Poly(ADP-ribose) drives pathologic α-synuclein neurodegeneration … · RESEARCH ARTICLE SUMMARY NEURODEGENERATION Poly(ADP-ribose) drives pathologic a-synuclein neurodegeneration

RESEARCH ARTICLE SUMMARY◥

NEURODEGENERATION

Poly(ADP-ribose) drives pathologica-synuclein neurodegeneration inParkinson’s diseaseTae-In Kam, Xiaobo Mao, Hyejin Park, Shih-Ching Chou, Senthilkumar S. Karuppagounder,George Essien Umanah, Seung Pil Yun, Saurav Brahmachari, Nikhil Panicker, Rong Chen,Shaida A. Andrabi, Chen Qi, Guy G. Poirier, Olga Pletnikova, Juan C. Troncoso,Lynn M. Bekris, James B. Leverenz, Alexander Pantelyat, Han Seok Ko, Liana S. Rosenthal,Ted M. Dawson*, Valina L. Dawson*

INTRODUCTION: Parkinson’s disease (PD) isthe second most common neurodegenerativedisorder. Intracellular protein aggregates com-posed primarily ofa-synuclein lead to neuronaldysfunction throughout the nervous system,ultimately accumulating in structures calledLewy bodies and neurites. Loss of substantianigra pars compacta dopamine (DA) neuronsand dystrophic striatal projections account forthe major motor symptoms of PD, which in-clude a rest tremor, slowness of movement, ri-gidity, and postural instability. Other neuronalsystems are affected by pathologic a-synucleinand contribute to the nonmotor symptoms ofPD, which include anxiety, depression, sleepdisorders, autonomic dysfunction, constipa-tion, and cognitive impairment.

RATIONALE:During the pathogenesis of PD,monomeric a-synuclein assembles into higher-ordered structures that ultimately becomepathologic and drive neuronal cell death. Path-ologic a-synuclein can spread from cell to cell,contributing to the progressive pathogenesisof PD. What drives the abnormal assembly ofpathologic a-synuclein and the cell injury anddeath mechanisms that are activated by path-ologic a-synuclein are not known.

RESULTS: Recombinant a-synuclein pre-formed fibrils (PFFs), which are similar instructure to those found in PD, were usedto model pathologic a-synuclein both in vitroand in vivo. We investigated the cellular celldeath pathways that contribute to and drive

a-synuclein PFF–mediated neuronal cell death.Pathologic a-synuclein was found to activatenitric oxide synthase (NOS), causingDNAdam-age and poly(adenosine 5′-diphosphate–ribose)polymerase-1 (PARP-1) activation, leading tocell death via parthanatos. a-Synuclein PFFwas found to primarily kill neurons via par-thanatos, because necroptosis and autophagyinhibition had no effect on a-synuclein PFFneurotoxicity and there was only modest pro-tection by caspase inhibition. Neuron-to-neuron transmission of pathologic a-synuclein

and accompanying path-ology and neurotoxicityin primary neuronal cul-tures were completelyattenuated by clinicallyavailable PARP inhibitorsor by deletion of PARP-1.

a-Synuclein PFF–induced loss of DA neuronsand biochemical and behavioral deficits in vivowere significantly prevented by PARP inhi-bition or lack of PARP-1. PAR generated byPARP-1 activation also binds to a-synuclein,accelerating its fibrillization and convertingpathologic a-synuclein to a more misfoldedcompact strain with 25-fold enhanced toxic-ity. PAR-modified a-synuclein PFF–injectedmice showed accelerated disease progressionand phenotype compared to a-synuclein PFF–injected mice. Moreover, PAR levels were in-creased in the cerebrospinal fluid (CSF) in twoindependent patient cohorts and brains of PDpatients, providing evidence that parthanatosmay contribute to the pathogenesis of PD.

CONCLUSION:We identified PARP-1 activa-tion and the generation of PAR as a key me-diator of pathologic a-synuclein toxicity andtransmission. Activation of parthanatos is theprimary driver of pathologic a-synuclein neuro-degeneration. Inhibition of PARP and depletionof PARP-1 substantially reduces the pathologyinduced by the transmission of pathologic a-synuclein. In a feed-forward loop, PAR con-verted pathologic a-synuclein to a more toxicstrain and accelerated neurotoxicity both in vitroand in vivo. Consistent with the notion thatPARP-1 activation plays a role in PD patho-genesis, PAR levels were increased in theCSF and brains of PD patients. Thus, strat-egies aimed at inhibiting PARP-1 activationcould hold promise as a disease-modifyingtherapy to prevent the loss of DA neurons inPD and related a-synucleinopathies. Moreover,assessment of PAR levels in the CSF couldserve as a theranostic biomarker for disease-modifying therapies in these disorders.▪

RESEARCH

Kam et al., Science 362, 557 (2018) 2 November 2018 1 of 1

The list of author affiliations is available in the full article online.*Corresponding author. Email: [email protected] (T.M.D.);[email protected] (V.L.D.)Cite this article as T.-I. Kam et al., Science 362, eaat8407(2018). DOI: 10.1126/science.aat8407

PARP-1 is activated by a-synuclein PFF and PAR mediates cell death. Inhibition of PARP ordeletion of PARP-1 reduces a-synuclein PFF–induced cell death. In a feed-forward loop, PARcauses the formation of a more toxic a-synuclein strain, resulting in accelerated pathologica-synuclein transmission and toxicity.

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RESEARCH ARTICLE◥

NEURODEGENERATION

Poly(ADP-ribose) drives pathologica-synuclein neurodegeneration inParkinson’s diseaseTae-In Kam1,2,3*, Xiaobo Mao1,2,3*, Hyejin Park1,2,3*, Shih-Ching Chou1,4,Senthilkumar S. Karuppagounder1,2,3, George Essien Umanah1,2, Seung Pil Yun1,2,3,Saurav Brahmachari1,2,3, Nikhil Panicker1,2,3, Rong Chen1,2,3, Shaida A. Andrabi1,2†,Chen Qi1,2,5, Guy G. Poirier6, Olga Pletnikova7, Juan C. Troncoso2,7, Lynn M. Bekris8,James B. Leverenz9, Alexander Pantelyat2, Han Seok Ko1,2,3, Liana S. Rosenthal2,Ted M. Dawson1,2,3,4,10‡, Valina L. Dawson1,2,3,10,11‡

The pathologic accumulation and aggregation of a-synuclein (a-syn) underliesParkinson’s disease (PD). The molecular mechanisms by which pathologic a-syncauses neurodegeneration in PD are not known. Here, we found that pathologic a-synactivates poly(adenosine 5′-diphosphate–ribose) (PAR) polymerase-1 (PARP-1), and PARgeneration accelerates the formation of pathologic a-syn, resulting in cell death viaparthanatos. PARP inhibitors or genetic deletion of PARP-1 prevented pathologic a-syntoxicity. In a feed-forward loop, PAR converted pathologic a-syn to a more toxic strain.PAR levels were increased in the cerebrospinal fluid and brains of patients with PD,suggesting that PARP activation plays a role in PD pathogenesis. Thus, strategies aimedat inhibiting PARP-1 activation could hold promise as a disease-modifying therapy toprevent the loss of dopamine neurons in PD.

Parkinson’s disease (PD) is an age-relatedneurodegenerative disease in which a-synuclein (a-syn) deposits as fibrils inintracytoplasmic inclusions in structurestermed Lewy bodies and neurites (1).

Recombinant a-syn can be aggregated in vitroto form fibrils similar in structure to thosefound in vivo (2), and these a-syn preformed

fibrils (a-syn PFFs) can spread in a prion-likemanner: both in in vitro neuronal cultures andin vivo when injected into the mouse brainwith accompanying phosphorylation of a-synon serine-129, a marker of pathologic a-syn(3) and neurotoxicity (2, 4, 5). Although it isclear that aggregated a-syn underlies the pa-thology of PD, what drives abnormal aggre-gation of a-syn and the cell injury and deathmechanisms that are activated by this aggrega-tion are not yet known. Because poly(adenosine5′-diphosphate–ribose) (PAR) polymerase-1(PARP-1) and PAR play a major contributingrole in cell death relevant to neurologic dis-orders (6, 7), here, we evaluated a role forPARP-1 and PAR in pathologic a-syn–inducedneurodegeneration.

a-Syn PFF–induced neurotoxicity isPARP-1 dependent

To determine whether a-syn PFF induces theactivation of PARP, levels of PAR were mea-sured using a highly sensitive and specific PARmonoclonal antibody after administration ofa-syn PFF to primary mouse cortical neurons(Fig. 1). a-Syn PFF (1 mg/ml) induced PARP ac-tivation peaks between 3 and 7 days and re-mained elevated for up to 14 days (Fig. 1A). Theelevation of PAR was accompanied by neurondeath as assessed by propidium iodide (PI) stain-ing (Fig. 1, B and C). Treatment of cortical neu-rons with 1 mM of the PARP inhibitors [ABT-888(veliparib), AG-014699 (rucaparib), or BMN 673

(talazoparib)] prevented the a-syn PFF–mediatedPARP activation and cell death (Fig. 1, B to D).Consistent with known half-maximal inhibitoryconcentration values for inhibition of PARP-1 (5.2 nM ABT-888, 1.4 nM AG-014699, and 1.2 nMBMN 673) (8), 10 nM ABT-888, 1 nM AG-014699,or 1 nM BMN 673 partially prevented PARP-1autoribosylation activity in vitro (fig. S1, A and B).Complete inhibition was observed at higherconcentration of these inhibitors (fig. S1, A andB). These PARP inhibitors prevented a-syn PFF–induced cell death and PARP activation at con-centrations as low as 10 nM (fig. S1, C to E).They also reduced a-syn PFF–mediated phos-phorylation of a-syn at serine-129 (p–a-syn)(fig. S1, F and G) and a-syn aggregation (fig. S1,H and I), both of which are associated withpathology in a-synucleinopathies (4). BecausePARP-1 plays a major role in parthanatos (9, 10),we deleted PARP-1 from cortical neurons usingCRISPR-Cas9 via adeno-associated virus (AAV)transduction carrying a guide RNA against PARP-1(Fig. 1, E and F, and fig. S2A) (11) or used corticalcultures from PARP-1 knockouts (KOs) (Fig. 1,G and H, and fig. S2, B to F). Deletion or knock-out of PARP-1 prevented a-syn PFF–mediatedPARP activation and cell death (Fig. 1, E to H,and fig. S2, A and B). Knockout of PARP-1 alsoreduced p–a-syn immunostaining and a-synaggregation (fig. S2, C to F). Treatment of corticalneurons with the broad-spectrum caspase inhib-itor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) (Z-VAD) partiallyreduced a-syn PFF toxicity. The necroptosis in-hibitor necrostatin-1 (Nec-1) and the autophagyinhibitor 3-methyladenine (3-MA) had no effect,whereas the PARP inhibitor ABT-888 preventeda-syn PFF toxicity (fig. S2, G to M). BecausePARP inhibition and knockout of PARP-1 reducedthe accumulation of pathologic a-syn as indicatedby a reduction of p–a-syn immunostaining, weassessed cell-to-cell transmission of a-syn (5).Knockout of PARP-1 or PARP inhibition did notshow a difference in the levels of a-syn–biotinPFF in endosomal-enriched fractions (fig. S3,A to D) (5), indicating that PARP-1 did not affectthe uptake of a-syn PFF. However, knockoutof PARP-1 reduced the cell-to-cell transmission ofpathologic a-syn by inhibiting propagation ofa-syn PFF into recipient cells (fig. S3, E to G).

a-Syn PFF activates PARP-1 via nitricoxide–induced DNA damage

To determine how a-syn PFF activates PARP-1,we measured levels of damaged DNA and ac-tivation of nitric oxide synthase (NOS) (Fig. 2and fig. S4) (12–14). a-Syn PFF treatment in-creased NO levels in primary cultured neurons,whereas pretreatment with the NOS inhibitor,Nw-nitro-L-arginine methyl ester hydrochloride(L-NAME), prevented a-syn PFF–induced NOgeneration (Fig. 2A). NO levels also increasedin a-syn PFF–injected brain (Fig. 2B). In botha-syn PFF–treated primary cultured neuronsand a-syn PFF–injected brain, expression ofgH2A.X, a marker of DNA strand breaks (15),was increased (Fig. 2, C to G, and fig. S4A).

RESEARCH

Kam et al., Science 362, eaat8407 (2018) 2 November 2018 1 of 10

1Neuroregeneration and Stem Cell Programs, Institute forCell Engineering, Johns Hopkins University School ofMedicine, Baltimore, MD 21205, USA. 2Department ofNeurology, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA. 3Adrienne Helis Malvin MedicalResearch Foundation, New Orleans, LA 70130-2685, USA.4Department of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, Baltimore, MD21205, USA. 5Department of Neurology, Xin Hua Hospitalaffiliated to Shanghai Jiaotong University School of Medicine,Shanghai 200092, China. 6Centre de recherche du CHU deQuébec-Pavillon CHUL, Faculté de Médecine, UniversitéLaval, Québec G1V 4G2, Canada. 7Department of Pathology(Neuropathology), Johns Hopkins University School ofMedicine, Baltimore, MD 21205, USA. 8Lerner ResearchInstitute, Genomic Medicine, Cleveland Clinic, Cleveland, OH44195, USA. 9Lou Ruvo Center for Brain Health, NeurologicalInstitute, and Department of Neurology, Cleveland Clinic,Cleveland, OH 44195, USA. 10Solomon H. Snyder Departmentof Neuroscience, Johns Hopkins University School ofMedicine, Baltimore, MD 21205, USA. 11Department ofPhysiology, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA.*These authors contributed equally to this work.†Present address: Departmental of Pharmacology and Toxicology,School of Medicine, University of Alabama at Birmingham,Birmingham, AL 35294-6810, USA.‡Corresponding author. Email: [email protected] (T.M.D.);[email protected] (V.L.D.)

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a-Syn PFF resulted in substantial numbers ofneurons with DNA damage, whereas no suchdamage was detected by treatment with L-NAME(Fig. 2, C to E, and fig. S4, B to D). Consistentwith suppression of PARP-1 activation (Fig. 2C),a-syn PFF–induced cell death was prevented byL-NAME (fig. S4, E to G). Thus, a-syn PFF acti-vates NOS, leading to DNA damage and PARP-1activation. Moreover, a-syn PFF primarily killsneurons through parthanatos, and PARP-1 con-tributes to generation of pathologic a-syn.

PARP-1 activation mediates a-synPFF–induced loss of DA neurons

Because synthetic a-syn PFF killed primary cor-tical neurons via parthanatos, we sought to de-termine whether parthanatos plays a role in theloss of dopamine (DA) neurons after the intra-striatal injection of a-syn PFF (Fig. 3) (4, 5). Asingle intrastriatal injection of a-syn PFF (5 mg)induced PARP activation and increased PARlevels (Fig. 3A). Intrastriatal injection of a-synPFF into PARP-1 KO mice failed to increase PARlevels (Fig. 3A). As previously described (4), a

single intrastriatal injection of a-syn PFF leadsto an approximate 50% loss of DA neurons6 months after the injection in wild-type (WT)mice (Fig. 3, B and C). In contrast, a single in-trastriatal injection of a-syn PFF into PARP-1KO mice failed to induce DA cell loss (Fig. 3, Band C). WT mice were also fed a diet contain-ing the PARP inhibitor ABT-888 (125 mg/kg)and compared with mice given a control diet(Fig. 3, B and C). Mice treated with ABT-888exhibited significantly less loss of DA neuronsafter an intrastriatal injection of a-syn PFF com-pared to mice on the control diet (Fig. 3, B andC). ABT-888 also reduced the formation of a-synPFF–induced increase in PAR levels (fig. S5A).Tyrosine hydroxylase (TH) and DA transporter(DAT) levels were also reduced in WT mice inresponse to a-syn PFF, whereas the reduction inTH and DAT levels was prevented in PARP-1 KOor ABT-888–treated WT mice (fig. S5, A to C).The loss of DA neurons was accompanied bya reduction in striatal DA and its metabolitesin WT mice, but not PARP-1 KO or ABT-888–treated mice (Fig. 3D and fig. S5, F to H). As

previously described (4, 5), injection of intra-striatal a-syn PFF leads to a-syn pathology inDA neurons of WT mice (figs. S5, A, D, and E,and S6, A to D). a-Syn pathology was markedlyreduced in PARP-1 KO mice and ABT-888–treated WT mice consistent with the absenceand reduction of neurodegeneration, respectively.Intrastriatal injection of a-syn PFF caused deficitson the pole test (16), a sensitive behavioral mea-surement of dopaminergic function, in WT mice,whereas there were no deficits in PARP-1 KO andABT-888–treated WT mice (Fig. 3E and fig. S6E).Both forelimb plus hindlimb and forelimb gripstrength were also reduced in WT mice aftera-syn PFF injection, but not in PARP-1 KO orABT-888–treated WT mice (Fig. 3F and fig. S6F).Thus, the striatal a-syn PFF–induced loss of DAneurons is dependent on PARP-1.

PAR accelerates a-syn fibrillization

Because PAR causes liquid demixing of intrin-sically disordered proteins leading to their ag-gregation (17), experiments were performed todetermine whether PAR could seed and accelerate


Fig. 1. a-Syn PFF induces parthanatos inneurons. (A) Activation of PARP-1 ina-syn PFF–treated primary corticalneurons. The representative Western blotanalysis (top) and quantification (bottom)of the levels of PAR accumulation.Numbers to the left of the blot indicatemolecular mass in kDa. Bars representmeans ± SEM. One-way analysis of vari-ance (ANOVA) followed by Tukey’s post hoctest (n = 3 to 4). (B) Representative imagesof Hoechst and PI staining from primarycortical neurons pre-incubated with ABT-888 (1 mM), AG-014699 (1 mM), or BMN 673(1 mM) for 1 hour and further incubated witha-syn PFF (5 mg/ml) for 14 days. Scale bars,20 mm. (C) Quantification of cell death.Bars represent means ± SEM. Two-wayANOVA followed by Tukey’s post hoc test(n = 4). (D) Inhibition of a-syn PFF–inducedPAR accumulation was determined byWestern blot analysis. (E) Representativeimages of Hoechst and PI staining fromprimary cortical neurons transducedwith AAV containing single-guide RNA con-trol (AAV-sgCon) or AAV–sgPARP-1 andfurther incubated with a-syn PFF for14 days. Scale bar, 20 mm. (F) Quantifica-tion of cell death. Bars representmeans ± SEM. Two-way ANOVA followed byTukey’s post hoc test (n = 3). (G) Repre-sentative images of Hoechst and PI stainingfrom WT or PARP-1 KO primary corticalneurons and further incubated with a-synPFF for 14 days. Scale bars, 20 mm.(H) Quantification of cell death. Barsrepresent means ± SEM. Two-way ANOVAfollowed by Tukey’s post hoc test (n = 3).*P < 0.05, **P < 0.005, ***P < 0.0005.

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Fig. 2. Increased NO levels and damagedDNA by a-syn PFF. (A) NO (nitrite + nitrate) levelsin primary cortical neurons preincubated withL-NAME for 1 hour and further incubated with a-synPFF for 3 days. Bars represent means ± SEM.Two-way ANOVA followed by Tukey’s post hoc test(n = 8). (B) NO (nitrite + nitrate) levels inSNpc of a-syn PFF–injected mice. Bars representmeans ± SEM. Unpaired Student’s t test (n = 8).(C) Representative immunoblots (left) andquantification (right) of gH2A.X and PAR levelsin primary cortical neurons preincubatedwith L-NAME for 1 hour and further incubatedwith a-syn PFF. Bars represent means ± SEM.Two-way ANOVA followed by Tukey’s posthoc test (n = 4). (D) Representative images ofgH2A.X (green) and MAP2 (red) in primarycortical neurons preincubated with L-NAME for1 hour and further incubated with a-syn PFF. Scalebars, 20 mm. (E) Quantification of gH2A.X-positiveneurons. Bars represent means ± SEM.Two-wayANOVA followed by Tukey’s post hoc test(n = 5). (F) Representative immunoblots (left)and quantification (right) of gH2A.X levels in theSNpc of a-syn PFF–injected mice. Bars aremeans ± SEM. Unpaired Student’s t test (n = 3).(G) Representative images (left) and quantification(right) of gH2A.X-positive cells (green) in theSNpcofa-synPFF–injectedmice.Scale bars, 20 mm.Bars are means ± SEM. Unpaired Student’s t test(n = 3). *P < 0.05, ***P < 0.0005.

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Fig. 3. a-Syn PFF–induced pathology isreduced by deletion of PAPR-1 or a PARPinhibitor, ABT-888, in vivo. (A) Representativeimmunoblots (left) and quantification (right) ofthe levels of PAR accumulation in the striatumof a-syn PFF–injected mice. Bars representmeans ± SEM. One-way ANOVA followed byTukey’s post hoc test (n = 4). (B) RepresentativeTH and Nissl staining of SNpc DA neurons ofa-syn PFF–injected WT, PARP-1 KO, and WTmice fed with ABT-888 at 6 months afterintrastriatal a-syn PFF or phosphate-bufferedsaline (PBS) injection. Scale bar, 400 mm.(C) Stereological counts. Data aremeans ± SEM. Two-way ANOVA followed byTukey’s post hoc test (n = 5 to 7 mice pergroup). (D) DA concentrations in the striatumof WT, PARP-1 KO, and WTmice fed withABT-888 at 6 months after intrastriatal a-synPFF or PBS injection measured by high-performance liquid chromatography (HPLC).Bars represent means ± SEM. Two-way ANOVAfollowed by Tukey’s post hoc test (n = 5 to10 mice per group). (E and F) One hundredeighty days after a-syn PFF injection, thepole test (E) and grip strength test (F) wereperformed in WT, PARP-1 KO, or WTmicefed with ABT-888. Data are the means ± SEM.Two-way ANOVA followed by Tukey’s post hoctest (n = 6 to 8 mice for PARP-1 KO, n = 11to 30 mice for ABT-888). *P < 0.05,**P < 0.005, ***P < 0.0005.

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a-syn aggregation. Recombinant a-syn was in-cubated at 37°C and agitated in the presenceand absence of 5 nM PAR, concentrations thatare observed in brain tissue (9). High–molecularweight forms of a-syn in the absence of PARwere observed as early as 4 hours of incubation,and a-syn continued to fibrillize with time (Fig. 4A).Different molecular weight forms of a-syn wereobserved at 72 hours. In the presence of PAR,the fibrillization of a-syn was markedly accel-erated with different molecular weight formsof a-syn being observed as early as 24 hoursof incubation (Fig. 4A). PAR accelerated thefibrillization of a-syn as indicated by thioflavinT fluorescence, whereas PAR alone had noeffect (Fig. 4B). PAR caused the fibrillization

of a-syn at lower temperatures than requiredfor a-syn fibrillization in the absence of PAR(fig. S7A). At 12 hours, a-syn fibrils were ob-served in the absence of PAR and were fullyformed and extensive after 72 hours of agitationand incubation at 37°C (Fig. 4C). In contrast,in the presence of PAR, a-syn was extensivelyfibrillated at 12 hours and was more extensivelyfibrillated at 36 and 72 hours (Fig. 4C). Wemonitored the concentration dependence offibrillization at 36 hours. PAR (1 nM) enhanceda-syn fibrillization, with peak aggregation oc-curring at 5 nM PAR, whereas 20 nM PAR didnot appreciably increase a-syn fibrillization(fig. S7, B and C). a-Syn was not PARylatedby PARP-1 activity, but it bound to PAR through

its N-terminal domain (fig. S8, A to C). In thea-syn PFF–injected mouse brain, about 20% ofa-syn was PAR bound (fig. S8, D and E). BecausePAR is a highly negatively charged molecule, wetested the effects of another highly charged poly-mer, PolyA. a-Syn failed to interact with poly-adenylic acid (PolyA), and it had no effect ona-syn fibrillization (fig. S7, D to F). ADP ribosemonomer also failed to interact with a-syn andhad no effect on a-syn fibrillization (fig. S7, Dto F). To determine whether endogenous PARformation accelerates a-syn fibrillization, pri-mary mouse cortical neurons overexpressing WThuman a-syn after AAV–a-syn transduction weretreated with a toxic dose of N-methyl-D-aspartate(NMDA). In WT cultures, NMDA treatment


Fig. 4. PAR accelerates a-syn fibrilliza-tion in vitro. (A) Acceleration of a-synfibrillization by PAR. Monomeric a-syneither with or without 5 nM purified PARwas incubated at 37°C for indicated times.Fibrillization of a-syn was detected byimmunoblotting using an a-syn antibody.Data are means ± SEM. Two-wayANOVA followed by Tukey’s post hoctest (n = 3). (B) The rate of formation ofa-syn fibrils either with or without PAR wasmonitored by thioflavin T (ThT) fluores-cence (n = 3). (C) Representative trans-mission electron microscopy (TEM) imagesfor a-syn fibrils. Scale bars, 200 nm.(D and E) Suppression of NMDA-induceda-syn fibrillization in (D) PARP-1 KO neu-rons and by (E) PARP inhibitors. Primarycortical neurons from WT or PARP-1 KOembryos were transduced with AAV–a-synand then further incubated with 500 mMNMDA for 5 min. ABT-888 (10 mM) orAG-014699 (1 mM) for 1 hour was pre-treated for 1 hour. a-Syn fibrillization wasdetected by Western blot (WB) analysis6 hours after NMDA treatment. (F) a-SynPFF or PAR–a-syn PFF was incubatedwith increasing concentrations of PK(0 to 2.5 mg/ml) and immunoblotted witha-syn antibody (top). Quantificationrepresents the ratio of cleaved touncleaved a-syn (bottom). Data aremeans ± SEM. (bottom). Unpaired two-tailed Student’s t test in each concentrationof PK (n = 3). (G) Representative immu-nostaining and quantification of p–a-syn(red) in primary cortical neurons treatedwith a-syn PFF or PAR–a-syn PFFfor 1, 4, and 7 days. Bars representmeans ± SEM. Two-way ANOVA followedby Tukey’s post hoc test (n = 3). Scale bar,20 mm. (H) Primary cortical neuronstreated with a-syn PFF or PAR–a-syn PFFwere sequentially extracted with 1%Triton X-100 (TX-100) (TX soluble)and 2% SDS (TX insoluble). Lysateswere subjected to immunoblotting usinga-syn, p–a-syn, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Bars represent means ± SEM. Two-way ANOVA followed by Tukey’s post hoc test (n = 3). ND,not detected. *P < 0.05, **P < 0.005, ***P < 0.0005. AU, arbitrary units; DAPI, 4′,6-diamidino-2-phenylindole.



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activated PARP and led to a concomitant ag-gregation of a-syn, whereas a-syn did not ag-gregate in PARP-1 KO cultures treated withNMDA (Fig. 4D). Exogenous administration ofPAR via BioPORTER (9) increased the aggrega-

tion of a-syn in both WT and PARP-1 KO culturestransduced with AAV–a-syn (fig. S9A). Thus, PAR,not PARP-1, can directly increase a-syn aggrega-tion. Two different PARP inhibitors, ABT-888 andAG-014699, prevented a-syn aggregation and

PARP activation in response to NMDA adminis-tration (Fig. 4E). In SH-SY5Y neuroblastoma cells,the potent PARP activator N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) enhanced the aggrega-tion of overexpressed WT or A53T a-syn, whereasMNNG had no effect in PARP-1 KO SH-SY5Ycells (fig. S9, B and C). Exogenous administrationof PAR increased the aggregation of a-syn inboth SH-SY5Y WT and SH-SY5Y PARP-1 KOcultures (fig. S9D). Two different PARP inhibitors,ABT-888 and AG-014699, prevented a-syn ag-gregation and PARP activation in response toMNNG administration in SH-SY5Y cells (fig. S9E).Thus, PAR seeds and accelerates a-syn aggregation.

PAR promotes formation of more toxica-syn PFF strains

To determine whether PAR changes the bio-physical properties of a-syn PFF, a series ofbiochemical analysis were conducted usinga-syn PFF and a-syn PFF in the presence ofPAR (PAR–a-syn PFF). First, proteinase K (PK)digestion of a-syn PFF was performed and moni-tored by a-syn immunoblots. a-Syn PFF andPAR–a-syn PFF showed very distinct bandingpatterns after PK digestion, with PAR–a-synPFF’s being more resistant to increasing con-centrations of PK (Fig. 4F). PAR–a-syn PFFpredominantly showed an undigested band ofa-syn (first band), with comparable digestedbands only at higher concentrations of PK,whereas a-syn PFF degraded into smaller frag-ments (second to fifth band) at lower concen-trations of PK (0.5 and 1 mg/ml), and thesebands became predominant at higher con-centrations of PK (1.5 to 2.5 mg/ml) (Fig. 4F).Epitope-specific antibodies to a-syn revealedthat PAR rendered the majority of the a-synregions resistant to PK digestion (fig. S10A).The resistance to PK digestion of PAR–a-synPFF suggests that PAR induces the formationof a distinct a-syn PFF strain with a more mis-folded and compact structure than a-syn PFF.We then compared the a-syn PFF– and PAR–a-syn PFF–induced neuronal cell death in culturedneurons. After 14 days of treatment, cell deathwas enhanced in cultures treated with PAR–a-syn PFF as compared to that with a-syn PFF(fig. S10B). PAR itself did not cause signifi-cant cell death even at a higher concentra-tion (20 nM) (fig. S10B). To further confirmthe potencies of neuropathology, p–a-syn im-munoreactivity was monitored after treatmentwith varying concentrations of a-syn PFF orPAR–a-syn PFF. P–a-syn immunoreactivity wasobserved at 20 ng of PAR–a-syn PFF at an equiv-alent level to 500 ng of a-syn PFF, suggesting thatthe PAR modification of a-syn PFF increasedtoxicity by 25-fold (fig. S10, C and D). PAR–a-syn PFF significantly increased p–a-syn immu-noreactivity at higher concentrations (fig. S10,C and D). P–a-syn immunoreactivity at differenttime points was also monitored in cultured neu-rons exposed to a-syn PFF or PAR–a-syn PFF. Inthe absence of PAR, a-syn PFF treatment leadsto barely detectable p–a-syn immunoreactivity1 day after treatment, whereas PAR–a-syn PFF


Fig. 5. PAR-a-syn PFF strains are more neurotoxic in vivo. (A) Representative TH and Nisslstaining of SNpc DA neurons of WTmice at 1, 3, and 6 months after instrastriatal PBS, a-syn PFF,PAR–a-syn PFF, or PAR injection. Scale bars, 400 mm. (B) Stereological counts. Bars represent means ±SEM.Two-way ANOVA followed by Tukey’s post hoc test (n = 5 to 8 mice per group). (C) Representativep–a-syn immunostaining in the SNpc of WTmice at 1, 3, and 6 months after instrastriatal PBS, a-synPFF, PAR–a-syn PFF, or PAR injection. Scale bar, 100 mm. (D) Quantification of p–a-syn levels. Barsrepresent means ± SEM.Two-way ANOVA followed by Tukey’s post hoc test (n = 5 to 8 mice per group).(E) DA concentrations in the striatum of PBS-, a-syn PFF–, PAR–a-syn PFF–, or PAR-injected mice at1, 3, and 6 months measured by HPLC. Bars represent means ± SEM.Two-way ANOVA followed byTukey’s post hoc test (n = 4 to 6 mice per group). (F and G) Behavioral abnormalities of PBS-, a-synPFF–, PAR–a-syn PFF–, or PAR-injected mice at 1, 3, and 6 months measured by the pole test (F, n = 9 to24 mice per group) and the grip strength test (G, n = 6 to 14 mice per group). Data are the means ± SEM.Two-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.005, ***P < 0.0005.



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treatment leads to detectable levels of p–a-synimmunoreactivity as early as 1 day after treat-ment and markedly enhances immunoreactivityat 7 days (Fig. 4G). Aggregated and phosphoryl-ated a-syn were detectable at 4 days, whereas inthe absence of PAR, these species of a-syn wereonly detectable after 7 days of treatment (Fig. 4H).In the presence of PAR, there was an increase inthe aggregated form of a-syn (Fig. 4H). We thencompared the cell-to-cell transmission of a-synPFF and PAR–a-syn PFF in neuronal culturestransduced with AAV–a-syn (fig. S11). The exo-somal secretion (18) of PAR-induced a-syn oligo-mers by NMDA treatment was significantlyincreased in WT neurons, but not in PARP-1KO neurons or in WT neurons with treatmentof a PARP inhibitor (fig. S11, A and B). PAR–a-syn PFF was enriched in the endosome-enrichedfraction, and propagation of pathologic p–a-synwas enhanced in recipient cells as compared tothose of a-syn PFF (fig. S11, C to F). Thus, PARconverts pathologic a-syn to a more misfoldedcompact toxic strain.

PAR–a-syn PFF strain is more neurotoxicin vivo

To determine whether the PAR–a-syn PFF strainexhibits enhanced neurotoxicity in vivo, a singleintrastriatal injection of PAR–a-syn PFF (5 mg)was compared to that of a-syn PFF (5 mg). Weobserved a trend toward the loss of DA neuronsipsilateral to the injection side of SNpc after1 month and a significant loss of DA neurons3 months after PAR–a-syn PFF injection, whereasa-syn PFF injection had no effect at these timepoints (Fig. 5, A and B, and fig. S12A). Six monthsafter PAR–a-syn PFF or a-syn PFF injection, therewas no significant difference in the loss of DAneurons (Fig. 5, A and B, and fig. S12A). Therewas no significant loss of DA neuron contra-lateral to the injection side at any time point

(fig. S12, B and C). PAR injection by itself hadno effect on DA neuron number (Fig. 5, A andB, and fig. S12A). PAR–a-syn PFF also accel-erated the loss of striatal DA and its metabo-lites, with significant reductions in DA and itsmetabolites 1 month after the PAR–a-syn PFFinjection in contrast to a-syn PFF (Fig. 5E andfig. S13). Three and 6 months after PAR–a-synPFF or a-syn PFF injection, there was no sig-nificant difference in the loss of DA and itsmetabolites (Fig. 5E and fig. S13). TH andDAT levels were also reduced after PAR–a-synPFF compared to a-syn PFF 3 months after theinjection, whereas there was no difference inthe degree of loss at 6 months (fig. S14, A and B).a-Syn pathology as assessed by immunostainingfor p–a-syn in DA neurons was increased byPAR–a-syn PFF compared to a-syn PFF at 3and 6 months after injection (Fig. 5, C and D).PAR–a-syn PFF caused a deficit on the poletest at 3 months consistent with loss of DA neu-rons and DA deficits at 3 months, whereas therewere no significant deficits in a-syn PFF–injectedor PAR-injected mice (Fig. 5F and fig. S14C).Both forelimb plus hindlimb and forelimb gripstrength were also reduced in PAR–a-syn PFF–injected mice but not in a-syn PFF–injected orPAR-injected mice at 3 months (Fig. 5G andfig. S14D). At 6 months, there was no significantdifference in the behavioral deficits induced byPAR–a-syn PFF or a-syn PFF (Fig. 5, F and G, andfig. S14, C and D). Thus, PAR–a-syn PFF is sub-stantially more neurotoxic than a-syn PFF in vivo.

Increased levels of PAR in the CSF ofpatients with PD

To determine whether PAR plays a role in pa-tients with PD, PAR levels were monitored inthe cerebrospinal fluid (CSF) of patients withPD versus controls (tables S1 and S2) using asensitive enzyme-linked immunosorbent assay

(ELISA) for PAR (fig. S15A). PAR levels wereelevated in patients with PD compared to con-trols in two independent patient cohorts (Fig. 6,A and E). One of the cohorts showed a positivecorrelation between PAR levels and either dis-ease duration or progression (Fig. 6, B to D andF to H). As previously reported, PAR levels wereincreased in the substantia nigra of patients withPD compared to controls (fig. S15, B and C, andtable S3) (19).

Discussion

Our results indicate that a-syn PFF kills neu-rons both in vitro and in vivo via activationof PARP-1 in a cell death process designatedparthanatos (20). Knockout of PARP-1 andinhibition of PARP prevents the neurodegen-eration and behavioral deficits initiated by anintrastriatal a-syn PFF injection. Activation ofparthanatos seems to be the primary driver ofa-syn PFF neurodegeneration because necrop-tosis and autophagy inhibitors had no effect ona-syn PFF neurotoxicity, and there is only modestprotection by caspase inhibition. It is known thata-syn PFFs induce inflammatory mediator activa-tion (21, 22), which likely contributes, in part, tocell death and accounts for the modest neuro-protection by the broad-spectrum caspase in-hibitor Z-VAD. It will be important in futurestudies to explore the role of caspase activa-tion, neuroinflammation, and neurodegenera-tion induced by a-syn PFF.For the studies reported here, we used a-syn

PFF (5 mg/ml) or PAR–a-syn PFF (5 mg/ml) forthe primary neuronal culture experiments, whichrepresents a concentration of about 340 nM inmonomeric equivalents. The actual concentra-tion of a-syn PFFs would be lower. Because a-synis thought to represent about 1% of soluble brainprotein (23), its monomeric concentration is esti-mated to be in the low micromolar range in the


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Disease duration (years) Hoehn & Yahr

Disease duration (years) Disease duration (years) Hoehn & Yahr

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Fig. 6. Increase of PAR levels in CSF of patients with PD. (A and E) Increase of PAR in CSF of patients with PD. The levels of PAR in CSF of healthycontrols (Con) (A, n = 31; E, n = 33) and patients with PD (A, n = 80; E, n = 21) were determined by PAR ELISA. Bars represent means ± SEM. Student’s t testwith Welch’s correction (A) and Mann-Whitney U test (E). *P < 0.05, ***P < 0.0005. (B and F) Correlation analysis between disease duration andHoehn & Yahr. (C and G) Correlation analysis between disease duration and PAR levels. (D and H) Correlation analysis between Hoehn & Yahr and PARlevels in the two independent patient cohorts used in (A) and (E). R2, correlation coefficient.



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brain. The concentration of higher-order speciesof a-syn, which is not known, would be lower inthe brain. For both the primary neuronal culturesand the intrastriatal injections, 5 mg of a-syn PFFor PAR–a-syn PFF was used for each experiment.Because the concentrations used for these andrelated studies are within the range of the con-centration of monomeric a-syn in the brain, theconcentration of a-syn PFF or PAR–a-syn PFF islikely to be within the range of the concentrationof higher-order species of a-syn in the brain. Pro-viding some specificity to these experiments isthe observation that the concentration of a-synPFF used here and in other studies has no toxicityin the absence of endogenous a-syn (2, 4).Recent studies have identified conformational

variants of a-syn strains that exhibit distinctneurotoxicity, seeding abilities, and propaga-tion, which contribute to different propertiesof a-synucleinopathies (24, 25). Given that a-synPFF induces PARP activation and PAR accumu-lation, PAR then accelerates a-syn fibrillizationand changes the biochemical properties of a-synPFF, converting it to a more toxic strain. Con-sistent with this notion, PAR–a-syn PFF showsan approximate 25-fold increase in a-syn aggre-gation and neurotoxicity compared to the paren-tal a-syn PFF. Moreover, PAR–a-syn PFF–injectedmice show an accelerated disease progression andphenotype compared to a-syn PFF–injected mice.In addition to PAR levels being increased in

cultured neurons and mouse brain, PAR levelsin PD are elevated in the substantia nigra andin the CSF. The elevation of PAR in the CSFand brains of patients with PD and evidenceof PARP activation in the substantia nigra ofpatients with PD suggest that PARP activationcontributes to the pathogenesis of PD throughparthanatos and conversion of a-syn to a moretoxic strain. In future studies, it will be impor-tant to determine whether the increase of PARin CSF from human PD correlates with diseaseseverity or progression. Moreover, it will be im-portant to determine whether it can serve as atheranostic biomarker for disease-modifyingtherapies. Because PARP inhibitors are currentlybeing used clinically as synergizing agents in thetreatment of cancer (26), they could be con-sidered for disease modification in PD (6).

Materials and methodsAnimals

C57BL/6 WT and PARP-1 KO mice were obtainedfrom the Jackson Laboratories (Bar Harbor, ME).The littermates of WT and PARP-1 KO mice wereused in experiments. All housing, breeding, andprocedures were performed according to the NIHGuide for the Care and Use of Experimental Ani-mals and approved by Johns Hopkins UniversityAnimal Care and Use Committee.

Preparation of a-syn PFF andPAR-a-syn PFF

Recombinant mouse a-syn proteins were puri-fied as previously described (5). After purifica-tion, bacterial endotoxins were removed usingToxineraser endotoxin removal kit (GeneScript).

a-syn PFF were prepared in PBS by constantlyagitating a-syn with a thermomixer (1,000 rpmat 37°C) (Eppendorf, Hamburg, Germany). After7 days of incubation, the a-syn aggregates werediluted to 0.1 mg/ml with PBS and sonicatedfor 30 s (0.5 sec pulse on/off) at 10% amplitude(Branson Digital Sonifier, Danbury, CT). Synthesisand purification of PAR polymer were performedas described (27). PAR-a-syn PFF was preparedby adding 5 nM or indicated dose of PAR ina-syn fibrillization reaction.

Stereotaxic injection of a-syn PFF

Two to 3-month-old WT and PARP-1 KO micewere deeply anesthetized with a mixture of keta-mine (100 mg/kg) and xylazine (10 mg/kg). PBS,a-syn PFF (5 mg), PAR-a-syn PFF (5 mg) or PARwas unilaterally injected into striatum (2 ml perhemisphere at 0.4 ml/min) with the followingcoordinates: anteroposterior (AP) = +0.2 mm,mediolateral (ML) = + 2.0 mm, dorsoventral(DV) = +2.8 mm from bregma. After the in-jection, the needle was maintained for an addi-tional 5 min for a complete absorption of thesolution. After surgery, animals were monitoredand post-surgical care was provided. Behavioraltests were performed 1, 3 and 6 months afterinjection and mice were euthanized for bio-chemical and histological analysis. For bio-chemical studies, tissues were immediatelydissected and frozen at -80°C. For histologicalstudies, mice were perfused with PBS and 4%PFA and brains were removed, followed by fix-ation in 4% PFA overnight and transfer to 30%sucrose for cryoprotection.

Thioflavin T (ThT) binding assay

a-syn fibrillization with or without PAR wasmonitored with ThT fluorescence. Aliquots of5 ul from the incubation mixture were takenat various time points, diluted to 100 ml with25 mM ThT in PBS, and incubate for 10 min atroom temperature. The fluorescence was recordedat 450 nm excitation and 510 nm emission usingSpectraMax plate reader (Molecular Devices,Sunnyvale, CA). The experiments were performedin triplicate.

Transmission electron microscopy(TEM) measurements

a-syn PFF or PAR-a-syn PFF were adsorbed toglow discharged 400 meshed carbon coatedcopper grids (Electron Microscopy Sciences,Hatfield, PA) for 2 min, quickly washed twicewith Tris-HCl (50 mM, pH 7.4), and floated upontwo drops of 0.75% uranyl formate for 30 s each.The grids were allowed to dry before imaging ona Phillips CM 120 TEM operating at 80 kV. Theimages were captured and digitized with anER-80 CCD (8 megapixel) by advanced micros-copy techniques.

Intracellular delivery of PAR

Purified PARwas intracellularly delivered usingBioPORTER (Genelnatis, San Diego, CA) accord-ing to the manufacturer’s instructions (9). PARpolymer was diluted to desired concentration

with PBS. The diluted solution was added tothe dried BioPORTER reagent and mixed gently,followed by incubation at room temperature for5min. The BioPORTER-PAR complex was addedto cell culture after a wash in serum-free mediaand incubated for 3–4 h at 37°C. Cultures weresubsequently used for experiments.

Tissue lysate preparation and Westernblot analysis

Human post mortem brain (Table S3) or mousebrain tissue were homogenized and prepared inlysis buffer [50 mM Tris-HCl (pH 7.4), 150 mMNaCl, 1 mM EDTA, 1% Triton x-100, 0.5% SDS,0.5% sodium-deoxycholate, phosphatase inhibi-tor mixture I and II (Sigma-Aldrich, St. Louis,MO), and complete protease inhibitor mixture(Roche, Indianapolis, IN)], using a Diax 900homogenizer (Sigma-Aldrich). After homogeni-zation, samples were rotated at 4°C for 30 minfor complete lysis, the homogenate was centri-fuged at 15,000 x g for 20 min and the super-natants were used for further analysis. Proteinlevels were quantified using the BCA assay (Pierce,Rockford, IL), samples were separated using SDS-polyacrylamide gels and transferred onto nitro-cellulose membranes. The membranes wereblocked with 5% non-fat milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1 h,probed using primary antibodies (Table S4)and incubated with appropriate HRP-conjugatedsecondary antibodies (Cell signaling, Danvers, MA).The bands were visualized by ECL substrate.

Immunoprecipitation (IP), overlay, andin vitro ribosylation assay of PAR

The deletion mutants of a-syn-GFP were gen-erated using site-directed mutagenesis (Agilent,Santa Clara, CA) according to the manufacturer’sinstructions. SH-SY5Y cells were transfected witheither a-syn-GFP or its deletion mutants byPolyfect (Qiagen, Hilden, Germany) for 36 hand then further incubated with 50 mM MNNGfor 15 min. The cells were washed 2 times withPBS and harvested with lysis buffer [50 mMTris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA,1% Triton x-100, 0.5% SDS, 0.5% sodium-deoxycholate, phosphatase inhibitor mixture Iand II (Sigma-Aldrich), and complete proteaseinhibitor mixture (Roche)]. The supernatantsafter centrifugation at 15,000 x g for 20 minwere used for measuring the protein concen-tration by BCA assay. The same amount ofproteins were incubated with anti-PAR anti-body (Table S4) overnight at 4°C, followed byincubation with PureProteome kappa Ig bindermagnetic beads (Millipore, Burlington, MA) for3 h at 4°C. The IP complexes were washed5 times with IP buffer and then denatured byboiling for 5 min after adding 2x Laemlli Bufferplus b-mercaptoethanol. For the PAR overlayassay, the equal amount of BSA or purifieda-syn protein was spotted onto a nitrocellulosemembrane. The membranes were washed oncewith TBS-T buffer and air-dried, followed byincubation with biotin-labeled PAR polymerfor 1 h at room temperature in TBS-T buffer.




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After washing 3 times in TBS-T buffer, the mem-branes were blocked with 5% non-fat milk for1 h, probed using anti-PAR antibody. The bandswere visualized by ECL substrate. For PARP-1in vitro ribosylation assay, 1 mg of recombinantPARP-1, activated DNA and NAD+ (Trevigen,Gaithersburg, MD) in the presence or absence ofindicated concentration of PARP inhibitors wereincubated in the PARP assay buffer (Trevigen)for 30 min at room temperature. For the sub-strate ribosylation assay of PARP-1, we used 2 or10 mg of purified a-syn and histone 2B (Abcam)protein under the same condition.

Endosome and exosome enrichment

Endosomes were enriched to detect internalizeda-syn-biotin PFF as previously described (5). Pri-mary cultured neurons from WT or PARP-1 KOwere incubated with a-syn-biotin PFF for1.5 h, followed by adding trypsin to remove themembrane-bound a-syn-biotin PFF. After wash-ing with PBS, neurons were harvested and lysedby aspirating a syringe 20 times in lysis buffer[250mMsucrose, 50mMTris-HCl (pH 7.4), 5mMMgCl2, 1mMEDTA, 1mMEGTA] with a completeprotease inhibitor mixture (Roche). The last pelletcontaining the endosomes were used for immu-noblot analysis after sequential centrifugation at1000 x g for 10 min, 16,000 x g for 20 min, and100,000 x g for 60 min at 4°C). Exosomes wereenriched to detect secreted a-syn as previouslydescribed (18). Culture supernatants of primarycortical neurons transduced with AAV-a-synfollowed by incubation with 500 mM NMDA for5 min were collected and spun at 300 x g for10 min to remove cells. The supernatants werethen sequentially centrifuged at 2000 × g for10 min, 10,000 × g for 30 min, and 100,000 × gfor 90 min at 4°C). The last pellet containingexosomes was washed once with PBS and cen-trifuged again at 100,000 × g for 90 min. Theremaining pellet was resuspended with lysisbuffer.

Cell culture, transfection, primaryneuronal culture and treatment

SH-SY5Y cells (ATCC, Manassas, VA) were cul-tured in DMEM containing 10% fetal bovineserum and penicillin/streptomycin at 37°C under5% CO2. The cells were transfected using PolyFectreagent (Qiagen) according to the manufacturer’sinstructions. Primary cortical neurons fromWT or PARP-1 KO embryos were prepared asdescribed previously (28). Briefly, the primarycortical neurons were cultured at embryonicday 16 in neurobasal media supplemented withB-27, 0.5 mM L-glutamine, penicillin and strep-tomycin (Invitrogen, Carlsbad, CA). ABT-888(1 or 10 mM), AG-014699 (1 mM), BMN 673 (1 or10 mM), Z-VAD (10 mM), Nec-1 (10 mM) or 3-MA(500 mM) were applied to neurons 1 h beforea-syn PFF treatment. The neuron culture mediawas replaced with fresh medium alone or in-cluding cell death inhibitors every 3-4 days.a-syn PFF was added at 7 days in vitro (DIV)and further incubated for indicated times fol-lowed by the cell death assay or biochemical

experiments. Primary neurons were infected withAAV9-control sgRNA or AAV9-PARP-1 sgRNA(ViGene Biosciences, Rockville, MD), and AAV-a-syn WT or AAV-a-syn A53T at DIV 4-5.

Cell death and viability assessment

Primary cultured cortical neurons were treatedwith 5 mg/ml of a-syn PFF or PAR-a-syn PFF for14 days. Percent of cell death was determined bystaining with 7 mM Hoechst 33342 and 2 mMpropidium iodide (PI) (Invitrogen). Images weretaken and countedby aZeissmicroscope equippedwith automated computer assisted software(Axiovision 4.6, Carl Zeiss, Dublin, CA). Afteradding Alamar Blue (Invitrogen), cell viabilitywas determined by fluorescence at an excitationwavelength 570 nm and an emission wavelength585 nm (29).

Microfluidic chambers

Triple compartmentmicrofluidicdevices (TCND1000)were obtained from Xona Microfluidic, LLC(Temecula, CA). Glass coverslips were preparedand coated as described, before being affixed tothe microfluidic device. Approximately 100,000WTor PARP-1 KOneuronswere plated per cham-ber individually. At 7 DIV, 5 mg/ml of a-syn PFFwas added into chamber 1. To control for direc-tion of flow, a 50 ml difference in media volumewasmaintained between chamber 1 and chamber2 and chamber 2 and chamber 3 according to themanufacturers’ instructions. Neurons were fixedon day 14 after a-syn PFF treatment using 4%paraformaldehyde in PBS. The chambers werethen processed for immunofluorescence stainingwith a p-a-syn antibody (Table S4).

Behavioral tests

The behavioral deficits in a-syn PFF injected WTor PARP-1 KO mice, a-syn PFF injected mice fedABT-888, and a-syn PFF or PAR-a-syn PFF in-jected mice were assessed by the pole test andthe grip strength test 1 week prior to sacrifice ofthe different cohorts. All the experiments wereperformed by investigators who were blind togenotypes or treatment condition and randomlyallocated to groups.

Pole test

A metal rod (75 cm long with a 9 mm diam-eter) wrapped with bandage gauze was used asthe pole. Before the actual test, the mice weretrained for two consecutive days and each train-ing session consisted of three test trials. Micewere placed 7.5 cm from the top of the pole. Thetime to turn and total time to reach the base ofthe pole were recorded. The end of test wasdefined by placement of all 4 paws on the base.The maximum cutoff time to stop the test andrecording was 60 s. After each trial, the mazewas cleaned with 70% ethanol. In Fig. 3E, weused 4 males and 4 females for WT mice in-jected with PBS, 4 males and 3 females for WTmice injected with PFF, 3 males and 3 femalesfor PARP-1 KO mice injected with PBS, and5 males and 2 females for PARP-1 KO miceinjected with PFF (Fig. 3E, left). We used 6 males

and 5 females for PBS injected mice fed withchow, 16 males and 9 females for PFF injectedmice fed with chow, 6 males and 6 females forPBS injected mice fed with ABT-888, and 15 malesand 10 females for PFF injected mice fed withABT-888 (Fig. 3E, right). In Fig. 5F, we used7 males and 7 females for PBS, 8 males and5 females for PFF, 13 males and 11 females forPAR-PFF, 6 males and 5 females for PAR-injected mice at 3 months, and 4 males and5 females for PBS, 5 males and 5 females forPFF, 7 males and 6 females for PAR-PFF, 5 malesand 4 females for PAR-injected mice at 6 months.

Grip strength test

Neuromuscular function was measured by deter-mining the maximal peak force developed by themice using a grip-strength meter (Bioseb, USA).Mice were placed onto a metal grid to grasp witheither fore or both limbs that are recorded as‘fore limb’ and ‘fore and hindlimb’, respectively.The tail was gently pulled and the force appliedto the grid before the mice lose their grip wasrecorded as the peak tension displayed in grams(g). In Fig. 3F, we used 4 males and 3 femalesfor WT mice injected with PBS, 3 males and3 females for WT mice injected with PFF, 3 malesand 3 females for PARP-1 KO mice injected withPBS, and 5 males and 2 females for PARP-1 KOmice injected with PFF (Fig. 3F, left). We used10 males and 8 females for PBS injected micefed with chow, 18 males and 9 females for PFFinjected mice fed with chow, 6 males and 6 fe-males for PBS injected mice fed with ABT-888,and 19 males and 11 females for PFF injectedmice fed with ABT-888 (Fig. 3F, right). In Fig. 5G,we used 3 males and 3 females for PBS, 3 malesand 3 females for PFF, 3 males and 3 females forPAR-PFF, 3 males and 3 females for PAR-injectedmice at 1 months, 4 males and 4 females for PBS,4 males and 4 females for PFF, 6 males and5 females for PAR-PFF, 4 males and 4 femalesfor PAR-injected mice at 3 months, and 5 malesand 4 females for PBS, 5 males and 5 femalesfor PFF, 8 males and 6 females for PAR-PFF,5 males and 4 females for PAR-injected miceat 6 months.

Dopamine and derivatives measurementusing HPLC

Biogenic amine concentrations were measuredby high-performance liquid chromatography withelectrochemical detection (HPLC-ECD). The stria-tum was rapidly removed from the brain, followedby weighing, then sonication in ice cold 0.01 mM ofperchloric acid containing 0.01% EDTA. The 60 ngof 3,4-dihydroxybenzylamine (DHBA) was used asan internal standard. After centrifugation at 15,000g for 30 min at 4°C, the supernatant was cleanedusing a 0.2 mm filter and 20 ml of supernatant wasanalyzed in the HPLC column (3 mm × 150 mm,C-18 reverse phase column, AcclaimTM PolarAdvantage II, Thermo Scientific) by a dual chan-nel coulochem III electrochemical detector (Model5300, ESA, Inc. Chelmsford, MA). The proteinconcentrations of tissue homogenates were mea-sured using the BCA protein assay kit (Pierce).




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Data were normalized to protein concentrationsand expressed in ng/mg protein.

Immunohistochemistry andimmunofluorescence

Mice were perfused with PBS and 4% PFA andbrains were removed, followed by fixation in4% PFA overnight and transfer to 30% sucrosefor cryoprotection. Immunohistochemistry (IHC)and immunofluorescence (IF) was performedon 40 mm thick serial brain sections. Primaryantibodies and working dilutions are detailedin Table S4. For histological studies, free-floatingsections were blocked with 10% goat serum inPBS with 0.2% Triton X-100 and incubated withTH or p-a-syn antibodies followed by incubationwith biotin-conjugated anti-rabbit or mouse anti-body, respectively. After three washes, ABC re-agent (Vector laboratories, Burlingame, CA) wasadded and the sections were developed usingSigmaFast DAB peroxidase substrate (Sigma-Aldrich). Sections were counterstained with Nissl(0.09% thionin). For the quantification, both TH-and Nissl-positive DA neurons from the SNpcregion were counted by an investigator whowas blind to genotypes or treatment conditionwith randomly allocated groups through opticalfractionators, the unbiased method for cell count-ing, using a computer-assisted image analysissystem consisting of an Axiophot photomicroscope(Carl Zeiss) equipped with a computer controlledmotorized stage (Ludl Electronics, Hawthorne,NY), a Hitachi HV C20 camera, and Stereo Inves-tigator software (MicroBright-Field, Williston, VT).The total number of TH-stained neurons andNissl counts were analyzed as previously described(16). For immunofluorescent studies, double-labeled sections with TH and p-a-syn antibodieswere incubated with a mixture of Alexa-fluor488- and 594-conjugated secondary antibodies(Invitrogen). The fluorescent images were acquiredby confocal scanning microscopy (LSM710, CarlZeiss). All the images were processed by the Zensoftware (Carl Zeiss). The selected area in thesignal intensity range of the threshold wasmeasured using ImageJ software.

Nitric oxide (NO) measurement

NO levels were measured using a NO assay kit(Abcam, Cambridge, MA) according to the man-ufacturer’s instructions. Briefly, the pellets of pri-mary neurons or SNpc tissues were washed withice-cold PBS, resuspended, and homogenized inice-cold assay buffer. The supernatant after cen-trifugation at 16,000 x g for 5 min at 4°C weredeproteinized and neutralized (pH 6.5-8) by add-ing 1M perchloric acid and 2M Potassium Hy-droxide. After centrifugation at 13,000 x g for15 min at 4°C, the supernatant was reacted withnitrate reductase and cofactors at room tempera-ture for 1 h to convert nitrate to nitrite. The amountof the azo compound converted from nitrite byGriess reagents were measured at OD540 nm.

Comet assay

The comet assay was performed according tothe manufacturer’s instructions (Trevigen). Briefly,

primary cortical neurons pre-treated with L-NAME(Sigma-Aldrich) followed by further incubationwith a-syn PFF were washed with ice-cold PBS(Ca2+ and Mg2+ free), harvested, and resuspendedin PBS at 1 x 105 cells/ml. After combining with1% low melting point agarose in PBS at 42°C,50 ml of the cell-agarose mixture was immedi-ately placed on the Comet slide at 4°C in thedark for 30 min. Slides were lysed in lysis buf-fer and immersed with alkaline unwinding solu-tion (200 mM NaOH, pH >13, 1 mM EDTA) for1 hour at room temperature. After electrophore-sis at 21 V for 30 min at 4°C, slides were rinsedtwice with dH2O), fixed with 70% ethanol for5 min, and then stained with SYBR green for5 min at 4°C. Images were captured using aZeiss epifluorescent microscope (Axiovert 200M)and analyzed with the tail positive cells (% oftotal cells) and tail length (the length from theedge of the nucleus to the end of the comet tail).

Proteinase K (PK) digestion of a-syn PFF

PK digestion was performed as previously de-scribed (30). Ten micrograms of a-syn PFF orPAR-a-syn PFF were mixed with 0.5 to 2.5 mg/mlof PK in PBS and incubated at 37°C for 30 min.The reaction was stopped by adding 1 mM PMSF,boiled with SDS-sample buffer for 5 min. Thebands of the PK digestion products were detectedby immunoblotting using epitope-specific a-synantibodies (Table S4).

Human CSF samples and PAR ELISA

Participants at the Johns Hopkins Universitysite of the NINDS Parkinson’s Disease Bio-marker Program (PDBP) underwent extensiveclinical and cognitive testing and a lumbar punc-ture annually. The CSF was centrifuged, aliquoted,and stored at -80°C within one hour of acquisi-tion. CSF samples were also obtained from theCleveland Clinic Lou Ruvo Center Brain HealthBiobank (CBH-biobank) under similar protocols.Two different clones (#19 and #25) of monoclonalanti-PAR antibody were used for PAR ELISA.Anti-PAR antibody (capture antibody, clone #19)(5 mg/ml) was coated on 96-well microtiter plate(NUNC, Cat #46051), various concentration ofpurified PAR (0-200 nM, positive control) andCSF samples from either normal or PD patientswere added to each well and incubated for 1 hat room temperature (RT). After washing theplates five times with PBST (0.05% Tween20 inPBS buffer), the biotinylated PAR antibody (detec-tion antibody, clone #25) was incubated for 1 hat RT. The color change was detected via HRP-conjugated streptavidin antibody (Thermo Sci-entific). Our assay can detect the PAR as low as3 pM and is saturated at 50 nM.

Statistical analysis

All data are represented as mean ± s.e.m. withat least 3 independent experiments. Statisticalanalysis was performed using GraphPad Prism 7.Differences between 2 means and among mul-tiple means were assessed by unpaired two-tailedstudent t test and ANOVA followed by Tukey’spost hoc test, respectively. The distribution of

data from human CSF samples were assessedwith D’Agostino & Pearson omnibus normalitytest and non-normally distributed data wereanalyzed with nonparametric test (Mann-Whitneytest). Assessments with P < 0.05 were consideredsignificant.

REFERENCES AND NOTES

1. M. Baba et al., Aggregation of a-synuclein in Lewy bodies ofsporadic Parkinson’s disease and dementia with Lewybodies. Am. J. Pathol. 152, 879–884 (1998). pmid: 9546347

2. L. A. Volpicelli-Daley et al., Exogenous a-synuclein fibrils induceLewy body pathology leading to synaptic dysfunction andneuron death. Neuron 72, 57–71 (2011). doi: 10.1016/j.neuron.2011.08.033; pmid: 21982369

3. H. Fujiwara et al., a-Synuclein is phosphorylated insynucleinopathy lesions. Nat. Cell Biol. 4, 160–164 (2002).doi: 10.1038/ncb748; pmid: 11813001

4. K. C. Luk et al., Pathological a-synuclein transmission initiatesParkinson-like neurodegeneration in nontransgenic mice.Science 338, 949–953 (2012). doi: 10.1126/science.1227157;pmid: 23161999

5. X. Mao et al., Pathological a-synuclein transmission initiated bybinding lymphocyte-activation gene 3. Science 353, aah3374(2016). doi: 10.1126/science.aah3374; pmid: 27708076

6. N. A. Berger et al., Opportunities for the repurposing of PARPinhibitors for the therapy of non-oncological diseases. Br. J.Pharmacol. 175, 192–222 (2018). doi: 10.1111/bph.13748;pmid: 28213892

7. T. M. Dawson, V. L. Dawson, Mitochondrial mechanisms ofneuronal cell death: Potential therapeutics. Annu. Rev.Pharmacol. Toxicol. 57, 437–454 (2017). doi: 10.1146/annurev-pharmtox-010716-105001 pmid: 28061689

8. M. Sisay, D. Edessa, PARP inhibitors as potential therapeuticagents for various cancers: Focus on niraparib and its firstglobal approval for maintenance therapy of gynecologiccancers. Gynecol. Oncol. Res. Pract. 4, 18 (2017). doi: 10.1186/s40661-017-0055-8; pmid: 29214031

9. S. A. Andrabi et al., Poly(ADP-ribose) (PAR) polymer is a deathsignal. Proc. Natl. Acad. Sci. U.S.A. 103, 18308–18313 (2006).doi: 10.1073/pnas.0606526103; pmid: 17116882

10. S.-W. Yu et al., Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor. Science297, 259–263 (2002). doi: 10.1126/science.1072221;pmid: 12114629

11. L. Swiech et al., In vivo interrogation of gene function in themammalian brain using CRISPR-Cas9. Nat. Biotechnol. 33,102–106 (2015). doi: 10.1038/nbt.3055; pmid: 25326897

12. A. A. Fatokun, V. L. Dawson, T. M. Dawson, Parthanatos:Mitochondrial-linked mechanisms and therapeuticopportunities. Br. J. Pharmacol. 171, 2000–2016 (2014).doi: 10.1111/bph.12416; pmid: 24684389

13. J. Zhang, V. L. Dawson, T. M. Dawson, S. H. Snyder, Nitric oxideactivation of poly(ADP-ribose) synthetase in neurotoxicity.Science 263, 687–689 (1994). doi: 10.1126/science.8080500;pmid: 8080500

14. M. Y. Kim, T. Zhang, W. L. Kraus, Poly(ADP-ribosyl)ation byPARP-1: ‘PAR-laying’ NAD+ into a nuclear signal.Genes Dev. 19, 1951–1967 (2005). doi: 10.1101/gad.1331805;pmid: 16140981

15. W. M. Bonner et al., gH2AX and cancer. Nat. Rev. Cancer 8,957–967 (2008). doi: 10.1038/nrc2523; pmid: 19005492

16. S. S. Karuppagounder et al., The c-Abl inhibitor, nilotinib,protects dopaminergic neurons in a preclinical animal model ofParkinson’s disease. Sci. Rep. 4, 4874 (2014). doi: 10.1038/srep04874; pmid: 24786396

17. M. Altmeyer et al., Liquid demixing of intrinsically disorderedproteins is seeded by poly(ADP-ribose). Nat. Commun. 6, 8088(2015). doi: 10.1038/ncomms9088; pmid: 26286827

18. E. Emmanouilidou et al., Cell-produced a-synuclein is secretedin a calcium-dependent manner by exosomes and impactsneuronal survival. J. Neurosci. 30, 6838–6851 (2010).doi: 10.1523/JNEUROSCI.5699-09.2010; pmid: 20484626

19. Y. Lee et al., Parthanatos mediates AIMP2-activated age-dependent dopaminergic neuronal loss. Nat. Neurosci. 16,1392–1400 (2013). doi: 10.1038/nn.3500; pmid: 23974709

20. L. Galluzzi et al., Molecular mechanisms of cell death:Recommendations of the Nomenclature Committee on CellDeath 2018. Cell Death Differ. 25, 486–541 (2018).doi: 10.1038/s41418-017-0012-4; pmid: 29362479

21. G. Codolo et al., Triggering of inflammasome by aggregateda-synuclein, an inflammatory response in synucleinopathies.




Dow

nloaded from

http://www.ncbi.nlm.nih.gov/pubmed/9546347http://dx.doi.org/10.1016/j.neuron.2011.08.033http://dx.doi.org/10.1016/j.neuron.2011.08.033http://www.ncbi.nlm.nih.gov/pubmed/21982369http://dx.doi.org/10.1038/ncb748http://www.ncbi.nlm.nih.gov/pubmed/11813001http://dx.doi.org/10.1126/science.1227157http://www.ncbi.nlm.nih.gov/pubmed/23161999http://dx.doi.org/10.1126/science.aah3374http://www.ncbi.nlm.nih.gov/pubmed/27708076http://dx.doi.org/10.1111/bph.13748http://www.ncbi.nlm.nih.gov/pubmed/28213892http://dx.doi.org/10.1146/annurev-pharmtox-010716-105001http://dx.doi.org/10.1146/annurev-pharmtox-010716-105001http://www.ncbi.nlm.nih.gov/pubmed/28061689http://dx.doi.org/10.1186/s40661-017-0055-8http://dx.doi.org/10.1186/s40661-017-0055-8http://www.ncbi.nlm.nih.gov/pubmed/29214031http://dx.doi.org/10.1073/pnas.0606526103http://www.ncbi.nlm.nih.gov/pubmed/17116882http://dx.doi.org/10.1126/science.1072221http://www.ncbi.nlm.nih.gov/pubmed/12114629http://dx.doi.org/10.1038/nbt.3055http://www.ncbi.nlm.nih.gov/pubmed/25326897http://dx.doi.org/10.1111/bph.12416http://www.ncbi.nlm.nih.gov/pubmed/24684389http://dx.doi.org/10.1126/science.8080500http://www.ncbi.nlm.nih.gov/pubmed/8080500http://dx.doi.org/10.1101/gad.1331805http://www.ncbi.nlm.nih.gov/pubmed/16140981http://dx.doi.org/10.1038/nrc2523http://www.ncbi.nlm.nih.gov/pubmed/19005492http://dx.doi.org/10.1038/srep04874http://dx.doi.org/10.1038/srep04874http://www.ncbi.nlm.nih.gov/pubmed/24786396http://dx.doi.org/10.1038/ncomms9088http://www.ncbi.nlm.nih.gov/pubmed/26286827http://dx.doi.org/10.1523/JNEUROSCI.5699-09.2010http://www.ncbi.nlm.nih.gov/pubmed/20484626http://dx.doi.org/10.1038/nn.3500http://www.ncbi.nlm.nih.gov/pubmed/23974709http://dx.doi.org/10.1038/s41418-017-0012-4http://www.ncbi.nlm.nih.gov/pubmed/29362479http://science.sciencemag.org/

PLOS ONE 8, e55375 (2013). doi: 10.1371/journal.pone.0055375; pmid: 23383169

22. W. Wang et al., Caspase-1 causes truncation and aggregationof the Parkinson’s disease-associated protein a-synuclein.Proc. Natl. Acad. Sci. U.S.A. 113, 9587–9592 (2016).doi: 10.1073/pnas.1610099113; pmid: 27482083

23. A. Iwai et al., The precursor protein of non-Ab component ofAlzheimer’s disease amyloid is a presynaptic protein of thecentral nervous system. Neuron 14, 467–475 (1995).doi: 10.1016/0896-6273(95)90302-X; pmid: 7857654

24. P. Brundin, R. Melki, Prying into the prion hypothesis forParkinson’s disease. J. Neurosci. 37, 9808–9818 (2017).doi: 10.1523/JNEUROSCI.1788-16.2017; pmid: 29021298

25. C. Peng, R. J. Gathagan, V. M.-Y. Lee, Distinct a-synucleinstrains and implications for heterogeneity amonga-synucleinopathies. Neurobiol. Dis. 109 (Pt. B), 209–218(2018). doi: 10.1016/j.nbd.2017.07.018; pmid: 28751258

26. C. J. Lord, A. Ashworth, PARP inhibitors: Synthetic lethality inthe clinic. Science 355, 1152–1158 (2017). doi: 10.1126/science.aam7344; pmid: 28302823

27. E. B. Affar et al., Immunological determination and sizecharacterization of poly(ADP-ribose) synthesized in vitro and invivo. Biochim. Biophys. Acta 1428, 137–146 (1999).doi: 10.1016/S0304-4165(99)00054-9; pmid: 10434031

28. T.-I. Kam et al., FcgRIIb-SHIP2 axis links Ab to tau pathologyby disrupting phosphoinositide metabolism in Alzheimer’sdisease model. eLife 5, e18691 (2016). doi: 10.7554/eLife.18691; pmid: 27834631

29. Y. Wang et al., A nuclease that mediates cell death induced byDNA damage and poly(ADP-ribose) polymerase-1. Science 354,aad6872 (2016). doi: 10.1126/science.aad6872; pmid: 27846469

30. J. L. Guo et al., Distinct a-synuclein strains differentiallypromote tau inclusions in neurons. Cell 154, 103–117 (2013).doi: 10.1016/j.cell.2013.05.057; pmid: 23827677

ACKNOWLEDGMENTS

We thank I.-H. Wu for graphic art assistance, H. Gu for collectingdata and writing assistance, N. Yoritomo and M. Gudavalli forassistance in patient recruitment and biospecimen collection, andthe patients and families that volunteer and participate in research.Funding: This work was supported by grants from the NIH/NINDS(P50NS38377, R37NS067525, NS082205, U01NS082133, andU01NS097049) and the JPB Foundation and by UO1 NS100610 andthe Jane and Lee Seidman Fund to J.B.L. We acknowledge thejoint participation by the Adrienne Helis Malvin Medical ResearchFoundation through its direct engagement in the continuous activeconduct of medical research in conjunction with the Johns HopkinsHospital and the Johns Hopkins University School of Medicineand the Foundation’s Parkinson’s Disease Programs M-2016. T.M.D. isthe Leonard and Madlyn Abramson Professor in NeurodegenerativeDiseases. Author contributions: Conceptualization: T.-I.K., V.L.D., andT.M.D. Methodology: T.-I.K., X.M., H.P., V.L.D., and T.M.D. Validation:T.-I.K., X.M., H.P., S.-C.C., and S.S.K. Formal analysis: T.-I.K., X.M.,H.P., S.-C.C., and S.S.K. Investigation: T.-I.K., X.M., H.P., S.-C.C., S.S.K.,G.E.U., S.B., S.P.Y., N.P., and C.Q. Resources: R.C., S.A.A., G.G.P., O.P.,J.C.T., L.M.B., J.B.L., A.P., H.S.K., L.S.R., V.L.D., and T.M.D. Writing—

original draft: T.-I.K., X.M., H.P., V.L.D., and T.M.D. Writing—reviewand editing: T.-I.K., H.P., V.L.D., and T.M.D. Visualization: T.-I.K., X.M.,and H.P. Supervision: V.L.D. and T.M.D. Funding acquisition: J.B.L.,V.L.D., and T.M.D. Competing interests: J.B.L. is a consultant forAcadia Pharmaceuticals, Avid Radiopharmaceuticals, Axovant,Bracco Radiopharmaceuticals, Eisai, GE Healthcare, andTakeda. T.M.D. is a member of the Board of Directors of theBachmann Strauss Dystonia and Parkinson’s Disease Foundation,is a member of the Executive Scientific Advisory Board of theMichael J. Fox Foundation for Parkinson’s Research, is aconsultant and advisor to Sun Pharma Advanced ResearchCompany Ltd., and serves on the advisory council of AligningScience Across Parkinson’s. These arrangements have beenreviewed and approved by the Johns Hopkins University inaccordance with its conflict of interest policies. T.-I.K., L.R.,S.A.A.,V.L.D., and T.M.D have filed a U.S. patent entitled“Detection of PAR in the CSF of Patients with Parkinson’sDisease.” Data and materials availability: All data are availablein the manuscript or the supplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/362/6414/eaat8407/suppl/DC1Figs. S1 to S15Tables S1 to S4

10 April 2018; resubmitted 13 August 2018Accepted 26 September 201810.1126/science.aat8407




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-synuclein neurodegeneration in Parkinson's diseaseαPoly(ADP-ribose) drives pathologic

and Valina L. DawsonC. Troncoso, Lynn M. Bekris, James B. Leverenz, Alexander Pantelyat, Han Seok Ko, Liana S. Rosenthal, Ted M. DawsonPil Yun, Saurav Brahmachari, Nikhil Panicker, Rong Chen, Shaida A. Andrabi, Chen Qi, Guy G. Poirier, Olga Pletnikova, Juan Tae-In Kam, Xiaobo Mao, Hyejin Park, Shih-Ching Chou, Senthilkumar S. Karuppagounder, George Essien Umanah, Seung

DOI: 10.1126/science.aat8407 (6414), eaat8407.362Science

, this issue p. eaat8407; see also p. 521Science-syn to a more toxic strain.αof

of PD patients indicates that PARP activation contributes to the pathogenesis of PD through parthanatos and conversion -syn PFF. An increase of PAR in the cerebrospinal fluid and evidence of PARP activation in the substantia nigraα−PAR

-syn PFF to a strain that was 25-fold more toxic, termedαinduced PARP-1 activation converted −-syn PFFαof PAR by 1 (PARP-1) and inhibition of PARP or knockout of PARP-1 protected mice from pathology. The generation−polymerase

ribose) (PAR)−-diphosphate′activated poly(adenosine 5−-synαBrundin and Wyse). They found that pathologic -syn PFF) model of sporadic PD (see the Perspective byα-syn preformed fibril (α studied the et al.understood. Kam

-syn) leads to neurodegeneration in Parkinson's disease (PD) remains poorlyα-synuclein (αHow pathologic -synuclein toxicityαPAR promotes

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Poly(ADP-ribose) drives pathologic α-synuclein neurodegeneration … · RESEARCH ARTICLE SUMMARY NEURODEGENERATION Poly(ADP-ribose) drives pathologic a-synuclein neurodegeneration