Fibril Growth and Seeding Capacity Play Key Roles in Α-Synuclein-Mediated Apoptotic Cell Death

Fibril growth and seeding capacity play key roles in α-synuclein-mediated apoptotic cell death

Anne-Laure Mahul-Mellier1#, Filip Vercruysse1#, Bohumil Maco1, Nadine Ait-Bouziad1, Mathias De Roo2, Dominique Muller2and Hilal A. Lashuel1*

1Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland.2Département des Neurosciences Fondamentales, Université de Genève, Faculté de Médecine, Centre Médical Universitaire, 1211 Genève 4, Switzerland.

# These authors contributed equally to this work.

*To whom correspondence should be addressed at:Laboratory of Molecular and Chemical Biology of Neurodegeneration, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne. Tel: +41 21 693 07 92, Fax: +41 21 693 17 80, Email:

Running title (less than 50 characters):

α-synuclein fibrilization promotes apoptosis

Keywords and abbreviations (3-6 keywords): α-synuclein, apoptosis, aggregation

Material and Methods

Cell culture

SH-S5Y5 neuroblastoma cell lines were maintained in 50% F-12, 50% MEM supplemented in 10% FCS, 100 µg/mL streptamycine, 100 U/mL peniciline (Life technologies, Switzerland).

Quantification of cell death by exclusion method in mammalian cell lines

SH-SY5Y neuroblastoma cell lines were plated in 24 well plates. Twenty-four hours after the plating, SH-SY5Y were treated with extracellular monomeric

α-syn, fibrillar α-syn, oligomeric α-syn or a mixture of monomeric and fibrillar α-syn or a mixture of monomeric and oligomeric α-syn. After two or four days of treatment, cell death was quantified by the vital dye exclusion method using Propidium Iodide (PI).

Briefly, the supernatant and the adherent cells were harvested and resuspended in PBS and PI (Millipore, Switzerland) was added to the cells at a final concentration of 2µg/mL. Cells were then analyzed by flow cytometry using Accuri C6 (BD Biosciences, Switzerland) and FlowJo software (Treestar, USA).

ThT measurement-cell culture media

M17 Neuroblastoma cellswere treated with extracellular monomeric α-syn, fibrillar α-syn or a mixture of monomeric and fibrillar α-syn and cell culture medium was sampled after 4 days of treatment. The presence of anti-parallel β-sheet structures was verified by ThT fluorescence by adding 10 % of ThT solution (500mM glycine pH 8.5, 100 µM ThT solution) and loaded as triplicate in black 384-well plate (Nunc, Switzerland). The plates were agitated under orbital shaking for 5 minutes to homogenize the samples and ThT fluorescence was measured with a TECAN spectrometer (Bucher Biotek, Switzerland) at an excitation wavelength of 450 nm and an emission wavelength of 485 nm.

NeuN Intensity

Hippocampal primary neurons plated in black 96 well plates were treated with extracellular with extracellular monomeric α-syn, fibrillar α-syn or a mixture of monomeric and fibrillar α-syn. 6 days post-treatment, cells were washed 3 times with PBS and then fixed with 4% PFA for 10 min at RT. Immunocytochemistry staining was performed as follows: after blocking with 3% BSA in 0.1% Triton X-100 in PBS (PBS-T), neurons were incubated O/N at 4⁰C with NeuN antibody. After 5 washes in PBS-T, cells were incubated with anti-mouse Alexa647. After 5 washes in PBS-T and a final wash in PBS, 50 µL of PBS were added per well, and the fluorescence of each well was measured by fluorescent top reading, with respective excitation and emission wavelength of 640nm and 670nm for Alexa647using a Tecan infinite M200 Pro plate reader (Tecan, Switzerland).

In VitroFibrillization Assay

Monomeric α-syn (M), fibrillar α-syn (F),α-syn mixture (M+F),or α-syn/Tau mixture (α-syn M + Tau F) or Tau mixture (Tau M+ Tau F) were incubated respectively at 20μM for the monomers only, at 2μM for the fibrils only or (18μM M + 2μM F) for the mixtures in an initial volume of 800 μL(10mM HEPES pH 7.4, 100mM NaCl, 2.5mM DTT) under constant agitation at 1,000 rpm for up to 144 h at 37°C. For each time-point, 7μL of each sample was diluted in 70μLof ThT fluorescence solution (10 μM ThT, 50 mM glycine, pH 8.5). For each condition, triplicate was prepared.

In parallel, sedimentation assays were performed to evaluate the amount of remaining soluble proteinas follows: at each time point, 20 μLof each sample was collected and centrifuged at 150,000g for 20 min at 4°C to pellet insoluble aggregates. 15µL of the supernatant was added with 15 µL of 2X Laemmli sample buffer (60 mM Tris pH 6.8, 3.6% w/v SDS, 20% v/v glycerol, 713 mM 2-mercaptoethanol, 0.004% w/v bromophenol blue) and10µlof each aliquots was loaded onto 15% polyacrylamide SDS-PAGE gels. After staining with Coomassie R-450 solution, gels were scanned and the relative amounts of soluble protein for each condition were evaluated by densitometry analysis with ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA).

Quantification of internalized α-syn in neuroblastoma cell lines by flow cytometry

M17 neuroblastoma cell lines were plated in 24 well plates. 24 hours after the plating, M17 were treated for 4, 8, 24 and 36 hours with Tris buffer (negative control), α-syn monomers (20 µM) fluorescently labelled with Alexa Fluor594 dye (MA594; 20 µM), sonicated α-syn fibrils fluorescently labelled with Oregon Green dye (OG-F; 2 µM) or α-syn mixture fluorescently labelled and composed of α-synmonomers (MA594; 18 µM) and sonicated α-syn fibrils (OG-F; 2 µM). To quantify only the proteins that were internalized into the cells, extracellular α-syn attached to the plasma membrane was removed by washing the M17 cells three times with 0.5 mg/mL heparin in PBS at 37°C and cells were then incubated with 0.1% trypsin for 15 minutes at 37°C as described by Richard et al1. Harvested cells were analyzed by flow cytometry [Accuri C6 (BD Biosciences, Switzerland)] and the mean fluorescence intensity per cell, which directly reflects the amount of internalized protein, was recorded in Fl1 channel for α-syn OG-F and in Fl2 channel for α-syn proteins labelled with Alexa Fluor594 dye. Using forward and side scatter, only healthy cells were considered for the analysis using FlowJo software (Treestar, USA).

Legends

Figure S1 Mixture of α-syn monomers and pre-formed fibrils but not individual α-syn species is toxic to SH-SY5Y neuroblastoma cell line

A-E. The human neuroblastoma SH-SY5Y cells were treated with Tris buffer (50 mM Tris pH 7.5, 150 mM NaCl; negative control), α-synmonomers (M), sonicated α-syn PFFs (F)or (M + F) for the indicated times.

A-C. Cells were harvested and stained with propidium iodide (PI). Cell death level is expressed as the percentage of cells with loss of plasma membrane integrity(PI positive cells) to the total cell number analyzed by FACS. D.Cells were harvested and caspase 3 activity was quantified by a caspase 3 activity assay using FACS.

E. Cells were fixed with PFA 4% and subsequently stained for the apoptotic cells (TUNEL, red) and nucleus was counterstained using Sytox Green (green). Scale bars = 10 µm.

A-D. Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (Tris versus α-syn treated conditions), *p<0.01, **p<0.001, *** p<0.0001.

Figure S2 Monomeric α-syn aggregates in cell culture media when seeded with α-syn pre-formed fibrils

ThT analyses of cell culture media from M17 treated with Tris buffer (Tris), monomeric α-syn (M), sonicated α-syn PFFs (F) or α-syn mixture (M + F) incubated for up to 4 days. Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (Tris versus α-syn treated conditions), *p<0.05, *** p<0.0001.

Figure S3 A mixture of α-syn monomers and pre-formed fibrils induces apoptotic cell death in M17 andSH-SY5Yneuroblastoma cell line

M17 cells (A) or SH-SY5Y cells (B) were treated for 2 or 4 days with Tris buffer (negative control) or α-syn mixture composed of α-synmonomers (M) and sonicated α-syn PFFs (F). Final concentration of α-synfibrils tested was 10% or 20% of the total α-syn concentration in the mixture. For each condition, cells were harvested and stained with propidium iodide (PI). Cell death was scored as follow: cell death level is expressed as the percentage of cells with with loss of plasma membrane integrity(PI positive cells) to the total cell number analyzed by FACS. Shown are the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (Tris versus α-syn treated conditions), **p<0.001, *** p<0.0001.

Figure S4 Mixture of α-syn monomers and fibrils exacerbates cell death in hippocampal primary neurons

Hippocampal primary neurons were plated in 96 wells plates and treated with Tris buffer (negative control), α-syn monomers (M), sonicated α-syn PFFs (F) or with a mixture of α-syn species composed of α-synmonomers and sonicated α-syn PFFs (M + F). After 6 days, cells were washed 3 times prior to fixation and immunostained against NeuN, a specific neuronal marker. Fluorescence intensity of the NeuN-positive cells was quantified by Tecan infinite M200 Pro plate reader(640nm/670nm). Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). n=5 for each experiment. One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (Tris versus α-syn treated conditions), * p<0.01, ** p<0.001.

Figure S5 Inhibiting fibril growth attenuates α-syn-induced toxicity in M17 neuroblastoma cell line.

A-B.M17 cells were treated for 4 days with Tris buffer (negative control) or (A) the indicated α-synspecies in presence of DMSO (negative control) or Tolcapone (aggregation inhibitor), (B) with β-synmonomers, a mixture of β-synmonomers and sonicated α-syn PFFs, sonicated Tau PFFs or a mixture composed of α-synmonomers and sonicated Tau PFFs.

For each condition in (A) and (B), cells were stained with SG. Cell death level is expressed as the percentage of cells with compromised cell membrane (SG positive cells) to the total cell number analyzed by Tecan infinite M200 Pro plate reader(487nm/519nm). Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed, ** p<0.001 for α-syn mixture +/- Tolcapone (A); Tris versus α-syn, β-syn or Tau treated conditions(B).

Figure S6 Comparison of α-syn fibrilization capacity in presence of Tau or α-syn fibrils seeds

A. ThT analyses of monomeric α-syn (α-syn M; 20 μM) incubated in vitrowith or without sonicated Tau fibrils (Tau F; 2 μM) or sonicated α-syn PFFs (α-syn F, 2 μM) for up to 144 hours.

B. Sedimentation assays were performed to evaluate the amount of remaining soluble protein for monomeric α-syn (20 μM) incubated in presence or absence of Tau fibrils (2 μM) or α-syn PFFs (2 μM).

A-B. Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (α-syn monomers only versus α-syn monomers +/- Tau or α-syn fibrils), *p<0.01, ** p<0.001.

C. EM analysis of the inhibitory effect of Tau fibrils on the aggregation of monomeric α-syn, monitored over 144 hours. Fibrils formation of the α-syn Malone(left column) or in the α-syn mixture (M + F) (middle column) is inhibited in the presence of Tau fibrils (right panel). Scale bars= 200 nm.

Figure S7 Labelling and characterization of monomeric and fibrillar α-syn

A. V16C-α-syn has a free cysteine that reacts with the maleimide compound of a small fluorophore via a Michael-type addition.

B-D.MALDI-TOF-MS analyses of V16C-α-syn labeled with oregon green (OG-α-syn) (B), with Alexa Fluor594 (α-synA594) (C) or with Alexa Fluor647 (α-syn647)(D),corresponding to the theoretical mass of 14’947 Da, 15’355 Da or 15’446 Da, respectively. The mass at ~7’473, 7’680 or 7’725 Da corresponds to the doubly-charged species.

E-G. Confirmation that the purified fractions of recombinant V16C-α-syn were fluorescently labeled with OG (E), with Alexa Fluor594 (F) or with Alexa Fluor647 (G). left: coomassie blue staining, right: fluorescent scan.

H-I. Characterization of OG-α-syn fibrils and α-syn647 fibrils by gel electrophoresis. More than 95% of the monomeric α-syn is converted to α-syn fibrils after 5 days of incubation, as shown after pelleting the non-soluble fibrils by centrifugation and analyzing the supernatant (sup) or the pellet before sonication (b.s) and after sonication (a.s).

Figure S8 α-syn mixture forms aggregates at the cell plasma membrane of hippocampal primary neurons

Hippocampal primary neurons were plated on coverslips (CS) and treated with labeled α-syn647monomers (M) (top row, white), labeled sonicated α-syn594 PFFs (F) (middle row, red) or α-syn mixture (α-syn647 M+ α-syn594F) (bottom row, white + red). After the indicated times, neuronal cells were washed 3 times prior to fixation and immunostained against MAP2, a specific neuronal marker (green). Scale bar = 10 µm.

Figure S9 α-syn mixture forms aggregates at the cell plasma membrane that are internalized into the M17 neuroblastoma cell line

A-D. M17 cells were treated with α-synmonomers (M), sonicated α-syn PFFs (F) or α-syn mixture (M+F).

A. At the indicated times, M17 cells were washed 3 times prior to fixation and then stained with wheat germ agglutinin membrane marker (WGA488, green) and immunostained against α-syn (red). Scale bar = 5 µm.

B. After4 days,M17 cells were washed 3 times prior to fixation and then stained with the proteostat aggregation dye (green) and immunostained against α-syn (red) and β-catenin (grey). Scale bars = 20 µm.

C. M17 cells were washed three times to wash out the excess of proteins not attached to the cells. M17 were then lysed directly in LB 2X and boiled for 10 min prior to be further analyzed by WB. α-syn protein level was detected as a main band around 15 kDa using specific antibody. Accumulation of α-syn at higher molecular weights was also observed and indicates the conversion of monomeric α-syn into stable oligomeric or fibrillar states. Actin was used as a loading control.

D. Densitometry quantification of WB. Protein level of α-syn was evaluated by densitometry quantification. Band intensities were normalized as follows: α-syn/actin. Bars represent the mean ± SD of three independent experiments. One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed (Tris versus α-syn treated conditions), *** p<0.0001.

E-F. M17 cells were treated for 4, 8, 24 and 36 hours with Tris buffer (negative control), α-syn monomers fluorescently labelled with Alexa Fluor594 dye (MA594; 20 µM), sonicated α-syn PFFs fluorescently labelled with Oregon Green dye (OG-F; 2 µM) or α-syn mixture fluorescently labelled (MA594+OG-F) and sonicated α-syn PFFs (OG-F). To quantify exclusively the proteins internalized into the cells, extracellular α-syn attached at the plasma membrane was removed by washing the M17 cells three times with 0.5 mg/mL heparin in PBS at 37°C and then incubating the cells with 0.1% trypsin for 15 minutes at 37°C, as described by Richard et al1. Harvested cells were analyzed by flow cytometry and the mean fluorescence intensity per cell, which directly reflects the amount of internalized protein, was recorded in Fl1 channel for α-syn OG-F (E) and in Fl2 channel for α-syn proteins labelled with Alexa Fluor594 dye (F). Only viable cells were considered for the analysis. Shown are the means of three independent experiments performed in triplicate for each condition. One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed *** p<0.0001 for α-syn OG-F or α-syn mixture (OG-F + MA594) versus Tris (E); for α-syn mixture (OG-F + MA594) versus α-syn MA594 (F).

Figure S10 Hippocampal neurons treated with Tris buffer exhibits a well-preserved plasma membrane

A. The IBIDI dishes with alphanumerical searching grids imprinted on the bottom glass were used to grow the primary hippocampal neurons. This allows to easily identify the neuron(s) of interest with the known alphanumeric coordinates (arrowhead) during each step of the CLEM procedure. Selected cells were imaged with confocal microscope (1) and both fluorescence (left panel) and phase contrast (right panel) images were taken. Then, the cells were processed for the electron microscopy (as described in Methods section) and embedded in resin (2). The left panel shows the selected neuron (arrowhead) after post-fixation with osmium tetroxide and en block staining with uranyl acetate and the right panel depicts the same neuron after resin embedding (arrowhead). Laser dissection microscope was then used to mark the selected neuron on the top surface of the cured resin block (3). Cured resin with imprinted pattern of the alphanumerical searching grids (on the top surface) was separated from the IBIDI dish (3-left panel) and the laser of the laser dissection microscope was used to etch the position of the selected neuron on the block surface, as outlined by dashed-line rectangle (3-right panel). This allowed to cut off the selected region from the resin disc and mount it on a dummy resin block to prepare the cutting face by trimming with a glass knife and a ultramicrotome. The final block face with selected neuron (arrowhead), from which ultrathin (serial) sections were cut is shown (3-right panel). 50-60 nm ultrathin sections through the neuron of interest were then imaged by TEM (4). Stitched low magnification overview image of the whole section showing the neuron of interest (arrowhead) surrounded by the etched rectangle (in white) created by the laser dissecting microscope and depicted by dashed-line rectangle.The small square size in (1-3) correspond to 50 µm × 50 µm. Scale bar in (4) = 10 µm.

B.Primary hippocampal neurons were seeded on IBIDI dishes. Cells were treated with Tris buffer (negative control) for one (1d), three (3d) or six days (6d). Cells were first imaged with confocal microscopy and the selected neurons were further process for TEM analysis. Shown are EM illustrations of the neurons of interest at 1d (top row), 3d (middle row) and 6d (bottom row). Control neurons incubated with Tris buffer exhibit well-preserved plasma membrane (highlighted in orange) and nuclear membrane (arrowheads) of the nucleus (nuc), mitochondria (m) and cisternae of endoplasmic reticulum (ER) throughout all time-points tested. Scale bars: 0.5 µm.

Figure S11 Mixture of α-syn monomers and fibrils initiates apoptosis in a caspase 8 dependent manner in M17 neuroblastoma cell line

A-B. M17 cells were treated for 4 days with Tris buffer (negative control), α-synmonomers (M), sonicated α-syn PFFs (F) or α-syn mixture (M+F)in presence of DMSO (A-B) or specific caspase inhibitors (C-F): IETD-fmk (caspase 8 inhibitor), LEHD-fmk (caspase 9 inhibitor), ZVAD-fmk a general caspase inhibitor or DMSO (as negative control). Caspase 8 activity (A, C), caspase 9 activity (B, D) or caspase 3 (E) was quantified by respectively a specific caspase 8 or caspase 9 activity assay usingFACS.

F. The level of cell death for each condition tested in (C-E) was also assessed using PI by FACS analysis.

A-F.Data shown represent the means of three independent experiments performed in triplicate for each condition (bars are means ± S.D.). One-way ANOVA test followed by a Tukey-Kramer post-hoc test were performed. A-B: (Tris versus α-syn treated conditions), *p<0.01, **p<0.001; C-F: (α-syn mixture + DMSO vs α-syn mixture + caspase inhibitors), *** p<0.0001 or (α-syn PFFs + DMSO vs α-syn PFFs + caspase inhibitors), #p<0.01.