β-synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with...

Department of Cellular Biochemistry and Human Genetics, Hebrew University-Hadassah Medical School, Jerusalem, Israel

The synuclein family of proteins consists of a-synuclein(aS), b-synuclein (bS) and c-synuclein, the cytoplasmicproteins expressed primarily in neurons. While aS and bS areco-localized in pre-synaptic nerve terminals of the CNS(Maroteaux et al. 1988), c-synuclein is expressed in the PNS[reviewed in (Clayton and George 1998)]. All three membersof the synuclein family have in their N-terminal region, arepetitive, highly conserved a-helical binding motif that issimilar to the class-A2 lipid-binding domains of theexchangeable apolipoproteins which mediates binding tomembrane phospholipids (George 2002).

Synucleinopathies are a group of neurodegenerativediseases including Parkinson’s disease (PD), dementia withLewy bodies (DLB) and multiple system atrophy [reviewedin (Duda et al. 2000)]. Abnormal aS cytoplasmic aggrega-tion and accumulation in Lewy bodies and Lewy neurites hasbeen implicated as the key pathogenic event in synuclein-opathies. Nevertheless, growing evidence point to a neuro-protective role for its homologue, bS, in synucleinopathies.

The findings that bS protein inhibit aS aggregation andfibril formation in vitro (Hashimoto et al. 2001; Uverskyet al. 2002; Park and Lansbury 2003) and furthermore, thatbS expression reduced aS aggregation, Lewy body formationand toxicity in vivo in mouse brains bigenic for aS and bS

(Hashimoto et al. 2001; Fan et al. 2006), have suggested aprotective role for bS in PD and the related synucleinopa-thies. Two mutations in bS, i.e. V70M and P123H, were thenfound in association with DLB (Ohtake et al. 2004). Over-expression of the mutated bS proteins in B103 neuroblas-toma cells resulted in the formation of bS-immunoreactive,eosinophilic cytoplasmic inclusions and enhanced lysosomalpathology (Wei et al. 2007). In this regard, bS accumulationswere documented in some synucleinopathies (Galvin et al.1999). bS is considered as a non-amyloidogenic (Masliahand Hashimoto 2002), natively unfolded protein (Bertoncini

Received July 29, 2008; revised manuscript received October 6, 2008;accepted November 4, 2008.Address correspondence and reprint requests to Ronit Sharon,

Department of Cellular Biochemistry and Human Genetics, HebrewUniversity-Hadassah Medical School, Jerusalem, Israel.E-mail: [email protected] used: ab, antibody; aS, a-synuclein; bS, b-synuclein;

ALA, 18:3 a-linolenic acid; BSA, bovine serum albumin; DEAE, Di-ethylaminoethyl; DLB, dementia with Lewy bodies; FA, fatty acid; IP,immunoprecipitation; MES, mesencephalic; MUFA, monounsaturatedfatty acid; MW, molecular weight; PD, Parkinson’s disease; PFU, plaqueforming units; PUFA, polyunsaturated fatty acid; SDS–PAGE, sodiumdodecyl sulfate–polyacrylamide gel electrophoresis; SEC, size exclusionchromatography; tg, transgenic; WT, wild-type.

Abstract

a-synuclein (aS) and b-synuclein (bS) are homologous pro-

teins implicated in Parkinson’s disease and related synuc-

leinopathies. While aS is neurotoxic and its aggregation and

deposition in Lewy bodies is related to neurodegeneration,

bS is considered as a potent inhibitor of aS aggregation and

toxicity. No mechanism for the neuroprotective role of bS has

been described before. Here, we report that similar to aS, bS

normally occurs in lipid-associated, soluble oligomers in wild-

type (WT) mouse brains. We partially purified bS and aS

proteins from whole mouse brain by size exclusion followed

by ion exchange chromatography and found highly similar

elution profiles. Using this technique, we were able to par-

tially separate bS from aS and further separate bS monomer

from its own oligomers. Importantly, we show that although

aS and bS share high degree of similarities, bS oligomeri-

zation is not affected by increasing cellular levels of poly-

unsaturated fatty acids (PUFAs), while aS oligomerization

is dramatically enhanced by PUFA. We show the in vivo

occurrence of hetero-oligomers of aS and bS and suggest

that bS expression inhibits PUFA-enhanced aS oligomeriza-

tion by forming hetero-oligomers up to a quatramer that do

not further propagate.

Keywords: a-synuclein, b-synuclein, polyunsaturated fatty

acids, protein oligomerization and aggregation.

J. Neurochem. (2009) 108, 465–474.

JOURNAL OF NEUROCHEMISTRY | 2009 | 108 | 465–474 doi: 10.1111/j.1471-4159.2008.05776.x

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 465–474 465

et al. 2007). Nevertheless, it was recently shown that certainfactors such as metals and pesticides cause its rapidfibrillation (Yamin et al. 2005).

The physiological role of all synucleins and bS inparticular is unknown. It was suggested that bS expressioninduces cell protectivity. bS was shown to act as an anti-apoptotic agent (da Costa et al. 2003) and induce cellprotection via increased Protein Kinase B activity (Hashimotoet al. 2004). Alternatively, purified bS was shown to restoreproteosomal activity inhibited by aS (Snyder et al. 2005).

We have recently reported that aS normally occurs inlipid-associated oligomers (Sharon et al. 2001, 2003b) andthat elevated cellular levels of polyunsaturated fatty acids(PUFAs) induce aS oligomerization, aggregation and itsdeposition in Lewy bodies (Sharon et al. 2003b; Assayaget al. 2007). We now report that similar to aS, bS occurs inlipid-associated oligomers in the cytoplasm. However, unlikeaS, bS oligomerization is not affected by the cellular fattyacids (FA) content. We further show the in vivo occurrence ofhetero-oligomers consisting of aS and bS and suggest thatbS expression inhibits PUFA-induced aS oligomerizationthrough the formation of hetero-oligomers that do not furtherpropagate.

Materials and methods

MiceThe WT C57Bl/6J (Jackson Laboratories, Bar Harbor, MN, USA)

and aS null mice C57Bl/6J (Specht and Schoepfer 2001) (Harlan

Laboratories, Rehovot, Israel) mouse lines were used. Holding and

breeding was carried out at the specific pathogen-free animal facility.

All protocols for animal use and experiments were reviewed and

approved by the committee for animal care and use of the Hebrew

University. Frozen brains of bS transgenic (tg) mice and their WT

controls (C57BL/6 · DBA/2) (Hashimoto et al. 2001) were gener-

ously provided by Prof. Eliezer Masliah (University of California).

Whole mouse brain fractionation was carried out as previously

described (Sharon et al. 2001). Briefly, whole mouse brains were

homogenized in 1 : 10 (wt/vol) homogenization buffer [20 mM

Hepes, pH 7.4, 1 mM MgCl2, 0.32 M sucrose, 43 mM 2-mercap-

toethanol, 1· protease inhibitor mix (Sigma, Rehovot, Israel)]. The

homogenate was centrifuged at 170 g for 10 min. The resultant

pellet (P1) was washed and respun at 370 g for 15 min. The washed

pellet was resuspended in homogenization buffer, and the suspen-

sion was brought to 2.1 M sucrose and spun at 180 000 g for 1 h.

The pellet (consisting of nuclei) and the lipid-rich fraction floating

on top of this sucrose cushion were collected. S1 supernatant was

centrifuged at 8000 g for 15 min to obtain P2 and S2. S2

supernatant was spun at 180 000 g. Protein concentrations were

assayed by the Bradford method (Bradford 1976).

Chloroform : methanol (2 : 1) extraction (Folch et al. 1957) ofhigh-speed supernatant (post 180 000 g) was performed as previ-

ously described (Sharon et al. 2001), with bS present at the interface

between organic and aqueous phases. For hexane extraction, one

volume of high-speed supernatant (15 lg protein) and three

volumes of hexane (100%) were mixed and rotated for 1 h and

then centrifuged at 2500 g for 10 min to separate the phases. bS was

present at the organic phase. The organic solvent was dried under

stream of N2 and the sample was re-suspended in 1· Laemmli buffer

and analysed by 10% sodium dodecyl sulfate–polyacrylamide gel

electrophoresis (SDS–PAGE) or native PAGE.

CellsThe mesencephalic cell lines, MES 23.5 and MN9D, which have

dopaminergic properties (Choi et al. 1991; Crawford et al. 1992)were stably transfected with human WT aS or human WT bScDNA [provided by Prof. George J.M., University of Illinois]

in the pCDNA 3.1. Alternatively, cells were viral transduced

with Lentivirus expressing either aS or bS or with both. For

the expression of either protein, aS or bS, we transduced with

25 000 plaque forming units (PFUs) of either one aS or bS and

additional 25 000 PFUs of a green fluorescent protein-expressing

virus. For the co-transduction of aS together with bS, each virus wastransduced at 25 000 PFUs, altogether 50 000 PFUs per 1 · 107

cells. Thirty-two hours post-transduction, the conditioning medium

was replaced and cells were incubated for 16 h with or without

FAs as previously described (Sharon et al. 2003b). Cells were

then (48 h post-transduction) collected and processed for analyses.

Conditioning living cells with FAs was carried out as previously

described (Sharon et al. 2001, 2003b). Briefly, subconfluent (75%)

cultures of MES or MN9D cells expressing human WT aS or bS or

both were cultured in Dulbecco’s modified Eagle’s medium

containing the essential nutrients for the cells (Crawford et al.1992, Choi et al. 1991) but without serum. Bovine serum albumin

(BSA)/FA complexes were added to the medium just before applying

it to the cultures. These complexes were prepared by mixing BSA

(FA-free; Sigma) with the indicated FA (at a molar ratio of 1 : 5) in

binding buffer [10 mM Tris–HCl (pH 8.0), 150 mM NaCl] at 37�Cfor 30 min. Sister cultures, used as controls, were incubated in

parallel with BSA but without FA. Cells were then collected and

high-speed supernatant (post 180 000 g) was prepared as above.

Oligomers detection and western blottingProtein samples of high-speed supernatant (post 180 000 g) were

incubated at 65�C for 16–18 h (Sharon et al. 2003b) prior to loadingon an 8–16% NuPAGE Bis-Tris (Invitrogen, Dublin, Ireland) or 10%

SDS–PAGE. For native electrophoresis we used the same buffers,

omitting SDS and b-mercaptoethanol throughout. Immunoblots

reacted with anti-bS antibodies: monoclonal anti-bS (1 : 5000,

612508; BD Transduction Laboratories, Franklin Lakes, NJ, USA);

polyclonal anti-bS (1 : 1000, AB5086, Chemicon, Temecula, CA,

USA); monoclonal anti-bS (1 : 1000, 328500; Zymed Laboratories

Inc., South San Francisco, CA, USA). For aS immunodetection

monoclonal Syn1 (1 : 1000; BD transduction laboratories) and

monoclonal LB509 (1 : 1000; 18-0215, Zymed–Invitrogen) were

used. We also used H3C antibody (generously provided by Prof.

George J.M., University of Illinois) for both aS and bS detection.

Immunoblots normalized with actin or tubulin levels on the same

blots.

ChromatographyPartial purification of aS and bS from WT and aS null mouse

brains was performed as described (Lee et al. 2002) with slight

Journal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 465–474� 2008 The Authors

466 | E. Israeli and R. Sharon

modifications. Briefly, 5 mg of 180 000 g centrifuged post-super-

natant in physiological buffer (Sharon et al. 2001) was loaded on

size exclusion column (Sephacryl S-100 16/60 cm; Amersham

Pharmacia, Uppsala, Sweden). The column was equilibrated with

Tris–Cl, 20 mM, pH 7.4, and 1 mL fractions were collected at a

rate of 0.2 mL/min. The fractions were tested for the presence of

aS and bS by western blot and the positive fractions were

combined and further purified on anion exchange column

(Diethylaminoethyl [DEAE] 1 mL column, Amersham; equilibra-

tion buffer: Tris–Cl, 20 mM, pH 7.4; elution at 0.1–0.4 M NaCl at

1 mL/min). The elution profiles of aS and bS to the specific

fractions were determined by western blotting reacted with specific

anti-aS or -bS antibodies.

ImmunoprecipitationSamples of semipurified aS and bS high-speed cytosols of whole

WT or aS null mouse brains subjected to purification by Sephacryl-

100 size exclusion chromatography (SEC fractions 56–95-pooled)

followed by DEAE fractionation (fractions 154–168) were used.

Samples containing 200 lg protein in 1 mL immunoprecipitation

(IP) buffer [Tris–Cl 20 mM, pH 7.4; 1· protease inhibitor mix

(Sigma); sodium fluoride 20 mM; phenylmethylsulfonyl fluoride

1 mM; EDTA 1 mM and EGTA 1 mM] were incubated at 65�Cfor 16 h. Next, 0.5 lg anti-aS antibody (ab) (Syn1, Transduction

Labs, Lexington, KY, USA) or 5 ll of anti-bS ab (Chemicon),

were added to the sample and rotated for 2 h at 22�C. Protein A/G

PLUS-Agarose beads (Santa Cruz, CA, USA) were pre-incubated in

10 lg rat liver protein extract for 16–18 h at 4�C to block non-

specific binding. The beads were then washed twice in IP buffer.

Binding was allowed for 2 h at 22�C and the A/G protein beads

were washed in IP buffer containing 0.05% SDS (wash 1) and

0.025% Tween 20 (wash 2). The proteins bound to the beads were

eluted in Laemmli buffer and boiled for 10 min at 100�C. The

eluent was loaded on a 10% SDS–PAGE and analysed by western

blotting using anti-aS or anti-bS antibodies in a reciprocal order to

the IP.

Results

b-Synuclein fractionation in whole mouse brainBecause of the high homology between bS and aS [reviewedin (Clayton and George 1998; George 2002)] and potentialinteractions between the two proteins, we initially usedaS)/) mouse brains to study bS protein. Whole aS)/)mouse brain was fractionated (see Materials and methods)and the distribution of bS in the subcellular fractions wasdetermined by quantitative western blotting with anti-bSantibodies [Chemicon (polyclonal) and Zymed (monoclonal)with similar results]. Approximately, 55% of the total bS wasfound to be cytosolic, i.e. detected in the high-speedsupernatant (post 180 000 g). A low portion of bS, c. 5%,was found in plasma membrane-enriched fraction and onlytraces of bS (c. 0.15%) in the high-speed membrane fraction.The remaining c. 40% was detected in the lipid-richfractions. No bS immunoreactivity was detected in thenuclei (Fig. 1a).

bS normally occurs in lipid-associated high molecularweight formsWe next analysed the soluble fraction of whole WT, aS)/)and bS+/+ mouse brains by non-denaturing gel electropho-resis followed by western blotting and probed with an anti-bS ab (see Materials and methods). Using these nativeconditions, we detected various bS-immunoreactive forms. Amonomer bS band migrating slightly lower than monomeraS band; higher molecular weight (MW) bS-immunoreactivebands detected in bS tg and aS)/) but not in WT mousebrain and lower MW bands (lower than the main bSmonomer) detected in the bS tg mouse brain, representspotential truncated bS forms (Fig. 1b). To test whethersimilar to aS, bS occurs as lipid-associated high MW forms,we next applied the methods used for aS oligomer detection,i.e. delipidation with organic solvents or heat treatment at65�C (Sharon et al. 2001, 2003a,b; Assayag et al. 2007).Heat delipidation at 65�C of the soluble fraction of aS)/)mouse brain dramatically enhanced the detection of variousbS immunoreactive forms on a western blot probed with fourdifferent specific anti-bS antibodies (see Materials andmethods), with indistinguishable result (Fig. 1c). Theimmunoreactivity appeared throughout the lane as a smearrepresenting various bS species with several main bands thataccording to their calculated MW may represent bS oligo-mers. Specifically, the monomer bS migrated at c. 19 kDa,the putative dimer at c. 39 kDa, trimer at c. 51 kDa andtetramer at c. 77 kDa. Quantifying bS signal detected by thewestern blot indicated twofold increase in bS monomeramount with the 65�C treatment and a dramatic increase inhigh MW bS forms. This immunoreactive profile was bS-specific, no immunoreactivity was detected with syn-1 anti-aS ab in the aS)/) mouse brain (not shown).

Enhanced detection of bS monomer and high MW formswere also observed upon extraction in organic solvents such aschloroform : methanol (2 : 1) (Folch et al. 1957) or hexane.bS monomer and high MW forms were detected in theinterface between the organic and the aqueous phases inchloroform : methanol extraction and in the organic phase inhexane extraction on an SDS–PAGE (Fig. 1c). Further,extraction in hexane affected the mobility of bS monomerfrom WT, aS)/) and bS+/+ mouse brains on a native gelappearing slightly higher after the hexane extraction (Fig. 1d).Interestingly, in contrast to its effect on aS oligomersdetection, incubating the soluble fraction with the FA-bindingresin, Lipidex-1000 (Hydroxyalkoxypropyl-Dextran type IV,Sigma), did not affect the detection of bS highMW forms on awestern blot (not shown). We next detected higher bS levelsand higher oligomers to monomer ratio in bS tg (Hashimotoet al. 2001) than in WT mouse brains (Fig. 1e and f).Together, the results suggest that similar to aS, bS occursin vivo in soluble assemblies that may represent bS oligomersand their detection is dramatically improved upon applyingcertain methods for delipidation.

� 2008 The AuthorsJournal Compilation � 2008 International Society for Neurochemistry, J. Neurochem. (2009) 108, 465–474

bS forms lipid-associated hetero-oligomers with aS | 467

0

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(a) (d)

(b) (e)

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Fig. 1 b-synuclein (bS) occurs in lipid-associated high molecular

mass forms in wild type (WT) a-synuclein (aS))/) and bS+/+ mouse

brains (a) Endogenous bS distribution in aS)/) mouse brain. Whole

aS)/) mouse brain was fractionated as in the study by Sharon et al.

(2001) and subjected to quantitative western blotting with anti-bS ab

(Chemicon, Temecula, CA, USA). Results are presented as the ratio

of the amounts of bS monomer (17 kDa) in each fraction to that of total

bS monomer in the starting homogenate (100%) run in parallel on

each gel (means ± SD of three brains). (b) Mouse endogenous and

human transgenic bS occurs in various molecular weight (MW) spe-

cies on native gel. Whole WT, aS)/) and bS+/+ mouse brains were

fractionated as in (a). Sample (15 lg protein) of the high-speed

supernatant (post 180 000 g) was analysed by 10% native polyacryl-

amide gel electrophoresis (PAGE). Western blot reacted with anti-bS

ab (BD transduction laboratories), in parallel, a sample of WT mouse

brain reacted with anti-aS ab (syn-1). (c) The detection of the various

bS forms is enhanced by treatments that remove lipids. Sample (15 lg

protein of aS)/) mouse brain) as in (b) was incubated at 65�C for

16 h; extracted in chloroform/methanol (2 : 1) or in hexane (see

Materials and methods) and analysed by 10% sodium dodecyl sulfate–

PAGE. Western blot reacted with anti-bS ab. (d) bS monomer elec-

trophoresis is affected by hexane. Protein samples of high-speed cy-

tosols from whole WT, aS)/) or bS+/+ mouse brains were extracted in

hexane and analysed by 10% native PAGE. A shift in the mobility of

the bS monomer is detected upon hexane extraction. (e) The relative

amount of high MW bS forms is higher in bS+/+ mice than in control

mice. High-speed cytosols of whole bS+/+ (Hashimoto et al. 2001)

and control mouse brains were treated at 65�C to enhance bS

detection as in (c). (f) Densitometric quantification of western blot in (e)

is presented in arbitrary units, as the ratio of bS high MW forms to

monomer.



bS oligomers co-purify with aS oligomersIn an attempt to further characterize bS oligomers incomparison with aS oligomers, we performed SEC of thehigh-speed soluble fraction (post 180 000 g) of WT andaS)/) fresh mouse brains (for aS and bS vs. bS alone in WTand aS)/) respectively), under non-denaturing conditionsi.e. physiological buffers. Using a Sephacryl S-100 column,we obtained a linear elution profile of standard proteins withMW of c. 6–77 kDa (Fig. 2a and b). The elution profiles ofaS and bS extracted from the cytosol of WT mouse brainswere similar to one another. Specifically, aS and bS eluted tothe same pick (fractions 56–95), recurrently appearing as a

double pick (Fig. 2c). The elution profile of bS from WT vs.aS)/) brains was identical (not shown), indicating that aSexpression does not affect the elution profile of bS. We nextanalysed the eluted fractions for the presence of monomerand oligomers of bS on native gel without delipidationtreatments. Using these native conditions, we detected bSmonomer alone at pick S1 and bS monomer together witholigomeric forms at pick S2 (Fig. 2d). The monomer bS atpick S1 appeared slightly higher than the monomer at pickS2. We next applied the 65�C treatment followed by SDS–PAGE electrophoresis to enhance the detection of bS andfound similar results, i.e. bS monomer alone was detected at

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(c) (d) (e)

(b)

Fig. 2 The elution profiles of b-synuclein (bS) and a-synuclein (aS) is

highly similar on Sephacryl-100 column. (a) Elution pattern of a

Sephacryl-100 size exclusion chromatography (SEC) column was

established using the indicated proteins as size markers. The column

was eluted with Tris–Cl, 20 mM, pH 7.4, at 0.2 mL/min. Fractions

containing protein (1 mL) were collected according to the UV detector.

Protein size markers: conalbumin, 77 kDa; fractions 49–89; bovine

serum albumin, 66 kDa, fractions 55–92; pepsin 34 kDa, fractions 77–

113 and insulin 6 kDa, fractions 135–180; (b) The elution pattern of the

protein size markers was linear on a semilog plot, indicating efficient

column separation. SEC conditions and protein size markers as in (a).

Graph presented as log molecular weight to Kav. (c) The elution profile

of high-speed supernatant (post 180 000 g) of whole WT mouse brain.

(5 mg protein starting sample). (d) Fractions from (c) were loaded on

native 10% polyacrylamide gel electrophoresis (PAGE) without any

further treatment and the polyvinylidene difluoride blot was stained

with anti-bS ab. Note that the bS monomer appears as a doublet (e)

bS and aS reactivity of the equivalent SEC fractions as in (d), treated

or untreated at 65�C before loading on 10% sodium dodecyl sulfate–

PAGE. Western blot reacted with anti-bS ab. Note, bS monomer alone

is eluted at pick S1 and monomer + oligomers are eluted at S2. Ve,

Elution volume; Vo, Void volume; Vt, total column volume.



pick S1, while bS monomer and oligomers were detected atthe S2 pick. The parallel profile of aS indicated the elution ofaS monomer and oligomers to the same fraction, inagreement with previous purification attempts (Sharon et al.2003b). Yet, lower amounts of aS oligomers are present atpick S1 than pick S2 (Fig. 2e). Therefore, although aS andbS eluted to the same fractions, the elution pattern wasdifferent, enabling the separation of bS (but not aS)monomer (S1) from its oligomeric forms (S2).

We next pooled fractions 56–95 (the entire fractions ofpicks S1 and S2) of WT mouse brains and subjected themto DEAE anion exchange chromatography (Fig. 3a).Overall, four picks were eluted from the DEAE columnand both aS and bS were eluted to the third pick(fractions 154–168, pick SD3). Importantly, bS, but notaS, was detected at the fourth pick, i.e. fractions 168–180(SD4), therefore, the DEAE column enabled the separationof bS from aS. Treating samples of the SD3 and SD4picks eluted from DEAE column for oligomers detectionagain revealed the elution of monomers and oligomers tothe same fractions (Fig. 3b). Similar elution profile for bSwas obtained in aS)/) mouse brain. In conclusion, aS andbS share a similarly high elution profile on a SephacrylS-100 and DEAE columns. However, some differences inelution profiles enable the separation of bS monomer fromits oligomers and from aS various forms.

18:3 a-linolenic acid induces oligomerization of purifiedbS in vitroThe similarities between aS and bS in chromatography(Figs 2 and 3) and the finding that their expression levelsare associated, led us to ask whether similar to aS, bSoligomerization is affected by FAs (Sharon et al. 2003b).Purified recombinant human bS protein (2 lM) wereincubated at 37�C with increasing concentrations of 18:3a-linolenic acid (ALA) for 1 h and an aliquot was analysedby SDS–PAGE (Fig. 4a), followed by immunoblot probedwith anti-bS ab. A bS monomeric band was detected in the

5

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050 100 150 200 250

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S S

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Fig. 3 b-Synuclein (bS) is separated from a-synuclein (aS) on a

Diethylaminoethyl (DEAE) column. (a) The elution profile of the entire

S1 and S2 picks (fractions 56–95) were loaded on a DEAE ion ex-

change column. (b) Fractions of the SD3 and SD4 DEAE picks were

treated at 65�C before loading on a 10% sodium dodecyl sulfate–

polyacrylamide gel electrophoresis. Western blot reacted with anti-bS

ab (BD transduction laboratories) and anti-aS ab (Syn-1). Note, bS is

eluted at both SD3 and SD4 picks, while aS is eluted at SD3 only. Actin

1x

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k D a

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78 55 45

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M W (a)

(b)

Fig. 4 Polyunsaturated fatty acids (PUFAs) induce the oligomeriza-

tion of human WT b-synuclein (bS) in vitro but not in living mesen-

cephalic cells. (a) Two micromolars of purified bS was incubated

without or with 18:3 a-linolenic acid at the indicated concentrations at

37�C for 1 h. Samples (75 ng protein) were western blotted with H3C

without any further treatment. Note that the presence of FA increased

the level of bS detection. (b) bS oligomerization is not affected by FA in

living mesencephalic cells. High-speed cytosols (post-supernatant of

15 lg protein centrifuged at 180 000 g supernatant) of human aS or

bS stable mesencephalic cells conditioned in the presence of the

indicated FA/bovine serum albumin (BSA) complexes (250/50 lM) in

serum-free medium. Samples were treated at 65�C overnight prior to

gel loading and blotting with H3C antibody. Sister cultures treated and

processed in parallel. 18:1, oleic acid; 18:2, linoleic acid; 18:3,

a-linolenic acid; 22:4, docosatetraenoic acid; MW, molecular weight.



absence of FA. However, in the presence of 10 lM of ALA,oligomerization of pure bS was promoted, enabling thedetection of bS dimer and trimer. Higher ALA concentrationsof 25 and 50 lM affected pure bS oligomerization to the sameextent as the low 10 lM concentration, and 100 lM ALAfurther enhanced bS oligomerization. Importantly, no heattreatment or organic solvents were applied to detect thepurified bS oligomers. Incubation of pure bS with ALA notonly enhanced oligomerization but also increased the totalimmunoreactivity, without affecting the monomer bS levels(Fig. 4a). The similarities in mobility on SDS–PAGE gels ofpurified recombinant and mouse endogenous bS reinforcedthe conclusion that bS high MW forms are oligomers.

PUFAs do not affect bS oligomerization in livingmesencephalic neuronsWe next tested whether altered cellular FAs content affectsbS oligomerization as previously carried out for aS (Sharonet al. 2003b). For this aim, we have conditioned naı̈ve, bS oraS over-expressing MES (stable) cells under standard serum-supplemented conditioning medium. Cells were then trans-ferred to serum-free medium supplemented with BSA only(at 50 lM) or with BSA and FA (at 50 and 250 lM for BSAand FA respectively) for 16 h and then harvested andfractionated. Samples of high-speed supernatant were pro-cessed for bS and aS oligomers detection using 65�Ctreatment and SDS–PAGE western blotting. In accord withour previous observations concerning FA-induced aS olig-omerization (Sharon et al. 2003b; Assayag et al. 2007),conditioning aS over-expressing MES cells in Dulbecco’smodified Eagle’s medium supplemented with monounsatu-rated fatty acid (MUFA) 18:1 had no detectable effect on aSoligomerization, however, induced aS oligomerization wasdetected with PUFAs 18:2; 18:3 or 22:4. Interestingly, noneof the FAs tested affected bS oligomerization in bS over-expressing cells cultured and treated in parallel (Fig. 4b).Specifically, we tested the effects of 18:0 saturated fatty acids(SFA, not shown), 18:1 (MUFA) and 18:2, 18:3 and 22:4(PUFAs) with no detectable effect on bS oligomerization(Fig. 4b). We therefore conclude that while purified bSoligomerization is enhanced by 18:3 PUFA in vitro (Fig. 4a),altered cellular PUFA levels do not affect bS oligomerizationin living MES cells.

bS expression inhibits PUFA-induced aS oligomerizationin living mesencephalic cellsFollowing upon the recent findings that bS expression inhibitsaS oligomerization and aggregation (Hashimoto et al. 2001;Uversky et al. 2002; Park and Lansbury 2003), we now askedwhether bS expression affects PUFA-induced aS oligomeri-zation. For this aim, we transduced naive MN9D cells eitherwith an aS or bS expressing viral vectors or both at 1 : 1 PFUratio (see Materials and methods). Following transduction, wereplaced the conditioning medium and treated the cells in

serum-free medium supplemented with BSA with or without18:3 PUFA (at 50 and 250 lM for BSA and 18:3 respectively)for 16 h. Cells were collected 48 h post-transduction and high-speed cytosols were processed for the detection of aS and bSoligomers by western blotting probedwith anti-aS and anti-bSantibodies, respectively. Induced aS oligomerization wasdetected in aS-transduced cells treated with 18:3 PUFA, withtwofold increase in oligomers tomonomer ratio. Note that 18:3treatment enhanced the levels of both aS monomer and

0

0.5

1.0

1.5

2.0

2.5

BSA PUFA BSA PUFA

S S + S

αS o

ligom

ers

to m

onom

er(r

atio

)

W e s t e r n b l o t : a n t i- S a n t i- S

T r u n s d u c t i o n :

BSA 18:3

BSA 18:3

BSA 18:3

BSA 18:3

S S S

S S S

15

25

37

50

75

(a)

(b)

Fig. 5 b-synuclein (bS) inhibits polyunsaturated fatty acids (PUFA)-

induced a-synuclein (aS) oligomers. (a) High-speed cytosols (15 lg

protein) of mesencephalic cells transduced with either a lentiviral

vector expressing aS, bS or both, conditioned and maintained in

parallel, in the presence of bovine serum albumin (BSA) or BSA/18:3

complexes (250/50 lM) in serum-free medium. Samples were treated

at 65�C overnight prior to gel loading and blotting with anti-aS or -bS

antibodies. (b) Densitometric quantification of aS signal detected at (a)

presented as the ratio of aS oligomers to monomer and normalized to

tubulin. A representative blot out of n = 3. Note, the bS high molecular

weight bands appearing at aS + bS and not in bS only transduced

cells, these bands may represent hetero-oligomers composed of aS

and bS.



oligomers (Fig. 5a). The PUFA-induced aS oligomerizationwas dramatically inhibited by bS expression (i.e. aS + bStransduced cells) with c. 85% inhibition (Fig. 5b). Importantly,in cells transduced with aS + bS, the 18:3 treatment enhancedthe formation of bS-immunoreactive high MW species, thataccording to their molecular mass may represent dimer, trimerand tetramer. These high molecular mass bS-immunoreactivebands are detected in the aS + bS cells, but not in the bS onlycells (Fig. 5a). Therefore, these bands may represent theputative hetero-oligomers consisting on aS and bS.

bS occurs in hetero-oligomers with aS in vivoWe next sought to find potential hetero-oligomers composedof aS and bS in vivo in WT mouse brains. The occurrence ofsuch hetero-oligomers could underlie the mechanism bywhich bS inhibits aS oligomerization in general and PUFA-induced aS oligomerization in particular (Figs 4b and 5a).For this aim we used pooled fractions of the entire SD3 pick(see Fig. 3a and b) containing both aS and bS of WT mousebrain and the corresponding fractions from aS)/) mousebrain and performed co-IP, using either anti-aS or -bSantibodies for the IP step and the reciprocal ab for the westernblotting. Three specific bands were detected on the westernblot, migrating at 36, 51 and c.70 kDa in WT but not in thecorresponding fractions from aS)/) mouse brain. The MWof these bands suggests that these bands represent heterodi-mer, heterotrimer and heterotetramer composed of aS and bS.The three specific bands were detected either with anti-aS abor anti-bS ab for IP. Importantly, a portion of aS and bSprotein is non-specifically attached to the agarose beads(lanes 5, 6 and 11, Fig. 6). This may explain the presence of a

bS monomer in aS)/) mouse brain immunoprecipitated withaS ab (lane 4, Fig. 6). We therefore conclude that bSnormally forms hetero-oligomers with aS in vivo.

Discussion

Using biochemical methods for fractionation in whole WT,aS)/) and bS+/+ mouse brains we detected various speciesof bS that according to their molecular mass and similaritiesto aS oligomers, may represent bS oligomers. The bSoligomers are enriched in the high-speed supernatant (post180 000 g) of mouse brains and can be detected usingphysiological buffers and non-denaturing gel electrophoresisfollowed by western blotting. However, the immunodetectionof the various bS species, including monomer and oligomers,is dramatically enhanced by heat treatment or extraction inorganic solvents such as chloroform/methanol or hexane,indicating that bS oligomers may associate with lipids. Thedetection of mouse endogenous bS as well as humantransgenic bS in the forms of oligomers in mouse brainssuggests that bS normally occurs in vivo in soluble, lipid-associated oligomers.

The purification attempts using Sephacryl-100 SEC fol-lowed by DEAE ion exchange chromatography resulted inhighly similar elution profiles for aS and bS proteins. Bothproteins co-eluted to the same fractions on the Sephacryl-100column and the following DEAE column, with the exceptionof pick ‘SD4’ of DEAE column elution profile, where bS butnot aS is detected. This result further emphasizes the highdegree of similarities between the two proteins not only at thelevel of sequence homologies but also at the levels of

ab S –/–

anti- S w/o ab

WT WT WTWT S–/– ConCon ab S–/–

anti- S w/o ab

S–/–

IP:

anti- S anti- SWB:

1 2 3 4 5 6 7 8 9 10 11 12

15

25

37

5075

1x

2x

3x

4x

Fig. 6 Hetero-oligomers composed of a-synuclein (aS) and b-synuc-

lein (bS) normally occur in wild type (WT) mouse brain. Immunopre-

cipitation of 1 mL of the pooled fractions of the entire SD3 pick (see

Fig. 3a and b) containing both aS and bS of WT mouse brain and the

corresponding fractions from aS)/) mouse brain. The sample was

heat-treated at 65�C prior to loading on protein A/G PLUS-Agarose

beads (Santa Cruz, CA, USA) that were pre-incubated in 10 lg rat

liver protein extract for 16–18 h at 4�C to block non-specific binding.

Con = sample of high-speed cytosol (15 lg) of whole WT mouse brain

treated at 65�C for oligomers detection; w/o (without) ab represents

the non-specific binding to the agarose beads; ab, [anti-aS (syn1)

and anti-bS] the antibody used for the immunoprecipitation (IP) step

loaded on the gel. > Indicates specific bands detected with anti-aS

and anti-bS antibodies representing heterodimer, trimer and tetramer,

respectively on the western blot (WB).



biochemical and biophysical properties. Importantly, acritical difference between the elution profiles of aS andbS was found using Sephacryl-100 chromatography. That isthe ability to separate between the different bS forms, i.e.monomer and oligomers. Specifically, only bS monomer wasdetected at the S1 fractions while bS monomer and oligomerswere detected at S2 fractions. This finding is important fortwo main aspects: (i) the purification method herein provideda way by which bS monomer can be separated from itsoligomers and (ii) the absence of bS oligomers in the S1 pickafter applying the protocol for enhanced oligomers detectionusing 65�C treatment strongly argues against the claim thatthe treatments used to enhance the oligomers detectionactually enhance artificial, in vitro oligomerization of thedenatured protein.

In our recent publications we reported different aspectsof aS interactions with FAs (Sharon et al. 2001, 2003a,b;Assayag et al. 2007) and related aS interactions with PUFAsto PD pathogenesis. Specifically, we showed that within thephysiological range of concentrations, PUFAs but notMUFAs of saturated fatty acids induce aS oligomerization,aggregation and Lewy-like inclusion formation in dopami-nergic cells (Sharon et al. 2001, 2003a,b; Assayag et al.2007). In accord, we detected higher long-chain PUFA levelsin PD and DLB brains than normal, age-matched controls(Sharon et al. 2003b). These high PUFA levels correlatedwith the accumulation of aS soluble oligomers in the samebrain regions. Based on these and additional results, wesuggested that high PUFA levels in the brain may act as arisk factor, inducing aS cytotoxicity. In this study we askedwhether similar to aS, bS responds to the presence of PUFAby induced oligomerization and aggregation. Based on thehigh degree of similarities between the two proteins and thefinding that bS, like aS, normally occurs in lipid-associatedsoluble oligomers, we expected to find that bS alsooligomerizes in the presence of PUFAs. The findings thatbS does not oligomerizes in the presence of PUFAs or otherFAs may underlie the biochemical difference between thetwo proteins resulting in two highly similar proteins withopposing effects on PD and the related synucleinopathies.

We show that bS occurs in vivo in lipid-associatedoligomers and that lipid extraction dramatically enhancesthe detection of bS oligomers. Yet, the lipid binding domainof bS differs from that of aS. The deletion of 11 residues inthe bS sequence dramatically affects the helical structure ofbS (Sung and Eliezer 2006). This may reduce the stability oflipid binding, including membrane phospholipids binding.For example, the missing 11 residues affects the degree ofhomology to the apolipoprotein A1 in the N¢ terminal regionof the bS protein, resulting in five apolipoprotein A1-likerepeats, while six such repeats are present in the aS sequences(Clayton and George 1998). Further, the sequence differencesbetween aS and bS result in a lower degree of similarities tothe FA-binding protein signature motif (Sharon et al. 2001).

In this regard, the detection of bS in purified ‘heavy’ myelinin mouse brains (Mor et al. 2003) and in Lipidex-1000extracts of DLB brains (Salem et al. 2007) suggests thatindeed bS interacts with lipids in vivo. Therefore, thedifferences in lipid-binding domains in bS and aS sequencemay indicate different affinities towards specific lipids andrepresent the core difference between aS and bS proteins.

In this regard, the finding that exposure to PUFAsenhances in vitro but not in vivo oligomerization of bS isworthy of note. This result highlights the potential of bS tonon-specifically react with lipid surface, represented by theFA micelles and oligomerize upon this interaction. Yet,further studies are needed to identify the physiological lipidpreferences of bS and the potential role of its post-translational modifications in lipid interactions.

The occurrence of a heterodimer consisting of aS and bSwas suggested before based on molecular modelling anddynamic simulation. It was further suggested that bS occursas a dimer that do not propagate. Using a cell-free system, bSwas shown to interfere with aS oligomerization (Tsigelnyet al. 2007). The results presented herein support thesepredictions and observations. Specifically, reduced levels ofPUFA-induced aS oligomerization were detected uponexpression of bS at a 1 : 1 ratio with aS. While a 1 : 1 ratiois closer to the physiological mouse brain levels (R. Sharonand E. Israeli, unpublished data), it is interesting to note that acomplete inhibition of aS fibrillation was previously reportedat 4 : 1 molar excess of bS (Uversky et al. 2002).

The formation of hetero-oligomers composed of aS andbS was enhanced upon exposure to PUFA. Based on theresults herein, we suggested that bS inhibits PUFA-inducedaS oligomerization by the formation of hetero-oligomers upto tetramer that do not further propagate. It is interesting tonote that hetero-oligomers composed of endogenous aS andbS proteins were also detected in WT mouse brains.Importantly, these hetero-oligomers were found in vivo, withendogenous aS and bS proteins and therefore suggest thepresence of such hetero-oligomers in normal, healthy animalconditions. This finding may represent the normal function ofbS that continually restrains aS oligomerization rate andtherefore, its toxicity. However, as both aS and bS oligomersinteract with lipids, this result may also suggest specificlipids preferences for the hetero-oligomers that may differfrom aS or bS oligomers.

In light of the critical role of aS in neurodegeneration, wehave tested the effect of bS, the intrinsic physiologicalinhibitor of aS toxicity. Indeed, increasing number ofevidences now suggests that bS interacts and regulates aS.The regulatory effect of bS on aS toxicity may result fromthe high degree of similarities between the two proteins, forexample, the high sequence homologies and a nearlyidentical subcellular distribution including enrichment inpre-synaptic vesicles [reviewed in (Clayton and George1999; George 2002)]. Therefore, the two proteins may share



similar but not identical, biochemical properties and undercertain conditions may compete for interacting molecules.Our discovery that similar to aS, bS normally occurs insoluble, lipid-associated oligomers highlights the potentialrole of lipids in PD and the related synucleinopathies andmay have important implications towards our understandingof the physiological role of these highly similar proteins.

Acknowledgements

This study was supported by the Israel Science Foundation (ISF),

Grant No. 1202/04.

References

Assayag K., Yakunin E., Loeb V., Selkoe D. J. and Sharon R. (2007)Polyunsaturated fatty acids induce {alpha}-synuclein-related path-ogenic changes in neuronal cells. Am. J. Pathol. 171, 2000–2011.

Bertoncini C. W., Rasia R. M., Lamberto G. R., Binolfi A., ZweckstetterM., Griesinger C. and Fernandez C. O. (2007) Structural charac-terization of the intrinsically unfolded protein beta-synuclein, anatural negative regulator of alpha-synuclein aggregation. J. Mol.Biol. 372, 708–722.

Bradford M. M. (1976) A rapid and sensitive method for the quantitationof microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

Choi H. K., Won L. A., Kontur P. J., Hammond D. N., Fox A. P., WainerB. H., Hoffmann P. C. and Heller A. (1991) Immortalization ofembryonic mesencephalic dopaminergic neurons by somatic cellfusion. Brain Res. 552, 67–76.

Clayton D. F. and George J. M. (1998) The synucleins: a family ofproteins involved in synaptic function, plasticity, neurodegenera-tion and disease. Trends Neurosci. 21, 249–254.

Clayton D. F. and George J. M. (1999) Synucleins in synaptic plasticityand neurodegenerative disorders. J. Neurosci. Res. 58, 120–129.

da Costa C. A., Masliah E. and Checler F. (2003) Beta-synuclein dis-plays an antiapoptotic p53-dependent phenotype and protectsneurons from 6-hydroxydopamine-induced caspase 3 activation:cross-talk with alpha-synuclein and implication for Parkinson’sdisease. J. Biol. Chem. 278, 37330–37335.

Crawford G. D. Jr, Le W. D., Smith R. G., Xie W. J., Stefani E. andAppel S. H. (1992) A novel N18TG2 · mesencephalon cell hybridexpresses properties that suggest a dopaminergic cell line of sub-stantia nigra origin. J. Neurosci. 12, 3392–3398.

Duda J. E., Lee V. M. and Trojanowski J. Q. (2000) Neuropathology ofsynuclein aggregates. J. Neurosci. Res. 61, 121–127.

Fan Y., Limprasert P., Murray I. V., Smith A. C., Lee V. M., TrojanowskiJ. Q., Sopher B. L. and La Spada A. R. (2006) Beta-synucleinmodulates alpha-synuclein neurotoxicity by reducing alpha-synuclein protein expression. Hum. Mol. Genet. 15, 3002–3011.

Folch J., Lees M. and Sloane Stanley G. H. (1957) A simple method forthe isolation and purification of total lipids from animal tissues.J. Biol. Chem. 226, 497–509.

Galvin J. E., Uryu K., Lee V. M. and Trojanowski J. Q. (1999) Axonpathology in Parkinson’s disease and Lewy body dementia hip-pocampus contains alpha-, beta-, and gamma-synuclein. Proc. NatlAcad. Sci. USA 96, 13450–13455.

George J. M. (2002) The synucleins. Genome Biol. 3, 1–6.Hashimoto M., Rockenstein E., Mante M., Mallory M. and Masliah E.

(2001) Beta-synuclein inhibits alpha-synuclein aggregation: apossible role as an anti-parkinsonian factor. Neuron 32, 213–223.

Hashimoto M., Bar-On P., Ho G., Takenouchi T., Rockenstein E., CrewsL. and Masliah E. (2004) Beta-synuclein regulates Akt activity inneuronal cells. A possible mechanism for neuroprotection in Par-kinson’s disease. J. Biol. Chem. 279, 23622–23629.

Lee D., Lee S. Y., Lee E. N., Chang C. S. and Paik S. R. (2002) Alpha-synuclein exhibits competitive interaction between calmodulin andsynthetic membranes. J. Neurochem. 82, 1007–1017.

Maroteaux L., Campanelli J. T. and Scheller R. H. (1988) Synuclein: aneuron-specific protein localized to the nucleus and presynapticnerve terminal. J. Neurosci. 8, 2804–2815.

Masliah E. and Hashimoto M. (2002) Development of new treatmentsfor Parkinson’s disease in transgenic animal models: a role forbeta-synuclein. Neurotoxicology 23, 461–468.

Mor F., Quintana F., Mimran A. and Cohen I. R. (2003) Autoimmuneencephalomyelitis and uveitis induced by T cell immunity to selfbeta-synuclein. J. Immunol. 170, 628–634.

Ohtake H., Limprasert P., Fan Y. et al. (2004) Beta-synuclein genealterations in dementia with Lewy bodies. Neurology 63, 805–811.

Park J. Y. and Lansbury P. T. Jr (2003) Beta-synuclein inhibits formationof alpha-synuclein protofibrils: a possible therapeutic strategyagainst Parkinson’s disease. Biochemistry 42, 3696–3700.

Salem S. A., Allsop D., Mann D. M., Tokuda T. and El-Agnaf O. M.(2007) An investigation into the lipid-binding properties of alpha-,beta- and gamma-synucleins in human brain and cerebrospinalfluid. Brain Res. 1170, 103–111.

Sharon R., Goldberg M. S., Bar-Josef I., Betensky R. A., Shen J. andSelkoe D. J. (2001) Alpha-synuclein occurs in lipid-rich highmolecular weight complexes, binds fatty acids, and showshomology to the fatty acid-binding proteins. Proc. Natl Acad. Sci.USA 98, 9110–9115.

Sharon R., Bar-Joseph I., Mirick G. E., Serhan C. N. and Selkoe D. J.(2003a) Altered fatty acid composition of dopaminergic neuronsexpressing alpha-synuclein and human brains with alpha-synuc-leinopathies. J. Biol. Chem. 278, 49874–49881.

Sharon R., Bar-Joseph I., Frosch M. P., Walsh D. M., Hamilton J. A. andSelkoe D. J. (2003b) The formation of highly soluble oligomers ofalpha-synuclein is regulated by fatty acids and enhanced in Par-kinson’s disease. Neuron 37, 583–595.

Snyder H., Mensah K., Hsu C., Hashimoto M., Surgucheva I. G., FestoffB., SurguchovA.,MasliahE.,MatouschekA. andWolozinB. (2005)Beta-synuclein reduces proteasomal inhibition by alpha-synucleinbut not gamma-synuclein. J. Biol. Chem. 280, 7562–7569.

Specht C. G. and Schoepfer R. (2001) Deletion of the alpha-synucleinlocus in a subpopulation of C57BL/6J inbred mice. BMC Neurosci.2, 11.

Sung Y. H. and Eliezer D. (2006) Secondary structure and dynamics ofmicelle bound beta- and gamma-synuclein. Protein Sci. 15, 1162–1174.

Tsigelny I. F., Bar-On P., Sharikov Y., Crews L., HashimotoM., Miller M.A., Keller S. H., Platoshyn O., Yuan J. X. and Masliah E. (2007)Dynamics of alpha-synuclein aggregation and inhibition of pore-likeoligomer development by beta-synuclein. FEBS J. 274, 1862–1877.

Uversky V. N., Li J., Souillac P., Millett I. S., Doniach S., Jakes R.,Goedert M. and Fink A. L. (2002) Biophysical properties of thesynucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins. J. Biol. Chem.277, 11970–11978.

Wei J., Fujita M., Nakai M., Waragai M., Watabe K., Akatsu H., Roc-kenstein E., Masliah E. and Hashimoto M. (2007) Enhancedlysosomal pathology caused by beta-synuclein mutants linked todementia with Lewy bodies. J. Biol. Chem. 282, 28904–28914.

Yamin G., Munishkina L. A., Karymov M. A., Lyubchenko Y. L.,Uversky V. N. and Fink A. L. (2005) Forcing nonamyloidogenicbeta-synuclein to fibrillate. Biochemistry 44, 9096–9107.



β-synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with...

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