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ΑLPHA-SYNUCLEIN IN THE CENTRAL NERVOUS SYSTEM AND FROM ERYTHROCYTES, MAMMALIAN CELLS AND E. COLI EXISTS PREDOMINANTLY AS A

DISORDERED MONOMER *

Bruno Fauvet1, Martial Mbefo Kamdem1, Mohamed-Bilal Fares1, Carole Desobry1, Sarah Michael2, Mustafa T. Ardah3, Elpida Tsika4, Philippe Coune5 , Michel Prudent6, Niels Lion6, David Eliezer7, Darren J. Moore4, Bernard Schneider5, Patrick Aebischer5, Omar M. El-Agnaf3, Eliezer Masliah2,

Hilal A. Lashuel1‡

From the 1Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland (EPFL), 2Department of Neurosciences, School of Medicine, University of California at San Diego, La Jolla, California 92093, 3Department of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, 4Laboratory of Molecular Neurodegenerative Research, Brain Mind Institute, EPFL, 5Neurodegenerative Disease Laboratory, Brain Mind Institute, EPFL, 6Service Régional Vaudois de Transfusion Sanguine, Route de la Corniche 2, 1066 Epalinges, Switzerland, 7Department of Biochemistry and Program in Structural Biology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA

*Running title: native brain-derived and recombinant α-syn exist as unstructured monomers.

‡ To whom correspondence should be addressed: Hilal A. Lashuel, Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Station 19, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. Tel: +41 21 693 96 91; Fax: +41 21 693 17 80; E-mail: hilal.lashuel@epfl.ch

SUPPLEMENTARY FIGURE S1. SDS treatment, but not heat denaturation induces a shift on α-syn mobility on NATIVE-PAGE. A: Coomassie Blue staining of recombinant α-syn wt boiled or not at 95°C for 10min as well as SDS treated samples. Only the SDS treated samples (0.5 and 1%) migrate faster than heated and non-treated samples. B: The same experiment was performed with cell lysates of stably-transfected SH-SY5Y cells expressing α-syn which showed the same pattern of mobility on NATIVE-PAGE like in A, as revealed by western blot using Syn-1 antibody. C: NATIVE-PAGE showing co-migration of α-syn from Wt mouse brain homogenates with recombinant α-syn spiked in α-syn KO brain homogenates. Heat denaturation induces the formation of high molecular weight smears of α-syn as revealed by western blot using the Syn-1 antibody.

SUPPLEMENTARY FIGURE S2. SDS-PAGE analyses of mouse and rat brain samples from Fig. 4. A: Analysis of mouse brain homogenates (10 µg of total protein per lane, 200 ng of samples). The blot was probed using the Syn-1 primary antibody, and and revealed using ECL (GE Healthcare) chemiluminescence detection reagent. ctx: cortex; BS: brain stem; TG: transgenic mice. B: Analysis of rat brain homogenates. The experiment was performed as in A. SN: Substantia Nigra. ST: Striatum.

SUPPLEMENTARY FIGURE S3. α-syn from wild-type mouse brain homogenates co-elutes with recombinant human α-syn monomer spiked in the lysate of α-syn-KO mice. ~150 µL of total brain homogenates (1 mg of total protein as determined by BCA assay) were loaded on Superdex 200 10/300 GL and eluted at 0.4 mL/min (elution buffer: 50 mM Tris pH 7.5, 150 mM NaCl) and collected into 0.5 mL fractions for analysis by Western Blot (Syn-1 primary antibody, 1:1000, overnight, 4°C). For

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“spiked” brain homogenates, 50 µg of wt human recombinant α-syn was added to the lysate before injection into the column. Left panels show absorbance chromatograms (dotted lines) and superimposed quantification data (continuous blue lines with circle symbols) of the band intensities corresponding to Western Blot analyses of the fractions (right panels). “NS” labels on the right of Western Blots indicate non-α-syn-specific bands (assigned as such because they appear in the same fractions of non-spiked α-syn-KO mouse samples; see below). A: Gel-filtration of wild-type mouse brain homogenates (black dotted line) and recombinant human α-syn monomer (blue dotted line, absorbance values x20 to display it on the same scale). B: The same experiment was performed with 50 µg of recombinant human α-syn spiked into a whole-brain homogenate obtained from α-syn-KO mice (black dotted line) superimposed with recombinant human α-syn monomer alone (blue dotted line). The same experiment was repeated without spiking recombinant α-syn (chromatogram not shown); the Western Blot (lower right panel) serves as a proof that the bands observed in the other blots between 40-70 kDa are indeed non-specific signals.

SUPPLEMENTARY FIGURE S4. α-syn from transfected HEK293 cells and SHSY5Y cells stably expressing α-syn co-elutes with recombinant monomer spiked in the lysate of untransfected cells. Cells were lysed under non-denaturing conditions as described above and protein content was estimated using BCA assays. ~500 µg of total cellular proteins were loaded on a Superdex 200 10/300 GL column and collected fractions were analyzed by Western blotting against α-syn. The elution pattern of α-syn from the cell lines was determined by performing a densitometric analysis of α-syn monomer-positive bands in the Western Blots (shown on the right panels). Recombinant α-syn injected alone was used as a control. In all left panels, black dotted lines indicate the absorbance chromatogram obtained for cleared whole-cell lysates. Blue dotted lines indicate the absorbance chromatogram of recombinant wt α-syn injected alone (values x20 so that it can be displayed on the same scale). Continuous blue lines with symbols represent the quantification of the α-syn monomer species detected in the corresponding Western Blots. A: Elution profile of α-syn from transiently transfected HEK293 cells overexpressing wt α-syn. B: Elution profile of recombinant wt α-syn (50µg) spiked in the lysate of untransfected HEK cells.

SUPPLEMENTARY FIGURE S5. Partial purification and characterization of RBC-derived α-syn could be achieved using the protocol from Bartels and colleagues, using differential ammonium sulphate precipitation, anion-exchanged chromatography, and gel-filtration chromatography as described (Bartels et.al., Nature 2011). Note that this purification was performed on a blood sample obtained from a different donor than the one shown in Supplementary Fig. S6. A: SDS-PAGE/Western Blot analyses of RBC α-syn after anion-exchange chromatography. Note that since the lysate was not depleted from Hb, it could not directly be probed by Western Blot, since Hb, which co-migrates with α-syn on SDS-PAGE, gives rise to intense non-specific signals. Different primary antibodies were used as indicated above the blots. Alexa680-conjugated secondary antibodies were used. B: Immunoprecipitation confirms that the ~46kDa bands seen in panel A indeed contains α-syn. The Syn-211 clone was used as capture antibody. 20 ng of recombinant α-syn samples were loaded as controls. SDS-PAGE and Western Blotting were done as in panel A; the membrane was probed with FL-140 primary antibody (1:1000, overnight at 4°C). C: SDS-PAGE / silver staining was performed at different stages of the purification of human α-syn from erythrocytes following Bartel’s protocol. α-syn is only detectable after anion-exchange chromatography, since before this step large amounts of hemoglobin that co-migrate with α-syn mask its signal. D: oligomer-specific ELISA assay applied on the ~46 kDa α-syn-positive complex described in panels A and B. The left ordinate axis shows the ELISA assay readings, while the right ordinate axis displays the total α-syn concentration. The RBC data was compared to signals obtained from untransfected HEK cells (the HEK cell data is the same as shown on Fig. 9C). E: Aminoacid sequence of human α-syn, with the unique peptides that unambiguously identified the protein highlighted in yellow, following a mass-spectrometry-based confirmation of the presence of α-syn in the ~46 kDa complex observed in RBC samples from the first RBC α-syn purification batch. Bands were cut from a silver-stained gel run using the same samples as the IP eluate from Fig. 7B, and digested with trypsin. Tryptic peptides were resolved

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and identified by LC-MS on an LTQ Orbitrap system (Thermo Scientific) followed by database searches using the Sequest and Mascot search engines. F: Typical CID fragmentation pattern from one of the unique α-syn peptides identified.

SUPPLEMENTARY FIGURE S6. Detailed purification protocol for RBC-derived human α-syn. Fractions corresponding to the green boxes in the chromatograms (left) were analyzed by SDS-PAGE/Western Blot and Coomassie (or sliver) staining (right panels) Fractions highlighted by the green boxes in the Coomassie-stained gels were pooled and subjected to the next step of purification. The corresponding pooled volumes are shown below in each case. Western Blot membranes were probed with the following primary antibodies: FL-140 (1:500); Syn-1 (1:1000), SA-3400 (1:2000); or N-19 (1:500) as indicated. A: Erythrocyte concentrates (ECs) from whole blood donations were prepared at the Lausanne blood bank (SRTS VD, Epalinges, Switzerland) according to local requirements (Council of Europe, Guide to the Preparation, Use and Quality Assurance of Blood Components. Recommendation n° R (95) 15 on the Preparation, Use and Quality Assurance of Blood Components, 14th ed.; Council of Europe Press: Strasbourg, France, 2008). ECs that did not satisfy quality criteria for transfusion, in this case wrong weight, were used here. Whole blood (450 ± 50mL) donation is collected in a plastic bag containing an anticoagulant (63mL of citrate phosphate dextrose, CPD). Then, red blood cells (RBC) are separated from white blood cells, platelets and plasma by centrifugation (3550g, 22°C, 14min) and mixed with 100 mL of storage media (sodium-adenine-glucose-mannitol solution). ECs are leukodepleted by filtration, washed twice by centrifugation and resuspension in 0.9% NaCl, before lysis by osmotic shock in PBS 0.1X. The lysate is then clarified by successive centrifugation and ultracentrifugation steps as indicated, followed by filtration (0.22 µm) in order to remove membrane debris before Hb depletion. B: IMAC-based hemoglobin depletion on a 20 mL Ni-NTA column (HisPrepTM FF 16/10) 400-500 mg of total protein (as quantified by a Bradford assay) were loaded at each injection. Middle panels show Western Blot analyses of flowthrough fractions from two different runs (lanes F1 and F2) and from the corresponding Hb-containing eluates (lanes H1 and H2), confirming that α-syn is present only in the Hb-depleted fraction. 50 ng of recombinant wt α-syn were loaded as control. The right panel shows qualitative assessment of Hb depletion by SDS-PAGE/Coomassie staining. Note that the second most abundant RBC protein carbonic anhydrase-1 (29kDa) is not depleted at this step. C: Anion-exchange chromatography (HiTrapTM Q HP 5 mL). The Hb-depleted fractions from 17 IMAC runs were combined, resulting in a ~350 mL solution that was purified in 3 injections. α-syn started to elute at a conductivity of ~30 mS/cm, and is present mainly in three fractions (1.5 mL each, framed by green bowes in the gels). Note that Western Blotting using Syn-1 as primary antibody revealed multiple co-eluting α-syn species consisting of the full-length protein and apparently truncated forms (top Western Blot), that were confirmed by probing the membranes with different antibodies (lower panels: immunoblots probed with the C-terminal-specific antibody SA-3400, and with the N-terminal antibody N-19), suggesting the band migrating in the middle is N-terminally truncated; however Coomassie staining showed that the full-length protein is the predominant species. Importantly, the IEX step also depleted the RBC lysates from CA-1 (see the 29 kDa band in the flowthrough lane in the Coomassie-stained gel. D: Hydrophobic Interaction Chromatography (HiTrapTM Phenyl FF (high sub.) 1 mL). Ammonium sulphate powder was added to the combined anion-exchange fractions so that the (NH4)2SO4 concentration of the injected sample matched that of the HIC binding buffer (i.e. 1.0 M). The pH of the combined anion-exchange fractions was also adjusted to 7.0 before injection into the HIC column. α-syn elutes at conductivity values between 120-80 mS/cm. 750 µL fractions were collected during the gradient elution. Fractions shown in the green boxes inside the gel panels were pooled for purification by gel filtration. E: Gel-filtration chromatography (SuperdexTM 200 10/300 GL), equilibrated with 50 mM Tris, 150 mM NaCl, pH 7.5. Combined HIC fractions were concentrated to 500 µL, and 2x 250 µL were injected into the gel-filtration column. 500 µL fractions were collected at a flow rate of 0.5 mL/min. α-syn elutes at ~14 mL. The SDS-PAGE gel shown on the right was revealed with silver staining. Fractions within the green frame were combined for further analyses.

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SUPPLEMENTARY FIGURE S7. The GST-α-syn fusion protein forms oligomers. Gel filtration/light scattering was performed as described before (see Fig. 1E) under reducing conditions (10 mM DTT were added to the sample). Continuous lines correspond to normalized absorbance (280 nm, left ordinate axis), and thin line segments correspond to the calculated molar masses for the corresponding absorbance peaks (right ordinate axis). The first peak (eluting at 11.5 mL) contains a species measured at 221 ± 7.5 kDa, suggesting a pentameric state, while the second peak, eluting at 13.3 mL, contained a 86 ± 11 kDa polypeptide, the mass of which is closest to a dimer (which is the natural form of free schistosoma japonicum GST). Note that the large experimental error in molecular weight measurement for the second peak is due to the partial co-elution of several species, as indicated by the close peaks on the absorbance trace.

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Supplementary Figure S1

A14

0C

wt

wt,

heat

ed

D

66

146

242

wt,

0.5%

SD

S

wt,

1% S

DS

M

A B

66

146

242

SH

-SY

5Y

α-sy

n

SH

-SY

5Y α

-syn

,boi

led

SH

-SY

5Y α

-syn

+ S

DS

MM + SDS

CN-PAGE / Western Blot (Syn-1)

CN-PAGE / Coomassie

C

CN-PAGE / Western Blot (Syn-1)

rec.

A14

0C

wt

KO KO wt

KO KO

rec. wt: - - + - - +

Unboiled Boiled

Mouse Brain Homogenates

146

242

480 kD

D

M

kD kD

6

Supplementary Figure S2

rec.

A14

0C

rec.

hum

an

A53

T-TG

(Ctx

)

A53

T-TG

(Bs)

Non

-TG

(Ctx

)

Non

-TG

(Bs)

10

20

3750

250

12

20

45

A B

N-A

c

rec.

hum

an

rec.

A14

0C

SN

non

-inj.

SN

inj.

ST

non-

inj.

ST

inj.

kDkD

A

8

20

60

α-syn(M)

NS

10.9 16.9

Elution Volume (mL)

B

8

20

60NS

α-syn(M)

11.4 16.8

Elution Volume (mL)

WB

den

sity

(A.U

.)

7

Supplementary Figure S3

NS

kD

kD

60

20

8

Elution Volume (mL), from α-syn-KO mouse brain10.9 16.9W

B d

ensi

ty (A

.U.)

0

WB

den

sity

(A.U

.)

A

B

Elution Volume (mL)

Elution Volume (mL) 8

Supplementary Figure S4

12.5 18.5

Elution Volume (mL)

20

40

α-syn (M)

kD

0

WB

den

sity

(A.U

.)

α-syn (M)

Elution Volume (mL)

6.0 17.0

20

60

kD

α-syn (M)

Supplementary Figure S5

A

IB: S

yn-2

11

IB: S

A34

00

IB: S

yn-1

8

12

2030

4560

RBC Prep. #1

kD

IB: FL-140

RB

C P

rep.

#1

Pre

-IP

Pos

t IP

IP e

luat

e

Rec

. A14

0C

Rec

. wt

IP: Syn-211

20

40

60

B

kD

C

P37840 (100 %), 14'459.3 DaAlpha-synuclein OS=Homo sapiens GN=SNCA PE=1 SV=16 unique peptides, 7 unique spectra, 7 total spectra, 71/140 amino acids (51 % coverage)

M D V F M K G L S K A K E G V V A A A E K T K Q G V A E A A G K T K E G V L Y V G S K T K E G V V H G V A T V A E K T KE Q V T N V G G A V V T G V T A V A Q K T V E G A G S I A A A T G F V K K D Q L G K N E E G A P Q E G I L E D M P V D PD N E A Y E M P S E E G Y Q D Y E P E A

b10+2H

b11+2Hb12-H2O+2H

b12+2H

b13-H2O+2H

b6-H2O

b6-H2O+1

y7-H2Ob15+2H

b16-NH3-H2O+2H

b16-H2O+2H

b16-H2O+2H+1

b16+2Hy8+1

b17-H2O+2H

b8+1

b17+2H

b18+2Hy18+2H

b2y2

y3

b3

y4

b4

y5

b5

y6

b6

y7

b7

y8

b8

b9

y9

b10

y10b11

y11 b12 b13 b14

Q V T N V G G A V V T G V T AQ A V A T V G T V V A G G V N

m/z

Rela

tive

Inte

nsity

0%

25%

50%

75%

100%

200 400 600 800 1000 1200 1400

1'927.04 AMU, +3 H (Parent Error: 0.47 ppm)

20

14

29

4366

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aste

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H4)

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igna

l (co

unts

per s

ec.)

HEK RBC

tota

l [α-

syn]

(ng

/mL)

Remove plasmaand buffy coat, thenremove leukocytes

(by filtration)Whole blood

(450 ± 50 mL)

Step 2

wash 2x with 0.9% NaCl(2 volumes), centrifuge,

Keep pellets

Lysis:Add 2 pellet volumes of PBS 0.1X + protease

inhibitors

Agitate1h, 4°C

Centrifuge17 000g, 30 min, 4°CKeep supernatants

Ultracentrifuge100 000g, 30 min, 4°C

Keep supernatants, filter (0.22µm)

Keep at 4°Cuntil hemoglobin

depletionVT =~ 180 mL

Step 1: Erythrocyte collection and lysisA

- Flowthrough: Depleted fraction- 100 mM imidazole eluate: Hb fraction

Step 2: Hemoglobin depletion

HisPrepTM FF16/10

Load 500-500 mg total protein

per injection

17 injections

Hbfraction

Depleted fraction Lysa

te

10

2030405070

120

160

Dep

lete

dFr

actio

n

Hb.

Frac

tion

VT = ~350 mL(combined depleted fractions)

8

12

20

45

100

rec.

wt

α-sy

n D

eple

ted

fract

ion

Hb.

Fr

actio

n

IB: FL-140

B

Step 3: Anion-exchange chromatographyC

HiTrapTM Q HP 5 mL

Load 1/3 of the depleted lysate

per injection(~116 mL, dilute

to 150 mL of binding buffer)

VT = 13.5 mL

1 2 3 4 5 6

14

20

29

43 66

212

IB: SA-3400(C-terminus)IB: N-19(N-terminus)

IB: Syn-11 2 3 4 5 6 IEX fractions: re

c. w

tα-s

yn

Flow

thro

ugh

Was

h

Gradient elutionincreasing [NaCl]

Step 4: Hydrophobic Interaction chromatographyD

HiTrapTM PhenylFF (High sub. )

1 mL

Dilute combinedIEX fractions to 50 mL in binding

buffer

1 injection

14

29

43

20

66 212

VT = 8 mL, concentrateto 500 µLIB: Syn-1

Flow

thro

ugh

Flow

thro

ugh

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

Gradient elutiondecreasing[(NH4)2SO4]HIC fractions:

Step 5: Gel-filtration chromatographyE

Superdex 20010/300 GL

Load 250 µL of concentratedHIC fractions

2 injections

12

20

45100

IB: Syn-1 Silver stainngVT = 4 mL

1 2 3 4 1 2 3 4

Supplementary Figure S6

SEC fractions:

% b

uffe

r B

UV

Abs

(A.U

.)

Time (min)

Time (min)

Con

duct

ivity

(mS/

cm)

UV

Abs

(10-

3 A.U

.)

Time (min)

Con

duct

ivity

(mS/

cm)

UV

Abs

(10-

3 A.U

.)

Time (min)

Con

duct

ivity

(mS/

cm)

UV

Abs

(10-

3 A.U

.)

10

Supplementary Figure S7

11