ΑLPHA-SYNUCLEIN IN THE CENTRAL NERVOUS SYSTEM AND · PDF file 1 ΑLPHA-SYNUCLEIN IN...
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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: [email protected]
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
“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
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