Asparagine endopeptidase cleaves α-synuclein and mediates pathologic activities in Parkinson s disease

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1 Asprgin nopptis lvs α-synulin n mits pthologi tivitis in Prkinson s iss Zhnto Zhng 2, Song Su Kng 2, Xi iu 2, Eun H Ahn 2, Zhohui Zhng 1, i H 3, P Mihl Iuvon 3,4, Du M Duong 5,6, Nihols T Syfri 5,6, Mtthw J Bnsky 7, Frri P Mnfrsson 7, ingjing Jin 8, Yi E Sun 8, Jin-Zhi Wng 9 & Kqing Y 2 Aggrgt forms of -synulin ply ruil rol in th pthognsis of synulinopthis suh s Prkinson s iss (PD). Howvr, th molulr mhnisms unrlying th pthogni ffts of -synulin r not ompltly unrstoo. Hr w show tht sprgin nopptis () lvs humn -synulin, triggrs its ggrgtion n slts its nurotoxiity, thus ling to opminrgi nuronl loss n motor impirmnts in mous mol. is tivt n lvs humn -synulin t N3 in n g-pnnt mnnr. is highly tivt in humn rins with PD, n it frgmnts -synulin, whih is foun ggrgt in wy ois. Ovrxprssion of th -lv -synulin 13 frgmnt in th sustnti nigr inus oth opminrgi nuronl loss n movmnt fts in mi. In ontrst, inhiition of -mit lvg of -synulin (wil typ n A53T mutnt) iminishs -synulin s pthologi ffts. Togthr, ths finings support s rol s ky mitor of -synulin-rlt tiopthologil ffts in PD. PD is hrtriz y th gnrtion of opminrgi nurons in th sustnti nigr (SN) prs ompt. Th tiology of PD pprs to multiftoril, involving oth gnti n nvironmntl omponnts. To t, svrl llulr mhnisms, suh s rrnt protin foling, oxitiv strss n mitohonril ysfuntion hv n implit in th vlopmnt n progrssion of this iss 1. Most PD ss r spori, n thir pthognsis is unlr. Howvr, svrl ss of fmilil PD hv n sri in whih mutnt gns hv n intifi s ing ustiv in this isorr. Ths gns inlu th unnt prsynpti protin α-synulin 2,3 n two omponnts of th protsom pthwy: 1 uiquitin C-trminl hyrols 4 n prkin 5. Th misfoling of α-synulin, th min omponnt of wy pthology, plys ky rol in th tiopthognsis of PD, us th isorr is hrtriz y th umultion of intrnuronl protin ggrgts (wy ois n wy nurits) 6. Svrl muttions in th gn noing α-synulin, inluing A3P, A53T n E64K, hv n intifi in ominnt forms of fmilil PD 2,3. Morovr, uplition or triplition of th wil-typ α-synulin-noing gn lso uss PD, thus initing tht n inrs α-synulin protin lvl is suffiint to us th iss. Furthrmor, singl-nuloti polymorphisms in th α-synulin-noing gn r ssoit with n inrs risk of vloping spori PD 7. Th potntil mhnisms of α-synulin in PD hv n xplor y trnsgni xprssion of humn α-synulin in vrious mols 8. Although ths stuis hv monstrt pthologil fft rsulting from trnsgni xprssion, inluing th formtion of inlusion ois, th molulr mhnisms involv in nurognrtion rmin osur. Th physiologil funtion of α-synulin is poorly unrstoo 11. α-synulin is wily xprss in th nrvous systm, whr it is foun in prsynpti nrv trminls, losly ssoit with prsynpti vsils 11. Th nurotoxiity of α-synulin my rlt to its oligomriztion n firilliztion 12 or my u to loss of funtionl protin, owing to th formtion of insolul ggrgts. Mounting vin inits tht C-trminl truntion promots α-synulin firilliztion 1316, in pross mit y numrous protss suh s lpin 17,18 n thpsin D 19,2. Howvr, whthr ths protss protolytilly lv α-synulin n mit thir pthologil ffts in n g-pnnt mnnr in PD tiopthology rmins unlr. Mmmlin (lso ll lgumin) is n nolysosoml ystin prots tht lvs protins ftr sprgin rsius. is synthsiz s n intiv zymogn, n its tivtion rquirs squntil rmovl of C- n N-trminl propptis t iffrnt ph 1 Dprtmnt of Nurology, nmin Hospitl of Wuhn Univrsity, Wuhn, Chin. 2 Dprtmnt of Pthology n ortory Miin, Emory Univrsity Shool of Miin, Atlnt, Gorgi, USA. 3 Dprtmnt of Ophthlmology, Emory Univrsity Shool of Miin, Atlnt, Gorgi, USA. 4 Dprtmnt of Phrmology, Emory Univrsity Shool of Miin, Atlnt, Gorgi, USA. 5 Dprtmnt of Biohmistry, Emory Univrsity Shool of Miin, Atlnt, Gorgi, USA. 6 Cntr for Nurognrtiv Disss, Emory Univrsity Shool of Miin, Atlnt, Gorgi, USA. 7 Trnsltionl Sin n Molulr Miin, Mihign Stt Univrsity, Collg of Humn Miin, n Hunstin Nurosin Cntr, Mry Hlth Sint Mry s, Grn pis, Mihign, USA. 8 Trnsltionl Cntr for Stm Cll srh, Tongji Hospitl, Dprtmnt of gnrtiv Miin, Tongji Univrsity Shool of Miin, Shnghi, Chin. 9 Pthophysiology Dprtmnt, Shool of Bsi Miin n th Collortiv Innovtion Cntr for Brin Sin, Ky ortory of th Ministry of Eution of Chin for Nurologil Disorrs, Tongji Mil Collg, Huzhong Univrsity of Sin n Thnology, Wuhn, Chin. Corrsponn shoul rss to K.Y. (ky@mory.u),.j. (lingjingjin@163.om) or J.-Z.W. (wngjz@mils.tjmu.u.n). iv 22 Dmr 216; pt 14 Jun 217; pulish onlin 3 July 217; oi:.38/nsm.3433 ntur struturl & molulr iology vn onlin pulition

2 thrshols 222. tivity is ontroll y th ystin prots inhiitor ysttin C 23. ntly, w hv rport tht nuronl is tivt y iosis uring xitotoxiity n ontriuts to nuronl poptosis y gring th DNs inhiitor SET 24,25. Morovr, -gnrt SET frgmnts inhiit protin phosphts-2a n nhn tu hyprphosphoryltion 26. W hv lso shown tht lvs TA DNA-ining protin 43 in rin smpls from humns with frontotmporl lor gnrtion 27. Most rntly, w hv foun tht lvs tu t rsius N255 n N368, thry miting formtion of nurofirillry tngls in humn rins with Alzhimr s iss (AD) 28. Furthr, w hv foun tht ts s δ-srts tht lvs APP t rsius N3 n N585, thry rgulting BACE1 frgmnttion of th C-trminl frgmnt of APP n rsulting in β-myloi gnrtion. Dltion of from tu P31S or 5XFAD trnsgni mi sustntilly llvits th pthologil n ognitiv fts 28,29. PD is hroni nurognrtiv iss, n ging is th mjor risk ftor for PD vlopmnt. W hv rntly rport tht protin lvls r inrs in th rin in n g-pnnt mnnr 28,29. Hn, it is possil tht might lso pross α-synulin in th rins of popl with PD uring ging. In th urrnt stuy, w tst this hypothsis n foun tht lvs α-synulin t N3 in th humn PD rin n promots α-synulin ggrgtion n nurotoxiity. Intrstingly, is tivt in n g-pnnt mnnr n ontriuts to n/or is onurrnt with α-synulin truntion in th SN. Ovrxprssion of th α-synulin 13 frgmnt in th ront SN inus opminrgi nurognrtion n rsultnt motor phnotyp, whrs lok of lvg of α-synulin (ithr wil typ or A53T mutnt) y mliorts ths α-synulin-mit pthologil ffts. Hn, our osrvtions monstrt tht ontriuts to th α-synulin pthogni tivity in PD. ESUTS Clvg of -synulin t N3 in PD n wy oy mnti To invstigt th protolyti prossing of α-synulin in PD, w onut protomi nlysis with immunopripitt α-synulin from PD rins. Notly, CMS/MS nlysis rvl n α- synulin ppti frgmnt lv t N3 from th prtil trypti pptis (Fig. 1). To tt th α-synulin N3 frgmnt, w gnrt nopitop ntioy (signt nti-n3) tht spifilly rogniz th lv α-synulin N3 frgmnt ut not th full-lngth protin. EISA msurmnts monstrt tht this ntioy ws highly sltiv for th α-synulin 13 frgmnt rthr thn intt α-synulin (Supplmntry Fig. 1). Wstrn lot nlysis with rominnt full-lngth () α-synulin n its frgmnt omprising rsius 13 onfirm tht this ntioy sltivly rogniz α-synulin 13 ut not α-synulin. Th ntioy lso immunopripitt th lv N3 frgmnt from PD rins (Fig. ). To onfirm tht α-synulin lvg ftr N3 in ours in popl with synulinopthy, w onut immunohistohmistry (IHC) ssys. IHC nlysis of rin stions with ntiαsynulin N3 lrly init tht α-synulin ws lv ftr N3 in rins with PD or wy oy mnti (BD) ut not in ontrol rins without synulinopthy. Th ntioy spifiity ws orroort with loking ppti (Fig. 1). Immunofluorsn (IF) stining onfirm tht th α-synulin N3 frgmnt ws prsnt s ggrgts in wy ois n synpti struturs (Fig. 1 n Supplmntry Fig. 1). Immunolotting with th α-synulin N3spifi ntioy rvl tht α-synulin N3 ws lvt spifilly in oth th solul n insolul frtions of SN tissus from PD rins, n its lvl ws muh highr thn tht osrv in ortil tissus from th sm sujts. By ontrst, α-synulin N3 ws srly ttl in oth rin rgions in ontrols without synulinopthy (Fig. 1f). mrkly, α-synulin N3 ws rily ttl in rins with BD ompr with ontrols. Ths finings wr furthr onfirm with nti-α-synulin ntioy. Intrstingly, ws tivt in th BD rin smpls, n this tivtion orrlt with α-synulin frgmnttion pttrns (Fig. 1g n Supplmntry Fig. 1). Thus, α-synulin N3 frgmnttion ourr prominntly in rins with PD or BD ut not in ontrols without synulinopthy. uts humn -synulin t rsiu N3 is th only rport mmmlin nzym tht lvs C trminl to sprgin. Hn, w hypothsiz tht is rsponsil for th lvg of α-synulin ftr N3. To tst this hypothsis, w trnsft N-trminlly -tgg humn α-synulin into HEK293 lls n inut ll lysts with kiny lysts (ph 7.4 or 6.) prpr from wil-typ or -knokout mi. A trunt α-synulin n ws sltivly osrv in wil-typ kiny lysts t ph 6. ut ws not osrv in -knokout kiny lysts or t ph 7.4. Th lvg of α-synulin ws ronstitut whn xognous tiv ws to th -knokout rtions (Fig. 2). nzymti tivity in th rtions ws onfirm (Fig. 2). -tgg α-synulin ws lso lv in mous rin lysts in th sm pttrn s tht in kiny lysts (Fig. 2). W lso tst th lvg of C-trminlly -tgg α-synulin in mous kiny n rin lysts, n foun th sm rsults (Fig. 2,). Whn α-synulin ws inut with wil-typ kiny lysts for 5, or min, th intnsity of th trunt α-synulin frgmnt inrs ovr tim t ph 6. (Fig. 2), thus initing tht in th tiv kiny lysts trunts α-synulin in tim-pnnt mnnr. Cys189 is ssntil for th prots tivity of, n lvg ftr N323 is oligtory for th mturtion of th prots 3. As xpt, α-synulin ws lv y wil-typ ut not y mutt (C189S or N323A), thry initing tht s nzymti tivity ws rsponsil for th osrv α-synulin protolyti lvg (Fig. 2f). is sltivly inhiit y th smll ppti AENK ut not th ontrol ppti AEQK 24. Aoringly, AENK ut not AEQK sltivly ntgoniz -α-synulin lvg y tiv in ospnnt mnnr (Fig. 2g). Furthrmor, rominnt tiv irtly lv rominnt α-synulin in tim-pnnt mnnr, n th rsultnt frgmnt ws rogniz y ntiα-synulin N3 ntioy. Th lvg ws lok y th inhiitor AENK (Fig. 2h,i). Togthr, ths rsults init tht α-synulin is irt iologil sustrt of. To trmin whthr thr r multipl -lvg sits in α-synulin, w onut mpping ssy with vrity of trunt α-synulin rominnt protins. α-synulin prou frgmnt of th sm siz s th 13 frgmnt, thus initing tht α-synulin ws ut nr rsiu 3 (Supplmntry Fig. 2). MS nlysis intifi th α-synulin N3 frgmnt from purifi mmmlin GST-α-synulin rominnt protins lv y tiv in vitro (Supplmntry Fig. 2). Bus sltivly lvs its sustrts ftr sprgin rsius, n humn α-synulin ontins thr sprgin rsius (N65, N3 n N122), w prpr thr α-synulin point mutnts with sprgin hng to lnin. Both N-trminl n C-trminl -tgg α-synulin N65A n N122A mutnts wr lv s ffiintly s wil-typ α-synulin y, whrs th N3A muttion ompltly olish lvg (Supplmntry Fig. 2,). Intrstingly, mous vn onlin pulition ntur struturl & molulr iology

3 rtils [M2H] y, z ions ltiv unn, y12 2, ions WB: Antiα-synulin N z z z z z114 z1 18 z z Anti-α-synulin N WB: Anti-N3 13 Anti-N WB: Antiα-synulin C [M3H]3 IP αsy n N 3 4 G A G SI A A A T G F V K K D Q G K N Ig G ll l ng th Fu 1 N3 Thioflvin S Mrg p-s129 Cortx MW (kd) 5 SN PD PD Solul frtion, WB: ntiα-synulin N3 Thioflvin S Bl BD p ok pt ing i l C f -lv α-synulin PD BD with loking ppti BD tro PD Positiv lls/visul fil on Mrg SDS frtion, WB: ntiα-synulin N3 Clv WB: nti-α-synulin (syn1) Pro- 5 Ativ WB: nti- 5 Pro- Ativ WB: nti- ro on t C.5 l, PD ort, x C on ort tro x l, S PD N,S N ro on t.5. C....5 tro on C WB: nti-α-tuulin. l, PD ort x, C or on t tro x l, S PD N,S N D. l 5 B on WB: nti-α-synulin (syn1) 5 C Clv.1 tro.2 l SDS frtion, WB: ntiα-synulin N3.3.2 D B Cytosol frtion, WB: ntiα-synulin N3 N3/α-synulin BD N3/α-synulin Clv /pro- g Clv /pro- WB: nti-α-tuulin -lv α-synulin m/z Figur 1 α-synulin is trunt ftr N3 in PD n BD rins. () MS/MS sptrum showing th lvg of α-synulin ftr N3 in rin smpls from sujts with PD. () Wstrn lot (WB) with nti-α-synulin ntiois. α-synulin or th α-synulin13 frgmnt wr tt y immunolotting with ntiα-synulin N-trminus (N) ntioy, ntiα-synulin C-trminus (C) ntioy n ntiα-synulin N3 ntioy. MW, molulr wight. () Immunopripittion (IP) of α-synulin (α-syn) from humn PD rin lysts with ntiα-synulin N-trminus or ntiα-synulin N3 ntiois. () Immunohistohmistry of th α-synulin N3 frgmnt in SN stions from popl with PD n ortx stions from popl with BD. Sl r, 5 µm. Br grph, quntifition of positiv lls in smpls. Dt r mn ± s..m.; n = 6 ptints; P <.5 y on-wy ANOVA. () Brin stions wr immunostin with ntiα-synulin N3 or ntiphospho (p)-s129-α-synulin (r) n thn stin with thioflvin S (grn), whih stins wy ois. Sl rs, 2 µm. Imgs r rprsnttiv of nin stions from thr sujts with BD. (f,g) Wstrn lots showing th prsn of th α-synulin N3 frgmnt in solul n insolul frtions of tissus from popl with PD (f) or BD (g), ut not g-mth ontrol tissus. α-tuulin, loing ontrol. Br grphs show quntifition s mn ± s..m.; n = 3 ptints in f, n = 5 ptints in g; P <.5 ompr with ontrol, y on-wy ANOVA in f n two-til Stunt s t tst in g. sults for n inpnnt ohort of smpls r shown in Supplmntry Figur. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. Sour t for grphs r vill onlin. ntur struturl & molulr iology vn onlin pulition

4 ominnt,, 5, ph ominnt tivity (AFU) ph Brin lysts ph ominnt ph ominnt / / WB: nti- WB: ntiα-synulin N WB: ntiα-synulin N3 WB: nti- / 7.4 / 6. / 6. / / / / / / / / / / / / / / / WB: ntiα-synulin C Full lngth Clv Full lngth Clv Clv Full lngth Clv Full lngth Clv Full lngth Clv f g ominnt tivity (AFU) 5 -α-synulin kiny lyst 5 min min min WB: nti- WB: nti- WB: nti-my -α-synulin kiny lyst ph 7.4 DMSO WB: nti-,, 5, ph AENK AEQK Brin lysts ph / / α-synulin-, WB: nti- My- WT C189S N323A ph 6. AENK / / 6. / 6. Full lngth Clv (ph) Full lngth Clv Full lngth Clv Full lngth Clv h i 5 min Intiv WB: nti-α-synulin WB: ntiα-synulin N3 Silvr stining Intiv Ativ Ativ DMSO min 2 min Intiv Ativ AENK 1 mm Intiv AENK mm AEQK mm Ativ WB: nti-α-synulin WB: ntiα-synulin N3 Silvr stining Full lngth Clv Full lngth Figur 2 α-synulin is sustrt of. () α-synulin lvg ssy in kiny lysts. N-trminl -tgg α-synulin ws inut with kiny lysts from wil-typ (/) or -knokout ( / ) mi t ph 7.4 or ph 6. t C for min. Wstrn lot (WB) showing α-synulin lvg t ph 6. (top) whn ws tivt (ottom). Br grph shows t s mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 ompr with th ph 7.4 group y on-wy ANOVA. AFU, ritrry fluorsn units. () α-synulin lvg ssy in rin lysts. N-trminlly -tgg α-synulin ws inut with rin lysts from wil-typ or -knokout mi t ph 7.4 or ph 6. t C for min. (,) Prossing of C-trminlly -tgg α-synulin in mous kiny lysts () n rin lysts (). () Tim-pnnt lvg of α-synulin y. (f) α-synulin lvg y wil-typ n mutnt. (g) Th protolysis of α-synulin is lok y AENK ppti ut not AEQK ppti (top). Th fft of AENK on ws onfirm y n nzymti tivity ssy (ottom). Grph shows mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 ompr with wil typ, y on-wy ANOVA. (h) ominnt hs high lvg tivity towr rominnt α-synulin. Th -gnrt α-synulin frgmnt ws rogniz y ntiα-synulin N3 ntioy. Silvr-stin gl shows ll th frgmnts. (i) inhiitor loks th lvg of rominnt α-synulin y. Silvr-stin gl shows ll th frgmnts. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. Sour t for grphs r vill onlin. AEQK (mm) Clv α-synulin os not ontin rsiu N3, ut humn α-synulin os. Aoringly, humn ut not mous α-synulin ws sltivly frgmnt y (Supplmntry Fig. 2,f). To onfirm th intrtion twn n α-synulin, w prform oimmunopripittion n foun tht α-synulin intrt with in humn PD rins (Supplmntry Fig. 3), whr it my lv α-synulin. Th protolyti tions in humn PD rin lysts wr sltivly lok y nti- ut not ontrol IgG, thus supporting th spifi prossing of α-synulin N3 y (Supplmntry Fig. 3). Immunofluorsn stining with humn rin slis rvl tht stritly ololiz with AMP th spifi mrkr for lysosoms in ontrol rins, whrs it lk out into th ytoplsm n ws not tightly ololiz with AMP1 in PD rins (Supplmntry Fig. 3). In, our sullulr frtiontion ssy lso monstrt tht th tivity of ws nhn in th ytoplsm in PD rins ompr with ontrols (Supplmntry Fig. 3). Clvg ssys with xognous α-synulin rominnt protins monstrt tht PD n BD rin ytosoli frtions isply muh strongr N3 lvg tivitis thn thos of ontrol ytosoli frtions, whrs th lysosoml frtions xhiit similr ffts mong ontrol, PD n BD rins (Supplmntry Fig. 3). Ths rsults suggst tht my lk from th lysosoms into th ytoplsm, whr it lvs α-synulin in synulinopthis. lvs -synulin inpnntly of post-trnsltionl moifitions Phosphoryltion of α-synulin t S129 n Y125 rgults α-synulin ggrgtion n toxiity 31. To tst whthr S129 phosphoryltion influns α-synulin lvg y, w onut n in vitro lvg ssy using rominnt α-synulin n S129-phosphorylt α-synulin. Th S129-phosphorylt n nonphosphorylt α-synulin wr lv in tim-pnnt mnnr with similr vn onlin pulition ntur struturl & molulr iology

5 SNCA-trnsgni mi Cortx WB: nti-α-synulin (syn1) Wil-typ mi Sn-knokout mi SN SN Cortx SN Cortx SN Ag (months) 5 WB: nti-n3 Clv/full lngth Ag (months) Cortx WB: nti-α-tuulin SNCA-trnsgni mi tivity (AFU) 4, 3, 2, Ag (months) rts, thus initing tht phosphoryltion of α-synulin t S129 os not intrfr with lvg (Supplmntry Fig. 4). α-synulin is lso phosphorylt t Y125 y th Sr fmily protin tyrosin kins Fyn 32. Nonthlss, oth wil-typ humn α-synulin n th Y125F mutnt xhiit omprl lvg rts y (Supplmntry Fig. 4). Morovr, phosphoryltion of α-synulin t Y125 y ovrxprssion of onstitutivly tiv or kins- Fyn mutnts i not fft its lvg rt, thus furthr onfirming tht Y125 phosphoryltion os not fft humn α-synulin lvg (Supplmntry Fig. 4,). W lso tst th lvg rt of th fmilil PD-ssoit A53T n A3P missns muttions n foun tht ths mutnts wr lso lv t th sm rt s wil-typ α-synulin (Supplmntry Fig. 4). A numr of protinss hv n implit in α-synulin lvg, inluing lpin, thpsin D n th protsom 33. To xplor thir potntil ffts on N3 frgmnttion, w us spifi inhiitors or prots-inhiitor oktil inhiiting srin protss, minpptiss, ystin protss n i protss. Notly, α-synulin lvg ws inhiit y only th ppti inhiitor AENK ut not y ny of th othr prots inhiitors or th prots-inhiitor oktil (Supplmntry Fig. 4f). Thus, ths rsults init tht is th mjor prots lving humn α-synulin t N3, n its protolyti tivity is inpnnt of othr post-trnsltionl moifitions. is tivt n lvs -synulin in n g-pnnt mnnr W hv rntly rport tht is sltivly uprgult n tivt in th rin in n g-pnnt mnnr 28,29. To xplor whthr this nzym trunts humn α-synulin in similr tmporl pttrn, w us mous trnsgni mol with th mous α-synulin-noing gn (Sn) rpl with humn vrsion, SN Cortx SN tivity (AFU) 4, 3, 2,, ortx n w prform immunolotting on SN n ortx-tissu smpls from mi t 1.5, 3, 5 n 7 months of g. W foun tht α-synulin ws lv t N3 in n g-pnnt mnnr. Frgmnttion ws lrly mor prominnt in th SN thn th ortx, in mnnr tightly oupl to th sptil n tmporl pttrns of tivity. In ontrst, no lvg signls wr tt in wil-typ or Sn-knokout SN tissus (Fig. 3). Thrfor, ths finings init tht spifilly lvs humn α-synulin in n g-pnnt mnnr, mor prominntly in th SN rgion thn in th ortx. W lso xtn ths stuis to wil-typ mi n foun tht tivity inrs in n g-pnnt mnnr, n th SN tivity ws muh highr thn tht in th ortx (Supplmntry Fig. 5). Intrstingly, w osrv th sm rsult in SN n ortx smpls from humn PD rins (Fig. 3). Hn, is pron to ing tivt in th SN vrsus th ortx. Clvg of -synulin y promots its ggrgtion n toxiity To trmin th fft of lvg of α-synulin on firilliztion, w monitor th kintis of filmnt formtion y α-synulin or th -riv α-synulin 13 frgmnt t iffrnt onntrtions, y using thioflvin T stining. W foun tht th α-synulin 13 frgmnt ggrgt muh fstr n to grtr xtnt thn i α-synulin (Fig. 4). Ultrntrifugtion ssys rvl tht mor ggrgt α-synulin 13 thn α-synulin ws prsnt in th pllt (Fig. 4). Eltron mirosopy onfirm th ggrgtion of α-synulin into firils (Supplmntry Fig. 6). Furthrmor, th α-synulin 13 frgmnt signifintly promot th ggrgtion of α-synulin vn t vry low onntrtions (Fig. 4). W lso tst th sing pity of firils from th α-synulin 13 frgmnt n tht from th full-lngth protin., SN PD, ortx PD, SN BD, ortx Figur 3 is tivt n lvs α-synulin in n g-pnnt mnnr. () Wstrn lot (WB) showing th prossing of α-synulin uring ging in humn SNCA-trnsgni mi. α-tuulin, loing ontrol. () Quntifition of -lv α-synulin in th ortx n SN of SNCAtrnsgni mi t iffrnt gs. Dt r mn ± s..m.; n = 3 mi pr group; P <.5 y on-wy ANOVA. () tivity in th ortx n SN in SNCA-trnsgni mi t iffrnt gs. Dt r mn ± s..m.; n = 5 mi pr group; P <.5 y on-wy ANOVA. AFU, ritrry fluorsn units. () tivity in th ortx n SN from PD, BD n g-mth ontrol tissus. Dt r mn ± s..m.; n = 4 iffrnt sujts for ontrol n BD smpls; n = 3 iffrnt sujts for PD smpls; P <.5 y on-wy ANOVA. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. Sour t for grphs r vill onlin. ntur struturl & molulr iology vn onlin pulition 5

6 Fluorsn intnsity (ritrry units) AAV- AAV-13 4, 3, 2, TH Fluorsn intnsity (ritrry units) W foun tht α-synulin 13 firils, ompr with α-synulin firils, s th α-synulin with muh highr pity (Fig. 4). Thus, ths finings suggst tht lvg of α-synulin my promot α-synulin ggrgtion. α-synulin truntion n oligomriztion r tightly ssoit with its nurotoxiity 12 ; thrfor, w onut opminrgi nuronl ll th ssys. As xpt, ovrxprssion of α-synulin inu poptosis of opminrgi nurons, n this fft ws mor pronoun with th α-synulin 13 frgmnt (Fig. 4). In ition, th α-synulin 13 frgmnt form mor ggrgts, s init y IF stining with n ntioy spifi to ggrgt α-synulin (Fig. 4f). Furthrmor, th α-synulin 13 frgmnt form mor unnt highr-molulr-wight spis thn i α-synulin (Fig. 4g). Hn, th -riv α-synulin N3 frgmnt is mor pron to ggrgtion n xhiits strongr nurotoxiity. IF stining n sullulr frtiontion ssys init tht most n trunt α-synulin loliz in th ytosoli frtion (Supplmntry Fig. 6,). Apoptosis (%) Full lngth 13 Numr of TH /MAP2 lls pr visul fil Fluorsn intnsity (ritrry units) Tim () Tim () Tim () Tim () Dy Full lngth 13 2, Full lngth Dy 1 # 13 Dy 2 Dy 4 Dy 6 Dy 1 Dy 2 Dy 4 Dy 6 S P S P S P S P S P S P S P S P S P S P 5 # Coomssi lu stining Full lngth, 2 µm Full lngth, 7 µm Full lngth, 35 µm 13, 2 µm 2,5 13, 7 µm 5 13, 35 µm 2, TUNE WB: nti-α-synulin (syn1) Mrg f IF: ntiggrgt α-synulin Full lngth WB: nti-α-synulin (syn1) Full lngth 13 g Totl Tim () -lv -synulin inus opmin nuronl loss n motor fiits in vivo To ssss th pthologil rols of th α-synulin 13 frgmnt in vivo, w us nossoit viruss (AAVs) xprssing ithr wil-typ α-synulin or th 13 frgmnt, whih wr livr y uniltrl injtion into th mous SN. Four months ftr th vtor livry, w prform IHC n IF stining on th rin stions to vlit trnsution. IHC nlysis with B59, n ntioy rognizing rsius 1122 of humn α-synulin, rvl tht α-synulin ws strongly xprss, whrs th α-synulin 13 frgmnt ws not tt (Supplmntry Fig. 7). By ontrst, IF with th spifi ntiα-synulin N3 ntioy monstrt roust immunortivity towr α-synulin 13 in rins injt with AAVs xprssing th α-synulin 13 frgmnt. In ition, som α-synulin N3 signl ws ttl in rins injt with humn α-synulin (Supplmntry Fig. 7), thus initing tht th humn α-synulin h n trunt t N3 in th SN. Immunolotting with th sm spifi ntiois sltivly Fluorsn intnsity (ritrry units) Fluorsn intnsity (ritrry units) 2, 5 5 Triton X- 13 WB: nti-α-synulin (syn1) SDS 13 : 13 (1:) : 13 (1:4) Full lngth ss 13 ss } Aggrgt Figur 4 lvg of α-synulin promots α-synulin ggrgtion n toxi ffts. () Thioflvin T ssy showing th kintis of ggrgtion of purifi rominnt α-synulin n th α-synulin 13 frgmnt t iffrnt onntrtions. Dt r mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 y two-til Stunt s t tst. () Ultrntrifugtion ssy. Th solul n ggrgt α-synulin ws nlyz y wstrn lotting (S, suprntnt; P, pllt). Th lots shown r rprsnttiv of thr inpnnt xprimnts. () Th α-synulin 13 frgmnt promots th ggrgtion of α-synulin. 7 µm α-synulin ggrgtion ws ssss with thioflvin T ssys in th prsn of 1: or 1:4 α-synulin 13 frgmnt. Dt r mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 ompr with ontrol, y on-wy ANOVA. () Sing of full-lngth α-synulin y firils of th α-synulin 13 frgmnt (lu) or firils of α-synulin (r). Dt r mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 ompr with ontrol; # P <.5 ompr with firils from α-synulin, y on-wy ANOVA. () Th nurotoxiity of α-synulin n th α-synulin 13 frgmnt. α-synulin (without tg) ws xprss in primry ultur SN nurons. Cll th ws trmin y TUNE stining n TH /MAP2 ll ounts. Sl r for mirogrphs, 2 µm. Dt shown in grphs r mn ± s..m.; n = 3 inpnnt xprimnts; P <.5 y on-wy ANOVA. (f) Immunostining showing th ggrgtion of α-synulin. Sl r, 2 µm. (g) Wstrn lot (WB) showing th ggrgtion of α-synulin 13 frgmnts into highr-molulr-wight spis. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. Sour t for grphs r vill onlin. 13 vn onlin pulition ntur struturl & molulr iology

7 5 TH/α-tuulin (% of ontrol group) SN, WB: B SN, WB: nti-α-synulin (syn1) SN, WB: nti-n Full lngth SN, SDS frtion WB: nti-α-synulin SN, WB: nti-α-tuulin 13 Stritum, WB: nti-th Stritum, WB: nti-α-tuulin rognizing humn α-synulin or th N3 truntion lso onfirm ths osrvtions (Fig. 5). As xpt, ovrxprssion of th α-synulin 13 frgmnt rsult in signifint loss of tyrosin hyroxyls (TH) xprssion in th stritum. In ontrst, th fft of α-synulin xprssion ws muh lss pronoun (Fig. 5). To invstigt th fft of th α-synulin 13 frgmnt on motor funtion, w prform pnl of hviorl tsts inluing rotro tsts, mphtmin-inu rottionl hvior tsts, ylinr tsts n gri ssys. Th rotro tst show rs ltny in mi injt with AAV-α-synulin 13 ompr with α-synulin (Fig. 5), thus initing tht th α-synulin 13 frgmnt triggrs muh mor svr opminrgi nuronl loss thn os. In grmnt with this fining, ovrxprssion of th α-synulin 13 frgmnt, s ompr with ovrxprssion, prou inrs mphtmin-inu rottionl symmtry n inrs forpw impirmnt (ylinr tst) (Fig. 5). Th gri tst lso monstrt n vint motor fiit in AAV-α-synulin 13 mi ompr with AAV- mi (Fig. 5). Togthr, ths hviorl tsts init tht th humn α-synulin 13 frgmnt inus mor svr motor impirmnt thn os th ountrprt. Quntifition of tny (s) Numr of ipsiltrl turns otro tst Amphtmin-inu rottion Cylinr tst IHC: nti-th Numr of TH lls in SN,, Contrltrl forlim ontts (% of totl ontts) 5, Stritl TH nsity (% of ontrol) TH immunortivity (strologil ll ounts of TH-positiv nurons n nsitomtry of TH-positiv stritl trminls) support ths finings: th SN n stritum in mi injt with AAVα-synulin 13, ompr with AAV-α-synulin, ontin signifintly fwr TH-positiv nurons n trmini (Fig. 5). Immunostining of th protins Ktrt slis init mor ggrgts in mi injt in th SN with AAV-α-synulin 13 ompr with AAV-α-synulin (Fig. 5). Finlly, HPC nlysis of stritl tissus show signifintly rs opmin onntrtions in mi injt with th AAV-α-synulin 13 frgmnt ompr with AAV-α-synulin (Fig. 5f). Hn, ths finings monstrt tht th -gnrt α-synulin 13 frgmnt is suffiint to inu PD-lik pttrn of nurognrtion. Knokout of mliorts -synulin s pthologil ffts W thn sought to furthr onfirm th rol of in th lvg of α-synulin n th susqunt opminrgi nuronl loss n motor fiits. To o so, w us AAVs to ovrxprss humn α-synulin in wil-typ n -knokout mi. Animls wr uthniz 4 months ftr th injtion, n IF nlysis onfirm 5 Totl stri gris Full lngth lv α-synulin Prots K, IHC: nti-α-synulin (syn1) Stritl opmin (ng/mg protin) 13 Gri tst Figur 5 Th α-synulin 13 frgmnt inus opminrgi ll th. () Wstrn lots (WB) showing th xprssion of α-synulin n th α-synulin 13 frgmnt in SN. α-tuulin, loing ontrol., lft;, right. () Wstrn lots showing th xprssion of TH in th stritum, Br grph, quntifition; t r mn ± s..m.; n = 3 mi; P <.5 y on-wy ANOVA. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. () Th α-synulin 13 frgmnt inus hviorl impirmnt, s monstrt y rotro tsts, mphtmin-inu rottion tsts, ylinr tsts n gri tsts. Dt r mn ± s..m.; n = 9 mi pr group; P <.5 y on-wy ANOVA. Sour t r vill onlin. () TH immunostining of th stritum n SN. ight, unis strologil ll ounts in th SN n totl nsity of stritl opminrgi trminls. Sl rs, 2 µm. Br grph, quntifition; t r mn ± s..m.; n = 5 mi pr group; P <.5 y on-wy ANOVA. () Immunostining showing th ggrgtion of α-synulin in SN slis ftr inution with µg/ml protins K. (f) Conntrtions of opmin in th stritl tissus, s trmin y HPC. Dt r mn ± s..m.; n = 4 mi pr group; P <.5 y on-wy ANOVA. Sour t for grphs r vill onlin. f 13 ntur struturl & molulr iology vn onlin pulition

8 Contrltrl si Ipsiltrl si / / Syn Syn SN, WB: B59 Full lngth 13 SN, WB: nti-α-synulin (syn1) TH/α-tuulin (% of ontrol group) rtils Stritum, WB: nti-th Stritum, WB: nti-α-tuulin 5 WT, 4 2 AAVs Syn Syn f 6 AAVs Syn Syn WT, syn /, Syn 5 Gri tst 2 g 5 AAVs Syn Syn WT, syn Prots K AAVs Syn Syn /, syn / 2 4 Cylinr tst 6 Totl stri gris Numr of ipsiltrl turns tny (s) Amphtmin-inu rottion 8 Syn AAVs Syn Syn Contrltrl forlim ontts (% of totl ontts) otro tst 5 / Stritl opmin (ng/mg protin) SN, WB: nti-α-tuulin 5 Mrg B59 5 TH 5 -lv α-synulin SN, WB: nti-n3 TH/ Prots K /, syn Prots K Numr of TH lls in SN, Stritl TH nsity (% of ontrol) IH: nti-th, 5, AAVs Syn Syn h 5 IHC: nti-α-synulin AAVs Syn Syn IF: nti-n3 Figur 6 Dltion of ttnuts α-synulin ggrgtion n opminrgi ll th in mi ovrxprssing humn α-synulin. () Immunofluorsn showing th xprssion of AAV- (grn) in TH-positiv nurons (r). Sl r, 2 µm. () Doul-ll immunofluorsn imgs showing th AAV-mit xprssion of humn α-synulin (grn) in TH-positiv nurons (r). Sl r, 5 µm. () Wstrn lots (WB) showing th xprssion n truntion of humn α-synulin (syn) in th SN n stritum of wil-typ (/) n knokout ( / ) mi., lft;, right. α-tuulin, loing ontrol. Br grph, quntifition; t r mn ± s..m.; n = 3 mi pr group; P <.5 y on-wy ANOVA. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. () Conntrtions of opmin in th stritl tissus, s trmin y HPC. Dt r mn ± s..m.; n = 4 mi pr group; P <.5 y on-wy ANOVA. () Efft of ltion on hviorl impirmnts inu y ovrxprssion of α-synulin, s shown y rotro tsts, mphtmin-inu rottion tsts, ylinr tsts n gri tsts. Dt r mn ± s..m.; n = 9 AAV--injt WT mi; n = 8 mi in th othr thr groups; P <.5 y on-wy ANOVA. (f) TH immunostining of th stritum n SN. Bottom, unis strologil ll ounts in th SN n th totl nsity of stritl opminrgi trminls. Sl rs, 2 µm. Br grphs, quntifition; t r mn ± s..m.; n = 5; P <.5 y on-wy ANOVA. (g) α-synulin ggrgtion in th SN in AAV-injt mi. Th rin slis wr stin with ntihumn α-synulin with or without prinution with protins K. Th numr of protins Krsistnt α-synulin inlusions in knokout mi ws only hlf of tht in wil-typ mi. Blk sl r, 2 µm. Whit sl rs, 5 µm. (h) IHC with ntiα-synulin N3 ntioy, showing th lvg of α-synulin in wil-typ ut not -knokout mi. Sl r, 5 µm. Sour t for grphs r vill onlin. vn onlin pulition ntur struturl & molulr iology

9 IHC: ntihumn-α-synulin (B59) IF: nti-n3 tny (s) A53T A53TN3A Prots K 5 Prots K otro tst Prots K Numr of ipsiltrl turns SN, WB: B59 Contrltrl forlim ontts (% of totl ontts) tht TH-positiv opminrgi nurons in th injt SN xprss or humn α-synulin (Fig. 6,). Immunolotting onfirm ths osrvtions. As prit, α-synulin ws sltivly lv t N3 y in wil-typ ut not knokout mi. Injtion of α-synulin virus inu signifint stritl TH-immunortivity loss. Dltion of prtilly rvrs th loss of stritl TH xprssion (Fig. 6). Similrly, th stritl opmin ontnt ws signifintly rs in wil-typ mi ompr with -knokout mi (Fig. 6). In grmnt with ths osrvtions, ovrxprssion of humn α-synulin inu mor svr motor fiits in wiltyp mi thn ontrol mi, s tt through th motor tsts sri ov. Agin, this fft ws signifintly llvit in knokout mi (Fig. 6). A quntittiv nlysis of TH-positiv nurons monstrt tht opminrgi nurons wr rstilly lost in th SN, with onomitnt nrvtion of th stritum, whn humn α-synulin ws ovrxprss in wil-typ mi, s ompr with ontrol mi. This nurotoxi fft ws rvrs whn ws plt (Fig. 6f). IHC with ntihumn α-synulin n IF nlysis with ntiα-synulin N3 show tht α-synulin ws sussfully ovrxprss in th SN rgions n tht α-synulin ws trunt t N3 in wil-typ ut not knokout mi (Fig. 6g,h). Th intrnuronl ggrgtion of α-synulin ws mor pronoun in wil-typ mi thn knokout mi, n th ggrgts wr prtilly rsistnt IHC: nti-th Totl stri gris 2 5 A53T A53T N3A SN, WB: nti-n3 Amphtmin-inu rottion SN, WB: nti-α-synulin (syn1) -lv α-synulin A53T A53T N3A A53T A53T N3A SN, WB: nti-α-tuulin Stritum, WB: nti-th Stritum, WB: nti-α-tuulin TH/α-tuulin (% of ontrol group) Cylinr tst 5 A53T A53T N3A A53T A53TN3 Numr of TH lls in SN Stritl TH nsity (% of ontrol),, 5, to protins K igstion (Fig. 6g). Furthrmor, th α-synulin ggrgts xhiit oimmunostining with n uiquitin, thus suggsting tht ths ggrgts wr wy oylik inlusions (Supplmntry Fig. 7f). Thrfor, ths t init tht th ltion of mliorts α-synulin frgmnttion, ttnuts its ggrgtion n llvits its nurotoxiity. Blok of -synulin (A53T) lvg y prvnts its pthologil tivitis Th A53T mutnt of α-synulin is link to utosoml-ominnt rly-onst PD. Trnsgni mi xprssing mutnt A53T show gpnnt formtion of intrnuronl α-synulin inlusions tht rpitult th fturs in humns with PD, n thy lso vlop svr n omplx motor impirmnt 34,35. To ssss whthr ths pthologil ffts us y th A53T mutnt r rlt to N3 lvg y, w gnrt n unlvl mutnt (A53TN3A) tht ws ovrxprss through AAVs in th SN in wil-typ mi. Four months ftr th AAV injtion, xprssion of humn α-synulin (A53T) or its unlvl mutnt ws onfirm with IHC. Protins Krsistnt α-synulin ggrgts wr mor unnt ftr xprssion of A53T s ompr with th unlvl mutnt (Fig. 7). IF nlysis with nti-n3 ntioy show tht A53T protins wr lrly frgmnt t N3; in ontrst, no lvg Gri tst A53T A53T N3A Stritl opmin (ng/mg protin) A53T A53T N3A A53T A53T N3A A53T A53T N3A Figur 7 Prsrvtion of opminrgi nurons in mi xprssing unlvl α-synulin A53TN3A. () Immunostining of α-synulin n th N3 frgmnt in th SN in mi. Th ggrgtion of α-synulin ws tt ftr th slis wr prinut with µg/ml protins K. Blk sl r, 2 µm. Whit sl rs, 5 µm. () Wstrn lots (WB) showing th xprssion n truntion of α-synulin A53T n A53TN3A in th SN n stritum., lft;, right. α-tuulin, loing ontrol. Br grph, quntifition; t r mn ± s..m.; n = 3; P <.5 y on-wy ANOVA. Blots shown r rprsnttiv of thr inpnnt xprimnts; unropp imgs r shown in Supplmntry Dt St 1. () Th N3A muttion ttnuts hviorl impirmnt inu y α-synulin A53T. Th mi unrwnt rotro tsts, mphtmin-inu rottion tsts, ylinr tsts n gri tsts. Dt r mn ± s..m.; n = for th A53TN3A group; n = 9 for th n A53T groups; P <.5 y on-wy ANOVA. () TH immunostining of th stritum n SN. ight, unis strologil ll ounts in th SN n totl nsity of stritl opminrgi trminls. Dt r mn ± s..m.; n = 5; P <.5 y on-wy ANOVA. Sl r, 2 µm. () Conntrtions of opmin in th stritl tissus, s trmin y HPC. Dt r mn ± s..m.; n = 4; P <.5 y on-wy ANOVA. Sour t for grphs r vill onlin. ntur struturl & molulr iology vn onlin pulition

10 of A53TN3A protins ws osrv (Fig. 7). tivity ssys init tht th rtio twn ytosoli n lysosoml tivitis inrs in mi injt with AAV-α-synulin wil typ n AAV-α-synulin A53T, s ompr with mi injt with AAV-, thus initing lysosoml lkg (Supplmntry Fig. 7g). Immunolotting vrifi tht oth mutnts wr highly xprss n tht th A53T protin ws trunt t N3, whrs th unlvl mutnt rmin intt. In grmnt with our finings ov, mi injt with A53TN3A-mutnt α-synulin virus, s ompr with A53T mi, wr prtilly spr from motor fiits, thus impliting lvg of α-synulin in movmnt isorrs liit y th A53T mutnt (Fig. 7). Aoringly, TH immunortivity in oth th SN n stritum ws signifintly lowr in A53T-xprssing rins thn in rins xprssing th unlvl mutnt (Fig. 7,). Finlly, stritl opmin onntrtions in mi injt with th unlvl mutnt wr signifintly highr thn thos in mi injt with th A53T mutnt (Fig. 7). Colltivly, ths t suggst tht lok of α-synulin (A53T) lvg y ntgonizs its ltrious pthologil tivitis, thus gin supporting PD-rlvnt -mit truntion of α-synulin. DISCUSSION is lysosoml prots tht lks into th ytoplsm in nurognrtiv isss 26. Hr w rport tht lvs humn α-synulin t N3 in n g-pnnt mnnr n triggrs α-synulin ggrgtion n nurotoxiity, thus ling to opminrgi nuronl loss n motor impirmnt. Notly, w foun tht is highly tivt in humn PD n BD rins ompr with g-mth hlthy ontrols, spilly in th SN. As onsqun, humn α-synulin is lv t N3, n this frgmnt is prsnt in wy ois. In grmnt with ths osrvtions, w show tht humn α-synulin ws lv t N3 in humn α-synulin trnsgni mi in n g-pnnt mnnr, n this tivity ws tightly oupl with th tmporl pttrn of tivity sltion. Th g-pnnt tivtion of inits tht xprssion is grully inrs, n/or nognous inhiitors, suh s ysttin C, r rs uring ging. Furthrmor, th ph in th rin grully rss uring ging 36, n this rs my liit tivtion. Thus, prosss α-synulin in n g-pnnt mnnr. Nxt, w provi xtnsiv vin monstrting tht irtly lvs humn ut not mous α-synulin t th N3 rsiu. Intrstingly, lthough humn α-synulin shrs 95% intity with its mous ountrprt, mous α-synulin os not hv n N3 rsiu. Notly, th A53T muttion in humn α-synulin uss ominnt forms of fmilil PD, whrs th fifty-thir position in wil-typ mous α-synulin is T (Supplmntry Fig. 2). Hn, mous α-synulin posssss iffrnt ky rsius from th humn vrsion, n ths rsius my xhiit istint iologil tivitis. Cysttin C is th nognous inhiitor of. Mounting vin implits ysttin C in th tiology of nurognrtiv isss inluing AD, strok n pilpsy. During ging, n lvt lvl n/or rs ysttin C lvl my mit th grul lvg of α-synulin n promot its ggrgtion uring th pthognsis of synulinopthis. Hr, w lso provi vin tht is srt from lysosoms into th ytoplsm in humn PD rins, whr it my lv α-synulin (Supplmntry Fig. 3). Ths osrvtions r onsistnt with finings from prvious rports initing tht th lysosoml pntrts into th ytoplsm in humn AD rins n lvs oth tu n SET, PP2A inhiitor, thry miting AD pthognsis 26. In ft, hs n foun to tiv in th nr-nutrl ph milius of th ytosol, nulus n ll surf 38. Post-trnsltionl moifitions inluing phosphoryltion, truntion n uiquitintion n fft th strutur n ggrgtion proprtis of α-synulin 39. C-trminl truntion promots α-synulin firilliztion, in pross mit y numrous protss inluing nurosin, lpin, thpsin D n mtlloprotins 12. Nvrthlss, th rols of ths protss in th pthognsis of PD rmin lusiv. Hr w provi xtnsiv iohmil vin monstrting tht lvs α-synulin t N3 in n g-pnnt mnnr. Notly, th lv humn α-synulin 13 frgmnt is mor pron to ggrgtion thn th full-lngth protin (Fig. 4) n forms wy oy-lik ggrgtions in th SN (Fig. 6, Supplmntry Fig. 7), thus proviing furthr vin of th rol of C-trminlly trunt α-synulin in th pthognsis of nurognrtiv isss inluing PD. W osrv som minor trunt α-synulin ns t ~12 kd in humn n mous rins (Figs. 5 n 7). This rsult is onsistnt with prvious finings of istint α-synulin truntions t sits ownstrm of N3, inluing t positions 119 (rf. 14), 121 (rf. 4) n 122 (rf. 18), in synulinopthis n in vivo mols. Unr iffrnt strsss or niml mols, vrity of protss inluing spss or lpins my tivt n mit α-synulin protolyti lvg t vrious sits, thus ontriuting to α-synulin ggrgtion n nurotoxiity. Nonthlss, lvg of α-synulin t N3 uring th ging pross provis n itionl mhnism ounting for s trimntl fft. To tst th hypothsis tht lvg of humn α-synulin ontriuts to PD pthognsis, w provi gin-of-funtion n loss-of-funtion vin (Figs. 57). As xpt, th humn α-synulin 13 frgmnt ws mor potnt thn α-synulin in provoking opminrgi nuronl loss n motor fiits (Fig. 5). Howvr, lthough ovrxprssion of humn α-synulin in wiltyp mi notly prou lr opminrgi nurognrtion, s ompr with tht in ontrols, fr fwr pthologil hllmrks wr osrv in -null mi (Fig. 6). This rsult suggst tht promots th nurotoxi fft of α-synulin. Th sm snrio ppli to th A53T mutnt, whih ws vn mor toxi. W monstrt tht lok of A53T α-synulin lvg y grtly supprss its pthologi tivitis in PD (Fig. 7). Furthrmor, th mount of α-synulin 13 frgmnts in humn PD SN tissu n mous SN injt with α-synulin virus ws omprl (Supplmntry Fig. 8), thus suggsting tht th mount in th humn PD SN ws suffiint to inu nurotoxiity. Togthr, our in vivo t support th hypothsis tht lvg of humn α-synulin promots th onst of PD pthologis. Ths finings notly init tht th α-synulin 13 frgmnt might t s th mjor pthologil triggr of opminrgi nuronl loss in PD. Thrfor, ths finings suggst tht my novl rug trgt n tht th inhiition of my hol promis s usful strtgy for trting PD. Mthos Mthos, inluing sttmnts of t vilility n ny ssoit ssion os n rfrns, r vill in th onlin vrsion of th ppr. Not: Any Supplmntry Informtion n Sour Dt fils r vill in th onlin vrsion of th ppr. Aknowlgmnts This work ws support y grnts from th Mihl J. Fox Fountion (grnt ID 111) to K.Y.; grnt from th Ntionl Nturl Sin Fountion (NSFC) of Chin (no ) to Zhnto Zhng; NSFC grnt (no. 8287) to K.Y. n J.-Z.W.; Ntionl Ky Bsi srh Progrm of Chin grnt (2CB94522) to Y.E.S.; n NSFC grnt (81333) to Y.E.S.; n grnts from th US Puli Hlth Srvi (P3EY636 n 1EY4864) to P.M.I. W thnk vn onlin pulition ntur struturl & molulr iology

11 th ADC t Emory Univrsity for proviing humn PD, BD n hlthy-ontrol smpls, n C. Wtts (Univrsity of Cmrig) for proviing nti-. AUTHO CONTIBUTIONS K.Y. oniv th projt, sign th xprimnts n wrot th mnusript. Zhnto Zhng sign n prform most of th xprimnts. S.S.K. n X.. prpr primry nurons n ssist with niml xprimnts. M.J.B. n F.P.M. provi lons n pkg virl vtors. D.M.D. n N.T.S. prform th mss sptromtry nlysis..h. n P.M.I. prform th HPC xprimnts n ritilly r n it th mnusript. Zhohui Zhng, E.H.A.,.J., Y.E.S., F.P.M. n J.-Z.W. sign th xprimnts, ssist with t nlysis n intrprttion n ritilly r th mnusript. COMPETING FINANCIA INTEESTS Th uthors lr no ompting finnil intrsts. prints n prmissions informtion is vill onlin t rprints/inx.html. Pulishr s not: Springr Ntur rmins nutrl with rgr to jurisitionl lims in pulish mps n institutionl ffilitions. 1. Mris, E., Dss, B., Collir, T.J., Korowr, J.H. & St-Collir, K. Th rol of lph-synulin in Prkinson s iss: insights from niml mols. Nt. v. Nurosi. 4, (23). 2. Polymropoulos, M.H. t l. Muttion in th lph-synulin gn intifi in fmilis with Prkinson s iss. Sin 276, (1997). 3. Krügr,. t l. Al3Pro muttion in th gn noing α-synulin in Prkinson s iss. Nt. Gnt. 18, 68 (1998). 4. roy, E., Boyr,. & Polymropoulos, M.H. Intron-xon strutur of uiquitin -trminl hyrols-1. DNA s. 5, 3974 (1998). 5. Kit, T. t l. Muttions in th prkin gn us utosoml rssiv juvnil prkinsonism. Ntur 392, 6568 (1998). 6. Spillntini, M.G. t l. α-synulin in wy ois. Ntur 388, (1997). 7. Olnow, C.W. & Brunin, P. Prkinson s iss n lph synulin: is Prkinson s iss prion-lik isorr? Mov. Disor. 28, 314 (213). 8. Mslih, E. t l. Dopminrgi loss n inlusion oy formtion in lph-synulin mi: implitions for nurognrtiv isorrs. Sin 287, (2). 9. Khl, P.J., Numnn, M., Ozmn,. & Hss, C. Physiology n pthophysiology of lph-synulin: ll ultur n trnsgni niml mols s on Prkinson s iss-ssoit protin. Ann. NY A. Si. 92, 3341 (2).. Fny, M.B. & Bnr, W.W.A. Drosophil mol of Prkinson s iss. Ntur 44, (2). 11. Burré, J. t l. Proprtis of ntiv rin α-synulin. Ntur 498, E4E6 (213). 12. shul, H.A., Ovrk, C.., Ouslti, A. & Mslih, E. Th mny fs of α-synulin: from strutur n toxiity to thrputi trgt. Nt. v. Nurosi. 14, 3848 (213). 13. i, W. t l. Aggrgtion promoting C-trminl truntion of lph-synulin is norml llulr pross n is nhn y th fmilil Prkinson s iss-link muttions. Pro. Ntl. A. Si. USA 2, (25). 14. iu, C.W. t l. A pripitting rol for trunt lph-synulin n th protsom in lph-synulin ggrgtion: implitions for pthognsis of Prkinson iss. J. Biol. Chm. 28, (25).. Hoyr, W., Chrny, D., Surmnim, V. & Jovin, T.M. Impt of th ii C-trminl rgion omprising mino is 9-14 on lph-synulin ggrgtion in vitro. Biohmistry 43, (24). 16. Murry, I.V. t l. ol of lph-synulin roxy-trminus on firil formtion in vitro. Biohmistry 42, (23). 17. Mishizn-Erz, A.J. t l. Distint lvg pttrns of norml n pthologi forms of lph-synulin y lpin I in vitro. J. Nurohm. 86, (23). 18. Mishizn-Erz, A.J. t l. Clvg of lph-synulin y lpin: potntil rol in grtion of firilliz n nitrt spis of lph-synulin. Biohmistry 44, (25). 19. Svlvr, D., Jing, P. & Yn, S.H. Cthpsin D is th min lysosoml nzym involv in th grtion of lph-synulin n gnrtion of its roxytrminlly trunt spis. Biohmistry 47, (28). 2. Tkhshi, M. t l. Oxitiv strss-inu phosphoryltion, grtion n ggrgtion of lph-synulin r link to uprgult CK2 n thpsin D. Eur. J. Nurosi. 26, (27). 21. Hlfon, S., Ptl, S., Vg, F., Zurwski, S. & Zurwski, G. Autotlyti tivtion of humn lgumin t sprti i rsius. FEBS tt. 438, (1998). 22. Gmlin, T.C. t l. Csps lvg of tu: linking myloi n nurofirillry tngls in Alzhimr s iss. Pro. Ntl. A. Si. USA, 32 (23). 23. Alvrz-Frnnz, M. t l. Inhiition of mmmlin lgumin y som ysttins is u to novl son rtiv sit. J. Biol. Chm. 274, (1999). 24. iu, Z. t l. Nuroprottiv tions of PIKE- y inhiition of SET protolyti grtion y sprgin nopptis. Mol. Cll 29, (28). 25. Mir, A., Pommt, J.M., Prohintz, A. & Allinqunt, B. SET protin (TAF1t, I2PP2A) is involv in nuronl poptosis inu y n myloi prursor protin ytoplsmi suomin. FASEB J. 19, (25). 26. Bsurto-Isls, G., Grunk-Iql, I., Tung, Y.C., iu, F. & Iql, K. Ativtion of sprginyl nopptis ls to Tu hyprphosphoryltion in Alzhimr iss. J. Biol. Chm. 288, (213). 27. Hrskowitz, J.H. t l. Asprginyl nopptis lvs TDP-43 in rin. Protomis 12, (212). 28. Zhng, Z. t l. Clvg of tu y sprgin nopptis mits th nurofirillry pthology in Alzhimr s iss. Nt. M. 2, (214). 29. Zhng, Z. t l. Dlt-srts lvs myloi prursor protin n rgults th pthognsis in Alzhimr s iss. Nt. Commun. 6, 8762 (2). 3. i, D.N., Mtthws, S.P., Antoniou, A.N., Mzzo, D. & Wtts, C. Multistp utotivtion of sprginyl nopptis in vitro n in vivo. J. Biol. Chm. 278, (23). 31. Chn,. t l. Tyrosin n srin phosphoryltion of lph-synulin hv opposing ffts on nurotoxiity n solul oligomr formtion. J. Clin. Invst. 119, (29). 32. Ellis, C.E., Shwrtzrg, P.., Grir, T.., Fink, D.W. & Nussum,.. α-synulin is phosphorylt y mmrs of th Sr fmily of protin-tyrosin kinss. J. Biol. Chm. 276, (21). 33. Byr, K. & Ariz, A. α-synulin posttrnsltionl moifition n ltrntiv spliing s triggr for nurognrtion. Mol. Nuroiol. 47, (213). 34. Gisson, B.I. t l. Nuronl lph-synulinopthy with svr movmnt isorr in mi xprssing A53T humn lph-synulin. Nuron 34, (22). 35., M.K. t l. Humn lph-synulin-hroring fmilil Prkinson s isslink Al-53 Thr muttion uss nurognrtiv iss with lphsynulin ggrgtion in trnsgni mi. Pro. Ntl. A. Si. USA 99, (22). 36. Forstr, B.P. t l. Ag-rlt hngs in rin nrgtis n phospholipi mtolism. NM Biom. 23, (2).. Guthir, S., Kur, G., Mi, W., Tizon, B. & vy, E. Prottiv mhnisms y ysttin C in nurognrtiv isss. Front. Biosi. (Shol. E.) 3, (211). 38. Dll, E. & Brnstttr, H. Strutur n funtion of lgumin in hlth n iss. Biohimi 122, 126 (216). 39. Ouslti, A., Fournir, M. & shul, H.A. ol of post-trnsltionl moifitions in moulting th strutur, funtion n toxiity of lph-synulin: implitions for Prkinson s iss pthognsis n thrpis. Prog. Brin s. 183, 1145 (2). 4. Wng, W. t l. Csps-1 uss truntion n ggrgtion of th Prkinson s iss-ssoit protin α-synulin. Pro. Ntl. A. Si. USA 113, (216). ntur struturl & molulr iology vn onlin pulition 11

12 ONINE METHODS Mi. Humn SNCAtrnsgni mi rrying trnsgn ontining th humn SNCA gn n knokout lll of th mous Sn gn wr from Jkson ortory (stok no. 238). Th -knokout mi on mix 129/Ol n C57B/6 kgroun wr gnrt s prviously rport 41. Only ml mi wr us in th xprimnts. Animl r n hnling ws prform oring to Emory Mil Shool guilins. Smpl siz ws trmin in Powr n Prision softwr (Biostt). Th mi wr rnomiz into iffrnt groups y using rnom numr tl. Invstigtors wr lin to th group llotion uring th niml xprimnts. Th protool ws rviw n pprov y th Emory Institutionl Animl Cr n Us Committ. Humn tissu smpls. Postmortm rin smpls wr isst from th frozn rins of fiv ontrol ss, BD ss, n PD ss otin from th Emory Alzhimr s Diss srh Cntr. Th stuy ws pprov y th iospimn ommitt t Emory Univrsity. PD n BD ss wr linilly ignos n nuropthologilly onfirm. Inform onsnt ws otin from ll sujts. Cll lins, trnsftion n inftion. HEK293 lls wr otin from th ATCC n tst for myoplsm ontmintion for us. Clls wr trnsft with plsmis noing wil-typ or point-mutnt -α-synulin, my-, my C189S, or my N323A y th lium phospht pripittion mtho. AAVs wr us to xprss wil-typ, mutnt n trunt α-synulin in primry nurons n SH-SY5Y lls. Th nurotoxiity ws nlyz through TUNE nlysis or DH ssys. In vitro -synulin lvg ssy. To ssss th lvg of α-synulin y in vitro, HEK293 lls wr trnsft with GST- or -tgg α-synulin DNA through th lium phospht pripittion mtho. 48 h ftr trnsftion, lls wr ollt, wsh on in PBS, lys in uffr (5 mm soium itrt, 5 mm DTT,.1% CHAPS, ph 5.5, n.5% Triton X-), n ntrifug for min t 14,g t 4 C. Th suprntnt ws thn inut with mous kiny lyst or rominnt t ph 7.4 or 6. t C for iffrnt tim points. To tst th ffts of thpsin, lpin n inhiitors on th lvg of α-synulin y, inhiitors wr us ginst thpsin (E64, Sigm- Alrih), lpins (AN, Sigm-Alrih) n (AENK ppti inhiitor n intiv ontrol AEQK). To msur th lvg of purifi α-synulin frgmnts y, GST-tgg α-synulin ws purifi with glutthion s. Th purifi α-synulin protin ws inut with rominnt protin (Novoprotin, 5 µg ml 1 ) in uffr (5 mm soium itrt, 5 mm DTT,.1% CHAPS n.5% Triton X-, ph 6.). Th smpls wr thn oil in 1 SDS loing uffr n nlyz y immunolotting. tivity ssy. Tissu homognts or ll lysts ( µg) wr inut in 2 µl ssy uffr (2 mm itri i, 6 mm N 2 HPO 4, 1 mm EDTA,.1% CHAPS n 1 mm DTT, ph 6.) ontining 2 µm sustrt Z-Al-Al-Asn-AMC (Bhm). AMC rls y sustrt lvg ws quntifi y msuring t 46 nm in fluorsn plt rr t C for 1 h in kinti mo for 5 min. Mss sptromtry nlysis. Protin smpls wr in-gl-igst with ng/µl Glu-C. Ppti smpls wr rsuspn in loing uffr (.1% formi i,.3% trifluoroti i n 1% tonitril) n lo onto 2-m nno- HPC olumn (intrnl imtr, 75 µm) pk with prosil-pur 12 C18-AQ 1.9-µm s (Dr. Mish) n lut ovr 2-h 15% uffr B rvrs-phs grint (uffr A,.1% formi i n 1% tonitril in wtr; uffr B,.1% formi i in tonitril) gnrt y Dionx SCnno UPC systm (Thrmo). Pptis wr ioniz with 2.-kV ltrospry ioniztion voltg from nno-esi sour on n Oritrp Fusion mss sptromtr (Thrmo). Dtpnnt quisition of MS sptr t 12, rsolution (FWHM), n MS/MS sptr wr otin in th Oritrp ftr ltron-trnsfr issoition (ETD) with supplmntl tivtion with high nrgy (EThD) for ppti msss orrsponing to th 3 n 4 hrg stts ( n , rsptivly) of th ppti GAGSIAAATGFVKKDQGKN. To intify -lvg sits in humn α-synulin, Protom Disovrr 2. (PD) ws us to srh n mth MS/MS sptr to omplt humn protom ts (NCBI rfrn squn rvision 62, with ntris) with ±-p.p.m. mss-ury thrshol n llowl lvgs t glutmts n sprgins. A proltor ws us to filtr th ppti sptrl mths (PSM) to fls isovry rt (FD) of <1%. All MS/MS sptr for puttiv -gnrt α-synulin lvg sits wr mnully inspt. Gnrtion of ntiois spifi to -gnrt -synulin (nti -synulin N3). Two rits wr immuniz with th ppti A- CVKKDQGKN-OH, whih inlu th nin mino is in α-synulin tht pr th -lvg sit t N3 s wll s n N-trminl ystin rsiu to llow for oupling to KH. Th rits wr riv oostr injtions four tims with th immunizing ppti with 3-wk intrvls twn injtions. Th ntisrum ws ffinity purifi y hromtogrphy with th immunizing ppti n ws thn sor to spnning ppti spnning th N3 lvg sit (A-CKDQGKNEEGAPQE-mi). Th titrs ginst th immunizing ppti wr trmin y EISA. Th mximl ilution giving positiv rspons with hromogni sustrt for horsrish proxis ws 1:3,. Th immunotivity of th ntisrum ws furthr onfirm y wstrn lotting n immunohistohmistry. Wstrn lot nlysis. Th mous rin tissu or humn tissu smpls wr lys in lysis uffr (5 mm Tris, ph 7.4, 4 mm NCl, 1 mm EDTA,.5% Triton X-, 1.5 mm N 3 VO 4, 5 mm NF, mm soium pyrophospht n mm soium β-glyrophospht, supplmnt with oktil of prots inhiitors), n ntrifug for min t 16,g. Th suprntnt ws oil in SDS loing uffr. Aftr SDSPAGE, th smpls wr trnsfrr to nitrollulos mmrn. Primry ntiois to th following trgts wr us: GST-HP (Sigm-Alrih, GEPN1236), α-tuulin (Sigm-Alrih, T926), HA (Snt Cruz, H3663), my (Snt Cruz, M4439), α-synulin N trminus (Snt Cruz, s-51498); nti-α-synulin (syn lon 42, BD Biosin, 6787), (lon 6E3, gift from C. Wtts), TH (Cll Signling Thnology, 2792), α-synulin C trminus (Cll Signling Thnology, 2642) n α-synulin B59 (Thrmo Fishr Sintifi, 182). Immunostining. Prffin-m humn rin stions or fr-floting mous rin stions wr trt with.3% H 2 O 2 for min. Stions wr thrftr wsh thr tims in PBS, lok in 1% BSA n.3% Triton X- for 3 min n inut ovrnight with nti-th (1:) ntioy, ntiα-synulin N3 ntioy (1:) or α-synulin B59 ntioy (1:5) t 4 C. Th signl ws vlop with Histostin-SP kit (Invitrogn). To tt th loliztion of th α-synulin 13 frgmnt in wy ois in humn PD rin stions, th stions wr inut with rit ntiα-synulin N3 primry ntioy n mous nti-α-synulin ntioy ovrnight t 4 C. Th slis wr wsh thr tims in PBS n inut with Txs ronjugt nti-mous IgG n FITC-onjugt nti-rit IgG for 1 h t room tmprtur. Th slis wr wsh thr tims in PBS, thn ovr with glss ovr with mounting solution n xmin unr fluorsn mirosop (Olympus). Th primry ultur nurons wr inft with AAVs noing full-lngth α-synulin or th α-synulin 13 frgmnt, n wr oul-stin for α-synulin/th, TUNE/TH or TH/N3. Th prntgs of TUNE /TH lls ovr TH lls wr lult. Th rsrhrs who prform immunostining n ll ounting wr lin to th group llotions. Coimmunopripittion. Th mous rin tissu smpls wr lys in lysis uffr (5 mm Tris, ph 7.4, 4 mm NCl, 1 mm EDTA,.5% Triton X-, 1.5 mm N 3 VO 4, 5 mm NF, mm soium pyrophospht n mm soium β-glyrophospht, supplmnt with oktil of prots inhiitors) n ntrifug for min t 16,g. Th suprntnt ws inut with ntiα-synulin ntioy n Protin A/Ggros ovrnight t 4 C. Aftr xtnsiv wshing with lysis uffr, th oun protins wr lut from th s y oiling in mmli smpl uffr n wr sujt to wstrn lot nlyss. In vitro ggrgtion of -synulin. Th ggrgtion of α-synulin ws inu s sri prviously 42. Brifly, purifi α-synulin protin (rppti, 5 mg ml 1 ) ws first inut with vhil or in for 1 h n thn ilyz ginst PBS, ph 7.. Th smpls wr inut t C with ontinuous shking for 3. Th ggrgtion kintis of α-synulin ws msur with thioflvin T stining. µl of 2 µm thioflvin T ws mix with 2 µl α-synulin protin ntur struturl & molulr iology oi:.38/nsm.3433