Interplay of LRRK2 with chaperone-mediated autophagy

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1 Interply of with hperone-medited utophgy Smnth J Orenstein,, Sheng-Hn Kuo,, Inmuld Tsset,,, Espernz Aris,, Hiroshi Kog,, Irene Fernndez-Crs, Etty Cortes,5, Lwrene S Honig,5, Willim Duer 6, Antonell Consiglio,7, Angel Ry 8, Dvid Sulzer,, & An Mri Cuervo, npg Nture Ameri, In. All rights reserved. Muttions in leuine-rih repet kinse () re the most ommon use of fmilil Prkinson s disese. We found to e degrded in lysosomes y hperone-medited utophgy (CMA), wheres the most ommon pthogeni mutnt form of, G9S, ws poorly degrded y this pthwy. In ontrst to the ehvior of typil CMA sustrtes, lysosoml inding of oth wild-type nd severl pthogeni mutnt proteins ws enhned in the presene of other CMA sustrtes, whih interfered with the orgniztion of the CMA trnslotion omplex, resulting in defetive CMA. Cells responded to suh - medited CMA ompromise y inresing levels of the CMA lysosoml reeptor, s seen in neuronl ultures nd rins of trnsgeni mie, indued pluripotent stem ell derived dopminergi neurons nd rins of Prkinson s disese ptients with muttions. This newly desried self-perpetuting inhiitory effet on CMA ould underlie toxiity in Prkinson s disese y ompromising the degrdtion of -synulein, nother Prkinson s disese relted protein degrded y this pthwy. Muttions in (or drdrin) re the most ommon identified use of fmilil Prkinson s disese, neurodegenertive motor disorder hrterized y the presene of α-synulein (α-syn)-ontining Lewy Body inlusions, prtiulrly in dopminergi neurons of the sustnti nigr tht die over the ourse of the disese. Most pthogeni Prkinson s disese muttions re loted in the kinse nd GTPse domins, nd n inrese in the kinse tivity of, suh s tht oserved in the G9S mutnt vrint, hs een proposed to e responsile for its neurotoxi effet. The speifi mehnisms y whih mutnt uses neuronl toxiity in Prkinson s disese, however, hve remined elusive. Dysregultion of ellulr proteostsis due to ltertions in the uiquitin-protesome nd the utophgi-lysosoml systems hs een desried in mny neurodegenertive disorders, inluding Prkinson s disese. Altertions in two of the most ommon forms of utophgy, mroutophgy nd CMA, hve een desried in Prkinson s disese 8. Severl studies hve reported possile intertions of with mroutophgy, proess tht involves sequestrtion of portions of ytosol in doule-memrne vesiles or utophgi vuoles tht then fuse with lysosomes 9. n lolize to the endosoml/lysosoml omprtments,, nd expression of mutnts in different experimentl models inreses the presene of utophgi omprtments. The interply of with mroutophgy my, however, e omplex, euse lthough knokout mie exhiit redued mroutophgy, t lest in kidney 5, prtil knokdown of in ultured kidney ells leds to n inrese in utophgi mrkers. In ontrst to the studies reporting effets of on mroutophgy, effets of on CMA re unknown. CMA involves the diret trnsport of ytosoli solule proteins ross the lysosoml memrne in seletive fshion 6,7. CMA sustrtes ontin pentpeptide motif 8 tht is reognized y the het shok ognte protein of 7 kd (hs7). Hs7 trgets sustrte proteins to the lysosoml memrne 9, where they intert with the lysosome-ssoited memrne protein type A (LAMP-A). One the sustrte inds, LAMP-A multimerizes to form the trnslotion omplex. LAMP-A dynmis re lso regulted y moiliztion in nd out of disrete lipid mirodomins where degrdtion of LAMP-A tkes ple,. Previous studies hve estlished onnetion etween CMA nd Prkinson s disese, s wild-type α-syn is sustrte for CMA 8, wheres pthogeni forms of this protein ind normlly to the lysosoml reeptor for CMA, therey preventing their own degrdtion nd the degrdtion of other CMA sustrtes 7,8,. Another Prkinson s disese ssoited protein, uiquitin C-terminl hydrolse L (UCH-L), interts with CMA omponents, nd this intertion is normlly enhned in UCH-L mutnt 5. Altertions in CMA my in ft e ommon feture in mny forms of Prkinson s disese, s post-mortem rin smples from Prkinson s disese ptients show redued levels of LAMP-A in the sustnti nigr 6. We noted tht the protein ers eight Deprtment of Developmentl nd Moleulr Biology, Alert Einstein College of Mediine, Bronx, New York, USA. Institute for Aging Studies, Alert Einstein College of Mediine, Bronx, New York, USA. Deprtment of Neurology, Columi University Medil Center, New York, New York, USA. Institute for Biomediine (IBUB), University of Brelon, Brelon, Spin. 5 Tu Institute, Columi University Medil Shool, New York, New York, USA. 6 Deprtment of Cell nd Developmentl Biology, University of Mihign, Ann Aror, Mihign, USA. 7 Deprment of Biomedil Siene nd Biotehnology, University of Bresi, Bresi, Itly. 8 Control of Stem Cell Poteny Group, Institute for Bioengineering of Ctloni (IBEC), Brelon, Spin. 9 Instituió Ctln de Reer I Estudis Avnçts (ICREA), Brelon, Spin. Center for Networked Biomedil Reserh on Bioengineering, Biomterils nd Nnomediine (CIBER-BBN), Brelon, Spin. Deprtment of Psyhitry, Columi University Medil Shool, New York, New York, USA. Deprtment of Phrmology, Columi University Medil Center, New York, New York, USA. These uthors ontriuted eqully to this work. Correspondene should e ddressed to A.M.C. (n-mri.uervo@einstein.yu.edu) or D.S. (ds@olumi.edu). Reeived 9 Otoer ; epted Jnury ; pulished online Mrh ; orreted online 7 Mrh (detils online); doi:.8/nn.5 9 VOLUME 6 NUMBER APRIL nture NEUROSCIENCE

2 npg Nture Ameri, In. All rights reserved. Figure is degrded in lysosomes. () Top: immunolots for the indited proteins from two independent experiments. Bottom: quntifition of hnges in totl (n = 6 independent experiments). SH-SY5Y ells were untreted or inuted with 5 µm ltystin (Lt), mm MA or mm NH Cl nd µm leupeptin (NL) for 9.5 h. () Top: representtive immunolots for the indited proteins from two independent experiments. Bottom: quntifition of reltive to tht in untreted smples (n = or independent experiments). Brin slies from mouse ortex, midrin or ereellum were inuted without dditions or with NL or MA for h t 7 C. (,d) Homogente (Hom) nd lysosoml frtions (Lys) isolted from SH-SY5Y ells () or the indited mouse rin regions (d), immunolotted for the indited proteins. (e) HEK9 ells expressing GFP-tgged nd trnsdued with lentivirus ontrol () or rrying shrna ginst LAMP-A (LA( )), inuted or not with NL for h nd immunolotted for the indited proteins. (f) Immunolots for nd LAMP-A of homogentes nd lysosome-enrihed frtions isolted from ontrol firolsts () or firolsts stly interfered for LAMP- A (LA( )), mintined in the presene or sene of serum. nd LAMP- (L-) re shown s loding ontrols of homogente nd lysosomes, respetively. All vlues re men + s.e.m.; P <.5, unpired t-test. Full-length lots nd gels in Supplementry Figure. puttive CMA motifs in its mino id sequene nd deided to determine whether might e CMA sustrte, nd how the most ommon muttion (G9S) ould ffet its intertion with CMA. We found tht frtion of ellulr ould e degrded y CMA, ut lso tht rnge of pthogeni mutnts, inluding the ommon G9S llele, s well s high onentrtions of wildtype, inhiited CMA. This inhiition ourred through new mehnism, involving lokde in the formtion of the CMA trnslotion omplex t the lysosoml memrne. Cells responded to the -medited lokge of CMA y inresing the mount of the essentil omponent of the trnslotion omplex, LAMP-A, phenomenon we lso oserved in the rins of trnsgeni mie nd in indued pluripotent stem ell (ipsc)-derived dopminergi neurons nd rins from -muttion Prkinson s disese ptients. One of the proteins ffeted y the inhiitory effet of on CMA ws α-syn. Thus, two of the dominnt muttions tht use Prkinson s disese onverge mehnistilly y inhiiting the norml degrdtion of ytosoli proteins y CMA. RESULTS n e degrded in lysosomes The ontriution of the lysosoml system to the degrdtion of itself hs not een systemtilly explored. On tretment of neurolstom ells with ltystin ( protesome inhiitor), -methyldenine (MA; to inhiit mroutophgy) or omintion of mmonium hloride nd leupeptin (to inhiit totl lysosoml proteolysis), we onfirmed tht portion of ellulr ws degrded y the protesome 7, ut we lso found tht prt of ws degrded in lysosomes, s intrellulr inresed when lysosoml proteolysis ws loked (Fig. ). Lysosoml degrdtion of did not our through mroutophgy, euse did not inrese on MA tretment (Fig. nd Supplementry Fig.,). Lysosoml degrdtion of lso ours in vivo, s we found mrked inrese in in slies of mouse ortex, midrin nd ereellum fter inution with NH Cl-leupeptin (Fig. ). In further support of these findings, we ould detet in lysosomes isolted from neuronl ells in ulture (Fig. ) nd from mouse nd rt rin regions (Fig. d nd dt not shown). The lk of inhiition of degrdtion y MA in the rin slies (Fig. ) nd the sene of in utophgosomes isolted from the rins of these nimls (Supplementry Fig. ) suggest tht mroutophgy LAMP- e (fold untreted) Hs7 GFP- LAMP-A Hom Lt MA NL None Lt MA NL Lys Hom Lys LA( ) NL NL d f Serum LAMP-A (fold untreted) Homogente Lysosome-enrihed does not ontriute sustntilly to degrdtion in rin, t lest under norml onditions. Insted, we found tht ells in whih LAMP-A, the CMA lysosoml reeptor, ws knoked down showed inresed intrellulr due, t lest in prt, to redued degrdtion y lysosomes, s shown y the lk of effet of NH Cl-leupeptin tretment on levels in these ells (Fig. e). Furthermore, the mount of ound to lysosomes isolted from LAMP-A knokdown ells ws muh lower thn in ontrol ells (Fig. f; notie tht persistent LAMP-A knokdown redued initil umultion, likely through tivtion of other proteolyti systems). We onlude tht frtion of intrellulr normlly is degrded in lysosomes, proly vi CMA. Seletive degrdtion of wild-type y CMA The presene in the mino id sequene of of eight puttive CMA trgeting motifs 8 (Fig. ) further inresed our onfidene tht CMA my ontriute to degrdtion of this protein. However, the most onvining test to onfirm protein s CMA sustrte is to diretly demonstrte its uptke into isolted lysosomes 9. We first used lysosomes isolted from rt liver to void possile interferene from the endogenous deteted in the rin lysosomes (Fig. d). Inution of purified with intt liver lysosomes untreted or pretreted with protese inhiitors showed tht ound to the lysosoml memrne nd ws seletively tken up y lysosomes (Fig. ). Uptke ws lulted s the inrese in lysosomessoited when lysosomes were pretreted with protese inhiitors. As noted for other CMA sustrte proteins, inresing onentrtions of ompeted with the lysosoml uptke nd LAMP-A Cortex Midrin Cereellum NL MA NL MA NL MA None NL MA Cortex Totl rin Hom Lys Hom Lys Hom Lys LA( ) + + Midrin Cortex LA( ) + + Cereellum Cereellum L- nture NEUROSCIENCE VOLUME 6 NUMBER APRIL 95

3 npg Nture Ameri, In. All rights reserved. Figure ehves s n typil CMA sustrte. () Amino id lotions of the puttive CMA trgeting motifs in. () Binding, ssoition nd uptke lulted from quntifition of in immunolots of lysosomes from strved rt livers, untreted or pretreted with protese inhiitors (PI) nd inuted with. Inset: representtive immunolot. LAMP-A is shown s lysosoml loding ontrol. Input (Inp), one-fifth of dded. n = 7 independent experiments. Uptke: differene etween ssoited with lysosomes with nd without PI. () Effet of inresing onentrtions of on the degrdtion of rdioleled pool of ytosoli proteins y intt lysosomes. Vlues re perentge of the degrdtion without (n = 5 or 6 independent experiments). (d) Assoition of to rt liver lysosomes in the presene of inresing onentrtions of. Vlues re perentge of the ssoited with lysosomes in the sene of (n = 6 independent experiments in duplite). Inset: representtive immunolot. Inp: µg of. (e,f) Assoition of nd RNse A (e) or nd (f) with PI-treted rt liver lysosomes inuted with inresing onentrtions of (e) or RNse A (f) or ovlumin (Ov). Left: representtive immunolots. Right: protein ssoition expressed s perentge of the vlue when inuted with lysosomes lone (n = 5 or 6 independent experiments). Inp: RNse A (. µg), (. µg) or ( µg). (g,h) Assoition of to lysosomes inuted with (g) or RNse A (h) t C (n = independent experiments). (i,j) Immunolots of rin lysosomes inuted with nd inresing onentrtions of (i) or RNse A (j). All vlues re men + s.e.m.; differenes with lysosomes inuted with the protein lone were signifint t P <.5, unpired t-test. Full-length lots nd gels in Supplementry Figure. degrdtion of pool of ytosoli proteins sustrtes for CMA (Fig. ) nd the inding to the lysosoml memrne of the wellestlished CMA sustrte glyerldehyde--phosphte dehydrogense () (Fig. d). We next ssessed the effet on CMA of the CMA sustrte proteins (Fig. e) nd rionulese A (RNse A; Fig. f) nd of negtive ontrol proteins tht lk ny lysosoml trgeting motif. To our surprise, nd in ler ontrst with the ompetition oserved etween onventionl CMA sustrtes ( nd RNse A shown here), CMA sustrtes did not ompete with s lysosoml ssoition ut insted mrkedly inresed the mount of ound to lysosomes in dose-dependent mnner (Fig. e,f). In ontrst, the non-cma sustrte proteins ovlumin (Fig. e,f) nd ylophilin A (Supplementry Fig. ) did not ffet the ssoition of with lysosomes. The inresed inding ws not due to proportionl inrese in uptke, s differenes etween smples treted or not with protese inhiitors were similr (Supplementry Fig. ) nd s inresed inding in the presene of (Fig. g) or RNse A (Fig. h) ws still oserved t low tempertures, whih do not llow trnslotion. We onfirmed tht this unusul inding of in CMA sustrte-dependent mnner lso ourred in lysosomes isolted Perentge of protein dded e RNAse A f i ANK LRR ROC COR Kinse WD 57 KEVFQ 769 QDVRK 79 QIRDK 555 QLVRE QKLIE 5KIRDQ 65LEKFQ 956DRLLQ LAMP-A Inp Binding Assoition Uptke Ov (µg) Inp Ov (µg) Inp 75 5 Ov (µg) Inp RNse A PI Protein ssoition (% untreted) Protein ssoition (% untreted) [ H] protein proteolysis (%) Ov (µg) Inp (µg) RNAse A j from rt rin nd tht this ehvior ws, gin, speifi to, s other CMA sustrtes produed the expeted ompetitive response in rin lysosomes (Fig. i,j). Immunolotting reveled tht the CMA sustrte nd were oth ound to the lysosomes nd tht enhned inding of to rin lysosomes ws not oserved in the presene of non-cma sustrtes suh s ovlumin (Fig. i). In summry, lthough showed hrteristis ommon to CMA sustrtes when presented lone to lysosomes, CMA sustrtes enhned, rther thn inhiited, inding of to lysosomes. These findings revel the existene of n interply etween nd CMA sustrte proteins t the lysosoml memrne tht enhnes inding of to this memrne ut not its trnslotion into lysosomes. Compromised degrdtion of mutnt The most ommon pthogeni muttion is G9S, sustitution in the kinse domin tht results in toxi gin of kinse funtion. Using -tet-on ell lines, in whih expression of wild-type () or G9S () n e tivted y doxyyline (Supplementry Fig. ), we indued expression y - to d g h Bound (% untreted) Bound (% untreted) 5 75 (µg) Inp..6. ssoition (% untreted) (µg) 5 5 RNse A VOLUME 6 NUMBER APRIL nture neuroscience

4 npg Nture Ameri, In. All rights reserved. LA( ) LA( ) NL MG MA NL MG MA Time (h) LA( ) LA( ) Inp PI PI GFP- LRKK LA 5 LA( ) Inhiitors.6 n.s LA( ) LA( ) Binding Assoition Uptke NL MG MA 5 5 Time (h) Time (h) d e f g Input Lysosom h (ng) (ng) 5 5 (ng) (ng) Brin Inp (µg) i.6 Inp 5 Inp (µg).8 GFP- LRKK Inhiition (%) ssoition (% untreted) ound (fold untreted) (% time ) Figure Differenes in the degrdtion of nd. () Top: immunolot for GFP of tet-on HEK9 ells expressing GFP-tgged or pulsed h with doxyyline nd, 8 h lter (no longer undergoing de novo synthesis), inuted without dditions ( ) or with mm NH Cl nd µm leupeptin (NL), 5 µm MG (MG) or mm MA. Bottom: perentge inhiition (n = independent experiments). () Sme ells s in, untreted () or ound (fold untreted) (% time ) degrdtion (% h ) None +PI (% of protein dded) ssoited (% untreted) ssoited (fold untreted) treted with RNA interferene for LAMP-A (LA( )), pulsed s in, sequentilly olleted nd immunolotted. Top left pnels: representtive immunolots for GFP. is shown s loding ontrol. Bottom left pnels, quntifition, orreted y levels of knokdown nd loding ontrol (n = or independent experiments). Top right: immunolot for LAMP-A. Bottom right: perentge of degrded per hour in the sme smples. () Binding, ssoition nd uptke lulted from quntifition of in immunolots of lysosomes from strved rt livers, untreted or pretreted with protese inhiitors (PI) nd inuted with or. Inset: representtive immunolot (n = 9 independent experiments). (d) Assoition of (5 µg) to strved rt liver lysosomes in the presene of inresing onentrtions of or. Inset: representtive immunolot n = 6. (e,f) Binding of nd to lysosomes in the presene of (e) or RNse A (f). Top: representtive immunolots. Bottom: lysosome-ound expressed s multiple of the mount ound when dded lone. Trends of men vlues (n = independent experiments); error rs indite rnge. (g) Immunolot of rt rin lysosomes (Lysosom) inuted with or s in. Inputs, one-tenth of protein dded. (h,i) Rt rin lysosomes inuted s in d nd e, respetively. Left: representtive immunolots. Right: quntifitions (n = or independent experiments). All vlues expressed s men + s.e.m.; differenes with untreted smples () nd etween nd () were signifint t P <.5; n.s., not signifint; ANOVA nd Bonferroni test. Full-length lots nd gels in Supplementry Figure. -h pulse of doxyyline nd followed the derese in the reominnt proteins 8 h fter the pulse to onfirm tht synthesis ws no longer ourring. As with endogenous in neurolstom ells (Fig. ), levels of the reominnt protein (Fig. ) nd of endogenous in these ells (Supplementry Fig. ) were inresed y protesome inhiitors (MG) nd lysosome inhiitors (NH Cl-leupeptin), inditing degrdtion of y oth pthwys. Levels of were lso higher fter ddition of protesome nd lysosome inhiitors, ut the effet of loking lysosoml degrdtion ws smller (Fig. ). Tretment with MA hd similr effet on s tretment with inhiitors of lysosoml proteolysis, suggesting tht the residul lysosoml degrdtion oserved for the mutnt ws likely tking ple minly y mroutophgy. We lso deteted higher sensitivity to MA in slies from rins of - trnsgeni mie thn from ontrol or - trnsgeni mie (Supplementry Fig. d). hd hlf-life of pproximtely 8 h, wheres the hlf-life of ws nerly douled (6 h) (Fig. ). Knokdown of LAMP-A redued the degrdtion of y 5% ut hd only modest effet on the degrdtion of (Fig. ). Together, these findings suggest tht the degrdtion of y CMA is ompromised nd tht the pprent swith of the mutnt protein towrd degrdtion y the protesome nd, for smll frtion, y mroutophgy is not suffiient to mintin the rtes of degrdtion normlly oserved for the protein. We then ompred the diret uptke of purified nd y isolted liver lysosomes fter verifying tht did not ompromise lysosoml integrity (Supplementry Fig. ) nd hd omprle suseptiility to degrdtion y the lysosoml hydrolses (Supplementry Fig. d). ound the lysosoml memrne with lower effiieny thn, nd its lysosoml uptke ws lso signifintly lower (Fig. ). Despite its lower CMA, exerted more pronouned inhiitory effet on uptke of other CMA sustrtes thn did (Fig. d). Notly, s inding to lysosomes ws lso inresed y CMA sustrte proteins, nd this effet ws more prominent thn tht nture NEUROSCIENCE VOLUME 6 NUMBER APRIL 97

5 npg Nture Ameri, In. All rights reserved. Figure Overexpression of proteins exerts n inhiitory effet on CMA. () Proteolysis in HEK9 ells stly trnsfeted with n empty plsmid () or plsmids expressing or under the ontrol of tet promoter, treted with doxyyline to tivte protein expression nd then leled with [ H]leuine for 8 h. After extensive wshing, protein degrdtion t the indited times ws mesured, in ells mintined either in the presene (+) or sene ( ) of serum, s the mount of id-preipitle rdiotivity (mino ids nd smll peptides) relesed into the medi. Vlues re expressed s perentge of rdiotivity in proteins (id preipitle) t time (n = independent experiments, eh in triplite). () The sme ells, trnsiently trnsfeted with KFERQ-PA-mCherry fluoresent reporter for CMA tivity. After phototivtion, ells were mintined for 6 h in the presene or sene of serum. Left: representtive mirogrphs; right olumn shows higher mgnifition imges of outlined regions. Arrows denote punt. Right: verge numer of punt per ell in eh ondition, mesured in >5 ells. () Cultured ventrl midrin dopminergi neurons from nd from - nd - trnsgeni mie, trnsdued with the sme reporter s in nd o-stined with ntiody to tyrosine hydroxylse (TH). Nulei re highlighted with DAPI (,6-dimidino--phenylindole) in lue. Left: representtive imges. Right: verge numer of punt per ell in >5 TH-positive ells. Sle rs, µm. All vlues re men + s.e.m.; differenes with () nd etween nd () were signifint t P <.5; unpired t-test in nd ANOVA nd Bonferroni test in,. of the protein (Fig. e,f). Studies with isolted rin lysosomes reveled similr redution in lysosoml inding nd uptke of (Fig. g), signifintly stronger inhiitory effet on CMA (Fig. h) nd slightly higher ssoition to lysosoml memrnes in the presene of CMA sustrtes (Fig. i) when ompred to. We ttriute the smll differenes etween liver nd rin lysosomes to the presene of endogenous in the rin lysosomes (Fig. g). Independent of these smll tissue-speifi differenes, overll these results indite tht is less effiiently degrded y CMA ut exerts greter inhiition over this pthwy. Inhiitory effet of on CMA To determine the ellulr onsequenes of higher ssoition of with the lysosoml memrne in the presene of CMA sustrtes (Fig. ), of the exertion of this inding for (Fig. e,f,i) nd of the greter inhiition y the mutnt on lysosoml uptke of CMA sustrtes (Fig. d,h), we nlyzed the sttus of the utophgi system in ells expressing nd. Long-lived proteins, whih re typilly degrded y the lysosoml system, were degrded more slowly when either form of ws overexpressed, lthough this inhiitory effet ws more pronouned for (Fig., left). Serum deprivtion, used to stimulte utophgy, did not inrese protein degrdtion in the ells expressing or, nd the inhiitory effet of remined more pronouned thn tht of the protein (Fig., right). To seprtely nlyze the impt of these proteins on the different utophgi pthwys, we first monitored levels nd degrdtion of mirotuule-ssoited protein light hin (LC), well-estlished mrker for mroutophgy. The inrese in ellulr LC oserved when lysosoml proteolysis is inhiited is inditive of the flux of sustrtes through mroutophgy. In ells expressing, LC flux trended slightly higher thn in ontrol ells (Supplementry Fig. ), whih, long with the fewer LC-positive punt (utophgosomes) oserved y immunofluoresene (Supplementry Fig. ), supported modest stimultion of mroutophgy y. LC flux in ells expressing ws lose to tht in ontrol ells, suggesting tht the derese in protein degrdtion oserved in these ells ws not due to inhiition of mroutophgy. Proteolysis (%) KFERQ-PA-mCherry KFERQ-PAmCherry Merged TH 6 Serum Time (h) Serum + Serum We then nlyzed CMA in the three groups of ells y trnsfeting them with phototivtle (PA) fluoresent reporter (KFERQ- PA-mCherry) tht provides visuliztion of CMA s hnge in the distriution of the fluoresent protein from the ytosol (diffuse fluoresent pttern) to lysosomes (puntte fluoresent pttern). Cells expressing nd oth showed mrked derese in the numer of fluoresent punt per ell in sl onditions in omprison to ontrol ells (Fig. ), nd they filed to indue CMA on removl of serum from the ulture medium (Fig. ). We onlude tht the derese in degrdtion of long-lived proteins oserved in ells expressing nd ws due, for the most prt, to their inhiitory effet on CMA. We found similr inhiitory effet on CMA for in neurons. Anlysis of CMA tivity with the sme fluoresent reporter in primry neuronl ultures reveled signifint redution in CMA in neurons from - trnsgeni mie in omprison to ontrol mie (Fig. ). Notly, lthough ompromise of lysosoml degrdtion ws not evident in rins of - trnsgeni mie (Supplementry Fig. d), we found tht CMA tivity ws signifintly redued in these neurons in omprison to those from ontrol mie (Fig. ). Together, these results indite tht oth nd high levels of inhiit CMA in neuronl nd non-neuronl ells. Effet of on CMA omponents Reent studies in Drosophil hve noted tht expression of results in perinuler lustering of endosoml nd lysosoml omprtments. Leling of lte endosomes nd lysosomes with LysoTrker or LAMP- (Supplementry Fig. 5,, respetively) onfirmed the desried norml expnsions of these vesiulr strutures not Proteolysis (%) Time (h) 5 Serum Serum + n.s. 6 Serum n.s. - - Averge punt per ell Punt per ell Punt per ell 6 8 n.s. 98 VOLUME 6 NUMBER APRIL nture neuroscience

6 npg Nture Ameri, In. All rights reserved. Figure 5 Interply of with CMA omponents. () Immunolots of rt liver lysosomes inuted with or lone or with mm GTP nd/or. Duplite smples re shown. Bottom: quntifition of lysosome-ound expressed s multiple of the mount ound without dditions (n = 6 independent experiments). () Effet of kinse inhiitor on,, or the kinse-ded mutnt D/A inuted with rt liver lysosomes in presene or sene of (5 µg). Right: quntifition expressed s multiple of the ssoition without dditions (n = 5 independent experiments). (,d) Trnsient trnsfetion of HEK9 ells with the my- or GFP-tgged onstruts indited t top left in. Full, full length; M, KFERQ-like motifs (M M8). () Binding to GST-hs7. Left, representtive immunolots; input lnes, onetenth of input mteril. NT, non-trnsfeted ells. Immunolot for hs7 is shown s loding ontrol. Top right: quntifition of ound to hs7. Where indited, smples were lso inuted with RNse A to ompete the KFERQ-medited inding of hs7 (n = or independent experiments). (d) Coimmunopreipittion (IP) of hs7 with nti-my or nti-gfp in the sme ells. Inputs (Inp), one-qurter of strting volume. (e) Binding or to rt liver lysosomes treted with the indited onentrtions of trypsin. LAMP- is shown to onfirm the integrity of the lysosomes. (f) Co-IP of LAMP-A with nti- in lysosomes inuted with or. Inp, one-qurter of strting smple; IgG, immunogloulin G. Vlues: LAMP-A reovered in the IP (IP LA) orreted for the mount of pulled down nd expressed s multiple of vlue. (g,h) Duplite LAMP-A immunolots of rt liver lysosomes inuted lone ( ) or with or (g) or with lysosomes from ontrol () or from - or - trnsgeni mie (h) nd sujeted to lue ntive eletrophoresis. Lysosoml (fold ) None + GTP My My GFP My My GFP Hs7 Kinse inhiitor (nm) None + GTP.5.. Arrow, 7-kD multimeri omplex. All vlues re men + s.e.m.; differenes ompred to none or untreted () or to or full-length protein () were signifint t P <.5; unpired t-test in nd ANOVA nd Bonferroni test in,. Full-length lots nd gels in Supplementry Figure. + None D/A D/A COR KIN ANKLRR ROC M5 M7 WD None 5 RNAse A M M M M M6 M8 RCK 5 COR K ROC Input + GST-hs7 5 Full RCK K COR ROC NT IP: Inp IP Inp IP LAMP-A IgG IP LA (fold ) Full RCK None GTP K + GTP COR ROC NT. LAMP-A Full RCK 7 kd kd K None GTP + GTP COR ROC GST-hs7 Perentge of input dded LAMP- Trypsin (µg) 5 5 f g h e 669 -ssoited (fold untreted) d Hs7. D/A Kinse inhiitor (nm) Kinse inhiitor (nm) Full RCK COR ROC Inp IP Inp IP Inp IP Inp IP My My My GFP only in ells expressing, ut lso in ells expressing. Although CMA does not our in endosomes, we nlyzed whether -indued hnges lso ffeted lysosomes tive for CMA (defined s those positive for oth hs7 nd LAMP-A; Supplementry Fig. 6). Although we did not note expnsion or lustering of CMA-tive lysosomes, immunofluoresene nd immunolot (Supplementry Fig. 6) reveled tht levels of LAMP-A were out twie s high in ells expressing either or s in ontrol ells. This ontrsted with the mrked derese in CMA tivity oserved in these ells (Fig. ) nd likely reflets ompenstory response of these ells similr to tht desried in other onditions with ompromised CMA, suh s ging or other models of Prkinson s disese 5 7. We did not find signifint hnges in the levels of LAMP-A in lysosomes isolted from ells in whih ws knoked down or y immunofluoresene nlysis of these ells (Supplementry Fig. 6), or in lysosomes isolted from -null mie (Supplementry Fig. 6d), suggesting tht the hnges in CMA oserved on expression of re more likely result of gin of toxi funtion rther thn loss of physiologil funtion. The lk of mrked hnges in the numer or distriution of CMAtive lysosomes led us to hypothesize tht the inhiitory effet of on CMA did not our t the level of formtion nd mturtion of the CMA lysosomes ut rther ws result of the diret intertion of with these lysosomes. To disset the inhiitory effet of nd tht of high levels of on CMA, we used the in vitro system with isolted lysosomes. GTP did not ffet inding of or to the lysosoml memrne (Fig. 5) or their inrese in the presene of the CMA sustrte (Fig. 5), inditing tht the GTPse tivity of does not modulte its inding to the lysosoml memrne. In ontrst, SU6656, seletive Sr-fmily kinse inhiitor previously used to lok the kinse tivity of (ref. 8), inresed inding of to lysosomes oth when the ws presented lone or in the presene of other CMA sustrtes (Fig. 5). Tretment with the inhiitor under the sme onditions did not ffet the lysosoml inding of kinse-ded form of, nd thus the hnges in inding were indeed onsequene of inhiition of its kinse tivity (Fig. 5). Inhiition of kinse nture NEUROSCIENCE VOLUME 6 NUMBER APRIL 99

7 npg Nture Ameri, In. All rights reserved. LAMP-A LAMP LAMP-A Control Control tivity did not ffet its lysosoml inding if the protein ws presented lone ut mrkedly enhned the -indued inding of to the lysosoml memrne (Fig. 5). We onfirmed tht tretment with the kinse inhiitor did not ffet lysosoml stility (Supplementry Fig. e) nd tht the did not mimi the - ehvior even t very high onentrtion of the inhiitor (Supplementry Fig. f). Overll, these results support the finding tht inding of to lysosoml memrnes inreses in the presene of other CMA sustrtes, nd more so if its kinse tivity eomes ompromised. To gin further insight into the regions of importnt for its CMA trgeting nd degrdtion, we investigted the relevne of the different CMA-trgeting motifs, the pentpeptide reognized y hs7, in its sequene (Fig. 5). Studies in other multi-motif CMA sustrtes hve demonstrted tht single motif is suffiient for lysosoml trgeting nd tht, lthough the presene of multiple motifs does not inrese effiieny of trgeting, hs7 often hs n order of preferene in the inding 7. However, one the most fvored motif(s) re eliminted, it is not unusul for hs7 to ind to one of the other motifs 9. For tht reson, we studied inding of this hperone to previously hrterized my- or GFP-tgged trunted forms of tht er different omintions of CMA trgeting motifs,. In vitro inding studies to glutthione S-trnsferse (GST)-tgged LAMP-A/ LAMP-A (punt per ell) LAMP- (punt per ell) Control Figure 6 Altered CMA mrkers in rins of Prkinson s disese ptients with the muttion. () Immunofluoresene for LAMP-A nd LAMP- of setions from the dorsl motor nulei of the vgus nerves of rins from two different unffeted ontrol () individuls nd two individuls with Prkinson s disese with the muttion. Left: imges from representtive fields. Right: numer of fluoresent punt per ell. Blk dots, vlues in individul ells; red lines, men. () Immunohistohemistry for LAMP-A in the sme smples. () Immunofluoresene for LAMP-A (red) nd (green) in the sme smples. Merged imges from the two fluorophore hnnels re shown (for ololiztion quntifition, see Supplementry Fig. 7). Sle rs, µm. Vlues re men + s.e.m.; P <.5, ANOVA nd Bonferroni test. hs7 reveled tht inding to the hperone of frgment tht ontins the ROC, COR nd kinse regions (RCK) nd tht ers the five entrl CMA motifs (M M7) ut is missing the two N-terminl (M nd M) nd the most distl C-terminl motifs (M8) ws omprle to inding of the full-length, rguing ginst involvement of these three motifs in hperone inding (Fig. 5). In ontrst, inding to hs7 of trunted form of ering only M to M6 (COR) ws mrkedly redued, suggesting tht either M or M7 ws importnt for hperone inding of (Fig. 5). We found tht M7, the motif in the kinse region of, ould not e seletively reognized y the hperone, t lest when this region ws presented lone. (Note tht the inding to hs7 of this produt ould not e ompeted with protein ering CMA-trgeting motif, suggesting nonspeifi inding; Fig. 5.) However, inding to hs7 ws preserved in trunted form of (ROC) ering M nd M. As we did not oserve inding to hs7 of the frgment ontining M M6, we onluded tht M, the motif in the ROC region of, ws strong ndidte for hs7 inding (Fig. 5). Coimmunopreipittion of hs7 with in ells expressing the vrious tgged trunted proteins onfirmed tht inding of hs7 to the frgment ering M nd M lso ourred in intt ells ut tht sene of M mrkedly redued the mount of hs7 reovered in the pull-down ssy (Fig. 5d). Although future detiled nlysis of the ontriutions of different motifs in LRKK inding to hs7 under different onditions is needed, these results support the requirement of M for hperone inding. We next nlyzed the nture of the inding of to CMA lysosomes nd demonstrted tht trypsiniztion of lysosoml memrnes grdully deresed inding of, supporting protein-to-protein inding of (Fig. 5e; LAMP- is shown to demonstrte tht trypsin did not reh the lysosoml lumen). The redued mount of deteted in lysosomes from ells in whih LAMP-A ws knoked down (Fig. f) suggested tht lysosoml inding of likely ourred through LAMP-A, nd pull-down experiments of lysosome-ound onfirmed its inding to LAMP-A (Fig. 5f). Of note, omprison of the mount of LAMP-A reovered in pulldowns for nd reveled tht, lthough the mutnt protein ound less effiiently to the lysosoml memrnes thn (Fig.,g), one ound to this reeptor its inding to LAMP-A ws more stle (Fig. 5f). We then nlyzed the effet of nd on LAMP-A. We hypothesized tht this tight inding to LAMP-A of might inhiit CMA y fvoring moiliztion of LAMP-A into speifi lipid mirodomins t the lysosoml memrne where it eventully undergoes degrdtion. However, inution of lysosomes with did not enhne reruitment of LAMP-A to the lysosoml lipid mirodomins, even when other CMA sustrte proteins were dded to the inution medium (Supplementry Fig. g,h nd dt not shown). We nlyzed the effet of on the orgniztion of LAMP-A into the multimeri omplex responsile for trnslotion of CMA sustrtes, whih n e visulized s single 7-kD nd y lue ntive eletrophoresis nd immunolotting for LAMP-A (Fig. 5g). We found tht oth proteins, t onentrtions tht inhiit CMA uptke, mrkedly deresed the mount of LAMP-A detetle in the multimeri omplex (Fig. 5g). Lysosomes from - trnsgeni mie lso showed lower undne of the LAMP-A trnslotion omplex thn lysosomes from ontrol mie (Fig. 5h). In the se of the - trnsgeni mie, there ws lso trend towrd lower levels of multimeri LAMP-A, lthough there ws more vriility from niml to niml thn in the se of the mutnt trnsgeni mie (Fig. 5h). Overll, these results support the VOLUME 6 NUMBER APRIL nture neuroscience

8 npg Nture Ameri, In. All rights reserved. Perentge of protein dded d 5 D/A I/T R/C D/A I/T R/C D/A I/T R/C 5 GFP- LA Hsp9 Hs7 Inputs (/) Binding D/A I/T R/C GFP IP Perentge of protein dded 5 D/A I/T R/C Inp IP Inp IP Inp IP Inp IP Inp IP 5 Lysosomes Uptke PI D/A I/T R/C e R/C I/T D/A D/A I/T R/C RNse A D/A I/T R/C RNse A 5 ound (fold unsupplemented) Punt per ell Perentge of totl re D/A I/T R/C LA LA Co-IP LAMP-A (times ontrol) D/A I/T R/C IP D/A I/T R/C R/C D/A I/T Figure 7 Intertion of other mutnt vrints with CMA. () Top: immunolot R/C D/A I/T of rt liver lysosomes inuted with,, D/A (kinse-ded), I/T or R/C mutnt, lone or in the presene of protese inhiitors (PI). Input lnes, one-qurter of input mteril. Bottom: quntifition of inding nd uptke (lulted s in Fig. ; n = or 5 independent experiments). () Left: immunolots of lysosomes inuted s in, lone or in the presene of inresing onentrtions of RNse A (left) or (right). Right: quntifition of ound, s multiple of the inding when inuted with lysosomes lone (n = independent experiments). () Top: oimmunopreipittion (o-ip) of LAMP-A (LA) with nti- from lysosomes inuted with proteins s in. Co-immunopreipitted frtions were immunolotted for LAMP-A nd. Input: immunolot for LAMP-A efore the ffinity purifition. Bottom: mount of LAMP-A oimmunopreipitted, expressed s multiple of the vlue with (n = or independent experiments). (d) Co-IP of LAMP-A with nti-gfp in tet-on HEK9 ells expressing GFP- proteins nd pulsed for h with doxyyline. Inputs (Inp), one-qurter of mteril dded. (e) The sme ells s in d nd ontrol ells trnsdued with KFERQ-PA-mCherry. Left: representtive imges. Nulei re highlighted with DAPI in lue. Right: Quntifition fter phototivtion nd 6 h of serum deprivtion (n > 5 ells). All vlues re men + s.e.m. Differenes with () or with the indited mutnts () were signifint t P <.5; unpired t-test in nd ANOVA nd Bonferroni test in e. Sle rs, µm. Full-length lots nd gels in Supplementry Figure. Co-IP Input notion tht the nd inhiition of LAMP-A multimeriztion is responsile for the redued CMA tivity oserved in non-neuronl nd neuronl ells overexpressing these proteins. Finlly, we onfirmed tht ltertions in LAMP-A were evident in the dorsl motor nuleus of the vgus (DMV), region onsidered to e the first highly trgeted re of the entrl nervous system in Prkinson s disese, from Prkinson s disese ptients with the muttion. Similrly to the upregultion of LAMP-A oserved in ultured ells when expression of ws tivted (Supplementry Fig. 6,), immunostining for LAMP-A reveled mrked inrese in LAMP-A in the ptients rins when ompred with tht in rins from ge-mthed ontrol individuls (Fig. 6,). This inrese ws lso detetle fter immunolot for LAMP-A of the sme rin smples (Supplementry Fig. 7). The inrese in LAMP-A does not seem mrker of generl upregultion of the lysosoml system in the Prkinson s disese rins, euse we did not detet signifint differenes in levels of LAMP-, more undnt lysosoml memrne protein, etween ontrol nd ptients rins (Fig. 6 nd Supplementry Fig. 7). In ddition, nd in greement with our findings in vitro, ssoition of with LAMP-A positive omprtments ws lso mrkedly inresed in the rins of the Prkinson s disese ptients (Fig. 6 nd Supplementry Fig. 7). These findings provide evidene for seletive ltertions in CMA in the rins of Prkinson s disese ptients with muttions. Intertion of other mutnts with CMA We next exmined three more mutnts, two in the kinse site nd one in the GTP-inding region, for possile differenes in their degrdtion y CMA nd impt on this utophgi pthwy. We seleted n dditionl muttion in the ROC site of (RC, or R/C ) nd muttion in the kinse site of (IT, or I/T ), oth desried in fmilil Prkinson s disese nture NEUROSCIENCE VOLUME 6 NUMBER APRIL

9 npg Nture Ameri, In. All rights reserved. Figure 8 Coinidene of nd α-syn t lysosomes enhnes their toxi effet on CMA. () α-syn immunolot of rt liver lysosomes inuted with or mutnt (A5T) α-syn lone ( ) or with or. Inp, input; Mon, monomers. Right: quntifition of α-syn oligomers, expressed s multiple of mounts in smples without (n = or 5 independent experiments). ( d) Degrdtion of rdioleled proteins (), inding of GST α-syn () nd levels of endogenous α-syn (d) in intt rin lysosomes (Lysos) from non-trnsgeni () nd trnsgeni mie. Bottom pnel in shows lower exposure to visulize monomers of α-syn. Homog, homogentes; olig, oligomers; I, input (n = independent experiments). (e) Ventrl midrin dopminergi neuronl ultures from ontrol () or - or - trnsgeni mie untreted or treted with RNAi for LAMP-A (LA( )). (f) Cololiztion etween α-syn nd totl LAMP- from ells in e (n > 5 tyrosine hydroxylse (TH)- positive neurons). Totl LAMP- is not redued in LA knokdown ells euse LAMP-B is upregulted. (g) ipsc lines from ontrols nd from ptients with -muttion Prkinson s disese, differentited for d nd immunostined for LAMP-A, α-syn nd TH. Merged hnnels (left) nd mgnified green nd red hnnels from oxed region (right). Arrows, ololiztion LAMP-A nd α-syn. (h) Perentge of LAMP-A + punt ololizing with α-syn in TH + ells (n > 5) for two ontrol nd two ipsc lines treted s in g. (i,j) ipsc lines s in g, differentited for or 75 d nd untreted or treted with RNAi for LAMP-A. (i) Immunofluoresene for LAMP-A, α-syn nd GFP in LAMP-A-RNAi ipsc lines differentited for d. Bottom: higher mgnifitions of oxed res. (j) Perentge of TH + ells showing ytoplsmi umultion of α-syn. n experiments using two ontrol nd two ipsc lines (n > 7 nd n > 5 TH + ells in nd, respetively). Sle rs, µm. All vlues re men + s.e.m. Differenes from or ontrol () or etween ontrol or knokdown ells () re signifint t P <.5; unpired t-test in,h nd ANOVA nd Bonferroni test in f,j. Full-length lots nd gels in Supplementry Figure. ptients nd shown to inrese dimeriztion, nd n experimentl muttion tht ltes kinse tivity (D99A, or D/A-) nd yields low dimer stility. We found tht, wheres I/T showed levels of inding nd uptke y isolted lysosomes omprle to those of the, inding nd in prtiulr uptke of R/C nd D/A were lower thn those of (Fig. 7). Notly, the mutnt forms with redued inding nd uptke y lysosomes showed more pronouned inrese in their inding to lysosomes in the presene of CMA sustrtes (suh s nd RNse A; Fig. 7). These results support tht the inility to trnslote into the lumen of lysosomes my enhne the sustrte-indued lysosoml inding of nd fvor its umultion in the memrne of these orgnelles. Pull-down experiments of the vrints ound to lysosomes in the in vitro system (Fig. 7) or oimmunopreipittion from ells expressing GFP-tgged forms of eh of these vrints (Fig. 7d) reveled tht the enhned intertion of with LAMP-A ws lso oserved for R/C- ut not for D/A, suggesting tht the enhned sustrte-dependent inding of this vrint to the lysosoml memrne my involve different mehnisms thn the persistent inding to LAMP-A of nd R/C. α-syn oligomers Mon e LA( ) f h j α-syn LAMP- ololiztion (%) α-syn ng Inp.9.6. α-synulein LAMP- 6 6 α-syn A5T Inp 6 6 TH LA α-syn ololiztion (% of LA punt) α-syn oligomers (fold no ) Merged LA( ) (µg) α-syn A5T α-syn (µg) g α-syn + TH + ells (% of TH + ells) Proteolysis (%) Control Intt lysosomes 6 As expeted from the effiient degrdtion of I/T- y CMA (Fig. 7) nd its lower ssoition to the lysosoml memrne in the presene of sustrtes (Fig. 7), we did not find signifint differenes from in the mount of this protein ound to LAMP-A (Fig. 7,d). To diretly nlyze the impt of these different mutnts on CMA in intt ells, we expressed these vrints in ells under the tet-regulted system desried efore (Supplementry Fig. ) nd used the fluoresent KFERQ-PA-mCherry reporter to quntify CMA tivity. In greement with the lower degrdtion of R/C nd D/A- y CMA (Fig. 7) nd their higher ssoition to lysosomes when in the presene of CMA sustrtes (Fig. 7), we found tht these two vrints signifintly redued CMA tivity to levels omprle to those oserved for (Fig. 7e). A redution in CMA tivity, lthough smller, ws lso oserved in ells expressing I/T, lthough in these the predominnt feture ws mrked inrese in the size of the lysosoml omprtments relted to CMA (Fig. 7e). Although the mening of these hnges in CMA lysosomes requires further investigtion, these findings show tht different muttions my inhiit CMA through different mehnisms. α- syn α- syn LAMP- A LAMP- A LAMP- A α- syn LA( ) Lysosomes (µg) LAMP-A/α-syn/TH/DAPI i Control d 75 d Anti-GST α-syn I α-syn/th/gfp/dapi DAPI TH Low Oligomers Mon exp d Mon α-syn Olig Hs7 LA Homog Lysos GFP DAPI GFP DAPI GFP DAPI GFP α-syn TH α-syn TH α-syn TH α-syn VOLUME 6 NUMBER APRIL nture neuroscience

10 npg Nture Ameri, In. All rights reserved. Consequenes of unusul interply etween nd CMA Beuse enhned inding to LAMP-A t the lysosoml memrne ws shred y two of the mutnts, inluding the most ommon muttion in fmilil Prkinson s disese ptients ( ), we set out to nlyze the onsequenes of this norml inding. We speulted tht, y loking the formtion of the reeptor omplex (Fig. 5g), nd high levels of my led to higher onentrtion of sustrte proteins ound to lysosomes t given time euse the inility of LAMP-A to multimerize does not ffet sustrte inding ut, rther, prevents their trnslotion into the lysosoml lumen. This high onentrtion my e prtiulrly detrimentl for pthogeni proteins tht tend to orgnize into norml multimeri omplexes nd oligomers. To test this possiility, we nlyzed the effet of on the lysosoml ssoition of α-syn nd pthogeni α-syn mutnt (A5T) tht we hve previously shown inds LAMP-A t the lysosoml memrne ut does not trnslote into the lumen 8. ompeted ssoition of monomeri α-syn to lysosomes (s ws the se for other CMA sustrtes) ut ws less effiient in ompeting the mutnt α-syn vrint (Fig. 8 nd Supplementry Fig. 8). Inresing onentrtions of deresed the presene of α-syn oligomers in lysosomes (likely y ompeting inding when still t the monomeri stge (Supplementry Fig. 8), wheres omintion of with the mutnt α-syn protein mrkedly inresed formtion of α-syn oligomers in dose-dependent mnner (Fig. 8). -indued formtion of oligomers of α-syn did not our when the two proteins were inuted lone, inditing tht oligomeriztion required their inding to lysosoml memrnes (Supplementry Fig. 8). These results suggest tht the oinidene t the lysosoml memrne of nd mutnt α-syn is suffiient to ggrvte the CMA defet., rther thn ompeting inding of the α-syn proteins, preserved or even enhned inding of monomeri α-syn to the lysosoml memrne (Supplementry Fig. 8), whih proly uses the oserved mrked inrese in formtion of oligomers of even the form of α-syn t the surfe of lysosomes (Fig. 8). did not indue oligomeriztion of α-syn proteins in the sene of lysosomes (Supplementry Fig. 8). The differenes etween the effets of nd on α-syn oligomeriztion re likely result of the different effets tht α-syn proteins hd on the lysosoml ssoition of nd. Wheres the omintion of forms of oth proteins did not interfere with lysosoml uptke of, omintion of with mutnt α-syn or mutnt with ny of the α-syn proteins mrkedly deresed the trnslotion of into lysosomes (Supplementry Fig. 8). Studies in neuronl ell lines otrnsfeted with α-syn nd proteins demonstrted signifint dereses in the degrdtion rtes of α-syn in the ells oexpressing (Supplementry Fig. 9). Overexpression of in these ells did not hnge the erly rtes of α-syn degrdtion, ut it did dely the degrdtion of this protein in the lte stges (Supplementry Fig. 9). Further experiments re needed to determine whether suh two-phse kinetis reflet n erly ompenstory tivtion of other proteolyti systems in the ells expressing or the mount of time neessry to umulte enough t the lysosoml memrne to ffet α-syn degrdtion. To determine whether the proposed norml oligomeriztion of α-syn t the lysosoml memrne nd the susequent ompromise of CMA lso ourred in lysosomes exposed to mutnt in vivo, we isolted lysosomes from non-trnsgeni nd - trnsgeni mie. Intt lysosomes isolted from - mie showed redution in their ility to tke up proteins vi CMA (Fig. 8). (Note tht this ssy repitultes inding, uptke nd proteolysis, ut, euse the proteolyti tivity of disrupted lysosomes ws omprle in oth groups (dt not shown), the oserved differenes re in inding nd uptke.) We onfirmed tht, in the se of α-syn, this redued CMA tivity fvored the formtion of the oligomeri omplex, similrly to wht we oserved in the in vitro experiments (Fig. 8). In ft, not only the exogenous α-syn ut lso endogenous α-syn ould e found ssoited to lysosomes from - mie, nd it orgnized there s irreversile oligomeri omplexes (Fig. 8d). We lso found signifint inrese in the ololiztion of α-syn with lysosoml mrkers (suh s totl LAMP; Fig. 8e,f) in neuronl ultures from rins of - nd - trnsgeni mie when ompred to neurons from ontrol mie. This enhned ssoition of α-syn to lysosomes is onsequene of its delivery to this omprtment y CMA euse knokdown of LAMP-A in these neurons redued ololiztion of α-syn with lysosomes (Fig. 8e,f). None of these mnipultions hnged the lredy miniml ololiztion etween nd LC (Supplementry Fig. ). A similr inrese in intrellulr levels of α-syn nd in the ssoition of this protein with lysosomes ws oserved in primry neuronl ultures from RG- trnsgeni mie (Supplementry Fig. ), whih ould e responsile for the redued CMA tivity oserved in these ells using the CMA fluoresent reporter (Supplementry Fig. ). Lstly, to determine whether similr ssoition of α-syn with lysosomes is lso present in neurons from Prkinson s disese ptients with the muttion, we differentited dopminergi neurons from indued pluripotent stem ells (ipsc) from suh individuls. We hve previously shown tht α-syn normlly umultes in these differentited neurons. After d of differentition, time when ptient ipsc-derived neurons do not show overt morphologil signs of neurodegenertion, only % of neurons positive for the dopminergi mrker tyrosine hydroxylse showed detetle mounts of α-syn in dopminergi neurons differentited from ipscs derived from helthy individuls, nd only smll perentge (less thn %) of these ells ontining α-syn showed ololiztion etween α-syn nd the lysosoml mrker. In ontrst, α-syn ws detetle in % of the neurons derived from the Prkinson s disese ptients, nd this protein ololized with LAMP-A in lmost 65% of them (Fig. 8g,h). To ddress the ontriution of hnges in CMA tivity to the normlly high levels of α-syn in the ptient-derived ells, we knoked down LAMP-A in differentited neurons t weeks nd 75 d (when signs of neurodegenertion re lredy evident in the ptient-derived neurons) y lentivirl trnsdution of the GFP-tgged short hirpin RNA ginst LAMP-A 5. Knokdown of LAMP-A t weeks of differentition mrkedly inresed the perentge of α-syn positive dopminergi neurons oth in ontrol nd in Prkinson s disese ptient ipsc derived neurons (Fig. 8i,j). Neurons from ultures infeted with ontrol lentivirus, or inluding the frtion of neurons in the sme ultures tht remined untrnsdued (Fig. 8i), did not show signifint hnges in their α-syn ontent. Notly, the inrese in ells showing α-syn umultion ws signifintly higher in the ptient-derived ells thn in ontrol, suggesting possile ompenstory upregultion of CMA in erly, presymptomti sttes (Fig. 8j). The umultion of α-syn in Prkinson s disese ptient derived ells fter CMA lokge ws often ssoited with mrked neurite shortening (Fig. 8i). Knokdown of LAMP-A in ells ultured for 75 d (when signs of neurodegenertion re lredy evident in ptient-derived ells ), lso inresed α-syn ontent in oth groups, ut the proportionl inrese ompred to untrnsdued ells ws lower in the ptient ells, likely inditing n lredy ompromised CMA of α-syn in these ells (Fig. 8i,j). nture NEUROSCIENCE VOLUME 6 NUMBER APRIL