Sigma, St.Louis, MO) or recombinant PARG was diluted to desired concen-tration with PBS. The diluted protein solution was added to thedried BioPorter reagent and allowed to rest at room temperaturefor 5 min, followed by gentle mixing. Serum-free medium wasadded to bring the BioPorter�PAR or BioPorter protein complexup to 250–300 �l. After a wash in serum-free media, the cellcultures were incubated with the BioPorter�PAR or BioPorter�protein complex for 3–4 h at 37°C. Cultures were subsequently usedfor experiments.

Cytotoxicity. Neurons (14 DIV) were exposed to PAR polymer orto NMDA (20). Viability was determined by computer-assisted cellcounting after staining of all nuclei with 7 �M Hoechst 33342(Invitrogen, Carlsbad, CA) and dead cell nuclei with 2 �M PI(Invitrogen). The numbers of total and dead cells were countedwith the Axiovision 4.3 software (Zeiss, Thornwood, NY).

Western Blotting. Cell lysates or subcellular fractions were size-separated through SDS�PAGE and processed for analysis by usinga Supersignal ChemiLuminiscence detection kit (Pierce, Rockford,IL) as described by the manufacturer (18). All primary antibodiesare previously characterized, including anti-PAR polyclonal anti-body (30) and rabbit anti-poly(ADP-ribose) glycohydrolase poly-clonal antibody (15).

Transduction of Cultured Neurons with Recombinant Adenovirus. Anexon-1-deleted WT PARG gene was subcloned into an adenoviralvector, and its expression was driven by a CMV promoter. To makea catalytically inactive mutant construct, two adjacent glutamicresidues at 756 and 757 were point-mutated to alanines, and theexon-1-deleted mutant PARG gene was cloned in the same adeno-viral vector as the exon-1-deleted WT gene. A GFP adenovirus wasused as a control. The viral constructs were diluted with MEMcontaining 2% serum and 21 mM glucose and added onto culturedneurons (12 DIV) or HeLa Cells. After 24 h, the medium waschanged into 10% serum-containing medium, and then the cellswere cultured for an additional day to allow PARG or GFPexpression. After 2 days, the cells were harvested for Western blotanalysis or subjected to NMDA administration. Cell death wasdetected after the insult by Hoechst 33342 and PI double stainingand computer-assisted quantification.

Isolation and ELISA of Intracellular PAR Polymer. Preparation andquantification of intracellular PAR polymer from cortical neuronswere performed as described (31). PAR polymer standards used inELISA were a complex form (average 60-mer ADP-ribose). Intra-cellular PAR polymer concentration was calculated based on theassumption that the diameter of neurons is 16.78 �m (32).

Focal Cerebral Ischemia. All procedures on mice were preapprovedby the Johns Hopkins University Animal Care and Use Committeeand conformed with the principles of laboratory animal research ofthe American Physiological Society. Transient MCAO was pro-

18312 � www.pnas.org�cgi�doi�10.1073�pnas.0606526103 Andrabi et al.

duced in mice by the intralumenal-filament technique as described(28). Mice were anesthetized with 5% halothane and maintained on1–1.5% halothane in O2-enriched air by using a face mask. Rectaltemperature was maintained at 36–37°C with a warming blanketand heating lamp. MCAO was produced by inserting a 6–0 nylonmonofilament, with the tip coated by cyanoacrylate glue, into theinternal carotid artery through a cut stump of the external carotidartery while the common carotid artery was occluded. A laserDoppler flowmetry probe placed on the skull over the lateralparietal cortex was used to verify MCAO over the first 10 min ofocclusion. Anesthesia was discontinued, and a neurological exam-ination was performed to verify functional deficits on a scale of 0–4:0 � no deficit; 1 � forelimb weakness; 2 � circling to affected side;3 � unable to bear weight on affected side; and 4 � no spontaneousmotor activity (33). To allow reperfusion, the mouse was brieflyanesthetized with halothane, and the filament was withdrawn. Thebrain was harvested 1 day after MCAO for analysis of infarctvolume by triphenyltetrazolium chloride staining of five coronalsections. Infarct size was expressed as a percent of the contralateralstructure after correcting for swelling (34).

Mouse Strains. PARP-1 KO and PARG KO mice have beenpreviously described. PARP-1 KOs are maintained on an outbredstrain of 129 SvEv, and 129 SvEv mice were used as controls (12).PARG KO mice are embryonic lethals and, for these studies,PARG�/� mice were crossed, and PARG�/� and age-matchedlittermate controls were used (15). PARG transgenic mice weregenerated by creating a transgenic expression construct containingthe cDNA of mouse PARG (gift from M. K. Jacobson, Universityof Arizona, Tucson, AZ) driven by the mouse PrP promoter(PrP-musPARG). A XhoI-SalI fragment containing mouse PARGcDNA was subcloned into the XhoI cloning site of MoPrP to yieldPrP-musPARG. The purified DNA fragment of PrP-musPARG wasmicroinjected into pronuclei of oocyte from a F1 hybrid of C57BL�

6J � SJL�J. The presence or absence of the transgene in theresulting animals and their progeny was determined by using tailDNA. The primers PrP-S � 5-CCTCTTTGTGACTATGTGGA-CTGATGTCGG-3, PrP-AS � 5-GTGGATACCCCCTCCCC-CAGCCTAGACC-3, and PARG3 � 5-CCAGCGCTATTTT-AGAAGCTGGCCAAGAGG-3 were used for PCR genotyping.The following PCR protocol was used: 94°C for 4.5 min, 30 cyclesof 94°C for 1.5 min, 72°C for 2 min, and a final extension at 72°Cfor 4 min.

Northern Blot Analysis. Total RNA was isolated with TRIzol (In-vitrogen) following the manufacturer’s instructions. Twenty micro-grams of total RNA was resolved in 1% agarose gel by using aNorthernMax-Gly kit (Ambion, Austin, TX) and transferred ontothe nylon membrane (Hybond-N; Amersham, Piscataway, NJ) andexposed to a phosphor screen (Perkin Elmer, Meriden, CT) over-night, and the screen was scanned with the cyclone imaging systemas described (15).

Immunohistochemistry and HeLa Cell Experiments. See SupportingMethods, which is published as supporting information on thePNAS web site.

Statistical Analysis. Statistical ANOVA was applied, followed bythe Turkey multiple-comparison test. Data are shown asmean � SD or SEM; P � 0.05 was considered statisticallysignificant.

We thank Ashok B. Ramalingam and Marc F. Poitras for the initialcharacterization of PARG transgenic mice, Suk Jin Hong for assistancewith adeno-PARG viruses, and Sika Zheng for assistance with adeno-GFP virus. This work was supported by National Institutes of HealthGrant NS39148 and the American Heart Association. T.M.D. is theLeonard and Madlyn Abramson Professor in NeurodegenerativeDiseases.

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Andrabi et al. 10.1073/pnas.0606526103.

Supporting InformationFiles in this Data Supplement:

Supporting Figure 5Supporting Figure 6Supporting Figure 7Supporting Figure 8Supporting Methods

Supporting Figure 5

Fig. 5. Delivery of purified PAR polymer into neurons. Detection of PAR polymer in neurons afterBioPorter-mediated delivery of purified PAR polymer. Neurons were treated with BioPorter reagent alone(Control), BioPorter + 40 nM PAR polymer (PAR 40), BioPorter + 80 nM PAR polymer (PAR 80) orBioPorter + a synthetic control polymer, Poly (A). Purified PAR, or synthesized Poly (A) was diluted withPBS, mixed with BioPorter reagent, and then added to neuronal cultures to a final concentration of 40 or 80nM in MEM. The neurons were cultured for 3 h to allow the polymers to be imported into neurons and thensubjected to confocal microscopy. Immunocytochemical staining indicates that PAR polymer PAR polymer(red) can be found in the neurons (shown in differential interference contrast) of PAR-delivered cultures butnot in control neurons or neurons treated with BioPorter + Poly (A). Neurons with higher magnification areshown as a separate panel on the right side of each treatment. (Scale bar, 20 mm.) These experiments havebeen replicated in separate experiments at least three times with similar results.

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Supporting Figure 6

Fig. 6. PAR polymer can induce the cell death in HeLa cells in a concentration-dependent manner, andPAR polymer-mediated cell death is caspase-independent. (A) PAR polymer was diluted in PBS, mixedwith BioPorter reagent to final concentrations of 50, 80, and 160 nM, and then applied to HeLa cells.BioPorter mixed with PBS buffer only was used as a negative control. Cells were incubated for 4 h to allowPAR polymer to be delivered into the cells and then washed and replenished with serum-containing normalculture medium. After 24 h, cell death was assessed with Hoechst/PI double staining and quantified by usingAxiovision 4.3 automated software (Carl Zeiss). At all concentrations, PAR polymer can induce significantcell death and shows concentration-dependent cytotoxicity. Data are the mean ± SD, n = 3; *, P < 0.05. (B)PAR polymer was delivered into the HeLa cells by using BioPorter in the presence and the absence of apancaspase inhibitor, z-VAD. BioPorter mixed with PBS buffer only was treated as a negative control. Celldeath was determined with Hoechst/PI double staining 24 h after PAR polymer delivery. z-VAD (100 mM)was applied 1 h before PAR polymer treatment and kept in the culture medium during and after PARpolymer treatment. PAR polymer can induce significant cell death, and z-VAD cannot reduce the PARpolymer toxicity in HeLa cells. Data are the mean ± SD, n = 3; *, P < 0.05.

Supporting Figure 7

Fig. 7. PAR Polymer Mediates MNNG-induced cell death in HeLa cells. (A) MNNG-induced cell death inHeLa cells is PARP-dependent but caspase-independent. HeLa cells were treated with 50 mM MNNG for15 min in the presence of a pancaspase inhibitor z-VAD (100 mM) or PARP inhibitor DPQ (30 mM). Celldeath was determined with Hoechst/PI double staining by using quantitative computer-assisted cell counting24 h after MNNG treatment. z-VAD or DPQ was applied 1 h before MNNG treatment and kept in theculture medium during and after MNNG treatment. DPQ, but not z-VAD, completely rescues HeLa cellsfrom MNNG cytotoxicity. Data are the mean ± SD, n = 3; *, P < 0.05. (B) MNNG-induced cell death ismediated by PAR polymer. HeLa cells were infected with an exon-1-deleted cytosolic form of PARGadenovirus (Av-cPARG WT) or GFP-control adenovirus (Av-GFP). In separate cultures, PAR polymer-specific antibody (PAR Ab), normal rabbit serum (NRS, negative control), or preimmune serum (PreIm)was delivered into HeLa cells by using BioPorter reagent. MNNG (50 mM) was applied to cell cultures for15 min after 48-h PARG overexpression or after 4-h PAR-specific antibody delivery. Cell death wasdetermined with Hoechst/PI double staining 24 h after MNNG treatment. PAR polymer-mediatedcytotoxicity in HeLa cells was significantly alleviated by PAR polymer-degrading PARG enzymeoverexpression or PAR polymer antibody neutralization. Data are the mean ± SD, n = 3; *, P < 0.05. Similarresults were obtained by FACS analyses of cell death and survival (data not shown).

Supporting Figure 8

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Fig. 8. Endogenous PARG knockdown enhanced the MNNG toxicity in HeLa Cells. (A) The level ofendogenous PARG protein was determined by immunoblot against PARG and one-dimensional zymogramassay, respectively. (B) Reduction of endogenous PARG leads to the significant increase in intracellularPAR polymer after MNNG treatment, as shown in the PAR immunoblot. These experiments (A and B) havebeen replicated in separate experiments at least three times with similar results. (C) MNNG toxicity isenhanced in PARG siRNA transfected HeLa cells compared with MNNG only or scrambled RNA (scRNA)treatment. Data are the mean ± SD, n = 3; *, P < 0.05.

Supporting Methods

Immunocytochemistry and Confocal Microscopy. Treated or untreated cortical neurons were washedtwice with ice-cold PBS before fixation with 4% paraformaldehyde (PFA). After blocking with 10% normaldonkey serum and 0.3% Triton X-100 in PBS, cells were incubated with the primary antibody against rabbitpolyclonal or anti-PAR polymer antibody (1) overnight at 4°C. Cells were washed with blocking solutionthree times and incubated with the secondary antibody conjugated with Cy2 or Cy 3 (Invitrogen) for 3 h atroom temperature. Nuclei were counterstained with DAPI (300 nM) (Invitrogen) for 10 min after secondaryantibody incubation and subjected to two rinses with PBS. Confocal microscopy was performed on a LSM510 Meta microscope (Carl Zeiss) equipped with argon, HeNe, and Chameleon lasers. Images were obtainedby scanning stained cells under ×40 or ×63 oil objectives. For each sample, at least 10 different regionswere scanned and >200 cells were examined for each data point in at least three separate and independentexperiments by two separate examiners.

HeLa Cell Culture. HeLa cells were split and plated on 24-well plates in DMEM containing 10% FBS(Invitrogen). The cells were cultured at 37°C in a humidified incubator containing 5% CO2.

Cell Viability. HeLa cells were exposed to MNNG for 15 min, and cell viability was determined 24 h laterby computer-assisted cell counting after staining of all nuclei with 7 mM Hoechst 33342 (Invitrogen) anddead cell nuclei with 2 mM propidium iodide (Invitrogen). The numbers of total and dead cells werecounted with the Axiovision 4.3 software (Carl Zeiss).

Preparation and Transfection of siRNAs. To knockdown endogenous PARG, siRNA oligomers weresynthesized by using Silencer siRNA construction kit (Ambion) according to the user's guide. The targetsequences used were as follows: human PARG, 5'AAGATGAGAATGGTGAGCGAA-3'. For negativecontrol, target sequences were scrambled as follows: scrambled human PARG, 5'-AAGGGGTGTAGATAACGAGAA-3'. HeLa cells (6 ´ 104) were plated 1 day before transfection in eachwell of 12-well tissue culture plates allowing cells to reach 40-60% confluence at the time of transfection.siRNA molecules were transfected with Oligofectamine (Invitrogen) at a final concentration of 60 nM (forPARG) for 72 h according to the user's guide.

One Dimensional PARG Zymogram Assay. A PARG activity gel assay was performed as described (2).In brief, proteins were separated on an SDS/PAGE gel incorporated with 32P-labeled automodified PARP-1without sample boiling. Renaturation was carried out by incubating the gel in at least 5 vol of renaturation

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buffer (50 mM sodium phosphate buffer, pH7.4, 50 mM NaCl, 10% glycerol, 1% Nonidet P-40, and 10 mMb-mercaptoethanol) at room temperature for 24 h with at least five buffer changes. PARG enzyme activitywas developed by incubating the gel in renaturation buffer at 37°C for 3 h. The gel was dried andautoradiographed on a PhosphorImager screen.

1. Affar EB, Duriez PJ, Shah RG, Winstall E, Germain M, Boucher C, Bourassa S, Kirkland JB, Poirier GG(1999) Biochim Biophys Acta 1428:137-146.

2. Brochu G, Shah GM, Poirier GG (1994) Anal Biochem 218:265-272.

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