Familial α-synuclein A53E mutation enhances cell death in response

Ganesh M. Mohitea, Ambuja Navalkara, Rakesh Kumara, Surabhi Mehraa, Subhadeep Dasa,c,. Laxmikant G. Gadhea, Dhiman Ghosha, Basil Aliasa, Vikas ...
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Familial #-synuclein A53E mutation enhances cell death in response to environmental toxins due to more population of oligomers Ganesh M Mohite, Ambuja Navalkar, Rakesh Kumar, Surabhi Mehra, Subhadeep Das, Laxmikant G Gadhe, Dhiman Ghosh, Basil Alias, Vikas Chandrawanshi, Aishwarya Ramakrishnan, Sarika Mehra, and Samir K. Maji Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00321 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 20, 2018

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biochemistry

Familial α-synuclein A53E mutation enhances cell death in response to environmental toxins due to more population of oligomers Ganesh M. Mohitea, Ambuja Navalkara, Rakesh Kumara, Surabhi Mehraa, Subhadeep Dasa,c, Laxmikant G. Gadhea, Dhiman Ghosha, Basil Aliasa, Vikas Chandrawanshib, Aishwarya Ramakrishnana, Sarika Mehrab, Samir K. Maji*,a a

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai,

India b

c

Department of Chemical Engineering, Indian Institute of Technology Bombay, Mumbai, India IITB-Monash Research Academy, IIT Bombay, Mumbai, India

* Correspondence should be addressed to Prof. Samir K Maji Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, India 400076. Telephone: +91-22-2576-7774. Fax: +91-22-2572 3480. E-mail: [email protected]

Keywords: Parkinson’s disease, α-synuclein, A53E mutant, oligomers, amyloid, cell death

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Abstract Amyloid formation of α-synuclein (α-Syn) and its familial mutations are directly linked with Parkinson’s disease (PD) pathogenesis. Recently, a new familial α-Syn mutation (A53E) was discovered, associated with an early-onset aggressive form of PD, which delays the α-Syn aggregation. When we overexpressed WT and A53E proteins in cells, neither of them showed toxicity or aggregate formation, suggesting merely overexpression may not recapitulate the PD phenotype in cell models. We hypothesized that cell expressing A53E mutant might possess enhanced susceptibility to PD associated toxicants compared to WT. When cells were treated with PD toxicants (dopamine and rotenone), cells expressing A53E showed more susceptibility to cell death along with compromised mitochondrial potential and increased generation of reactive oxygen species. The higher toxicity of A53E could be due to more oligomers load in cells as confirmed by dot blot assay using amyloid specific OC and A11 antibody and using in vitro aggregation study. The present cellular model suggests that along with familial mutation, environmental and other cellular factors might play a crucial role in dictating PD pathogenesis.

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Biochemistry

Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease after the Alzheimer’s disease. Although most of the PD cases are sporadic with unknown etiology, ~ 5% of cases are known to involve mutations in dominantly or recessively inherited genes causing a definitive form of PD.1 Various factors have been shown to cause PD, which include oxidative stress, mutations or overexpression of genes and exposure to environmental toxins.2,

3

These factors may act

independently or in association with each other inducing oxidative stress and subsequent death of dopaminergic neurons.2,

3

PD is pathologically characterized by the presence of proteinaceous

inclusions (Lewy bodies ‘LB’ and Lewy neurites ‘LN’) in the surviving dopaminergic neurons of the PD patients.4, 5 Immunostaining and biophysical characterization revealed the presence of α-synuclein (α-Syn) amyloid fibrils as a major component of these structures.4,

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Moreover, familial α-Syn

mutations (A30P, A53T, E46K, H50Q, G51D and A53E α-Syn) and α-Syn gene duplication/ triplication have been identified to be associated with familial PD.6-8 Discovery of familial mutants of α-Syn indicates the importance of point mutation in cause of PD pathogenesis.6 Studies on A30P, E46K, and A53T have shown that these mutations affect organelles which maintain cellular homeostasis (proteasomes, mitochondria and endoplasmic reticulum), and subsequently, cells become more susceptible to various death stimuli.9-13 It was observed that stress stimuli like dopamine, H2O2, proteasomal inhibitors, autophagy inhibitors, FeCl2, exogenous addition of fibrils and mitochondrial toxins such as rotenone induce more oxidative stress in α-Syn mutant expressing cells as compared to wild-type (WT) protein.9-13 Although the three previously discovered mutations (A30P, E46K, and A53T) are widely studied in vitro and in vivo, the other three newly discovered familial mutations (H50Q, G51D, and A53E) have not been explored regarding their mechanism of action. In this work, we have selected the most recently discovered A53E α-Syn mutation 7 and investigated its mechanism of cellular toxicity using SH-SY5Y cell line. The previous study of A53E mutation has shown that A53E aggregates slowly as compared to WT14 and their fibril shows similar cytotoxicity as of WT protein.15, 16 We hypothesized that cellular and environmental factors (PD associated toxins) may play a significant role in altered A53E toxicity in cells compared to the WT. In the present study, biophysical and cellular characterization of A53E α-Syn was performed. In this study, the effect of WT and A53E α-Syn overexpression was assessed in neuronal cell line SH-SY5Y in the presence and absence of stress inducers (dopamine and rotenone). We showed that overexpression of WT or A53E alone did not induce cytotoxicity and aggregate formation in the cells. However, in the presence of dopamine and rotenone, cells expressing A53E α-Syn showed decreased cell viability, increased production of reactive oxygen species (ROS) and reduced mitochondrial potential compared to WT and vector control cells. Since A53E mutation delays α-Syn aggregation, we hypothesized that higher amount of oligomer accumulation by A53E in presence of these toxicants 3

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could be one of the reasons for its higher toxicity. Indeed, the immunoblot analysis with amyloid specific OC17 and oligomers specific A11 antibody18 suggest high oligomer accumulation in A53E cells compared WT. In vitro α-Syn aggregation in the presence and absence of these toxins showed accelerated aggregation of both proteins in presence of rotenone whereas dopamine delayed the aggregation kinetics. Overall, A53E protein showed longer lag phase in absence and presence of toxicants compared to WT α-Syn under identical conditions. The in vitro studies indicate that due to slower aggregation kinetics by A53E mutant, effective population of oligomers might increase, which in turn increases cellular toxicity. The present data might explain the aggregation-toxicity relationship of new A53E α-Syn mutation for understanding the mechanism of familial PD pathogenesis due to A53E mutation.

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Biochemistry

Materials and Methods Chemicals and Reagents Most of the chemicals were purchased from Himedia and Merck unless stated. NotI, XbaI, and rapid ligation kit were purchased from Fermentas (Vilnius, Lithuania). Antibodies used in this study were following: human α-synuclein specific LB509 (catalog no- 180215, Invitrogen, USA), anti αsynuclein (catalog no-610786, BD Biosciences), phosphoserine 129 α-synuclein (Abcam catalog noEP1536Y) and β-tubulin (catalog no- T5293 Sigma). Amyloid fibril specific OC specific A11

18

17

and oligomers

antibodies were kind gift from Prof. Charles G Glabe,UC Irvine, USA. 5,5,6,6-

Tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) mitochondrial potential kit, dihydroethidium, and fluorophore labeled secondary antibodies were purchased from Invitrogen. Dopamine hydrochloride, rotenone, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), Geneticin (G418) and Triton X-100 were procured from Sigma, USA. Annexin V-FITC and PI apoptosis kit were purchased from BD Biosciences. Water was double distilled and deionized using a Milli-Q system (Millipore Corp., Bedford, MA). Protein expression and purification WT and A53E α-Syn were expressed in E. coli BL21 (DE3) strain and purified according to the established protocol.19, 20 Briefly, IPTG induced bacterial cells were pelleted down by centrifugation at 4000 g at 4°C for 30 min. It was resuspended in lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 150 mM NaCl). Proteolytic cleavage was prevented by addition of Protease inhibitor cocktail (Roche) . It was then sonicated using probe sonicator (Sonics & Materials INC, USA) at 40% amplitude and 45 pulse/min for 10 min, followed by heating at 95°C in water bath for 20 min. The suspension was then centrifuged at 10,000 rpm for 30 mins and the supernatant was collected. Nucleic acids were removed from the supernatant by addition of 10% streptomycin sulfate (136 μl/ml) and glacial acetic acid (228 μl/ml). Further, the solution was centrifuged at 12000 g for 30 min at 4°C and an equal volume of saturated ammonium sulfate was added to the supernatant. The solution was kept at 4°C for 1 hr for complete precipitation of α-Syn protein and then centrifuged at 12000 g for 30 min at 4°C. The pellet was resuspended in 50% ammonium sulfate solution and centrifuged again at 12000 x g for 30 min at 4°C. Finally, the pellet obtained was resuspended in 100 mM ammonium acetate (10 ml/lit growth of culture) followed by adding an equal volume of ethanol for protein precipitation. This step was repeated three times. The solution was centrifuged and redissolved in a minimum volume of ammonium acetate, lyophilized and stored at -20°C until further use. Preparation of low molecular weight (LMW) protein and amyloid fibrils formation Lyophilized protein was suspended in 20 mM Gly-NaOH, pH 7.4, 0.01% sodium azide. As the protein was partially soluble, therefore, a few drops of 2 N NaOH was added until the protein solution 5

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became clear. The pH was then adjusted to 7.4 by adding few drops of 2 N HCl. The solution was dialyzed overnight using 10 kDa MWCO mini-dialysis units (Millipore) against the same buffer at 4°C. LMW was isolated using 100 kDa cut off filters using centricon YM-100 filter (100 kDa MWCO, Millipore) according to the previously described method.19 To LMW, dopamine and rotenone, prepared in the same buffer were added independently in such a way that the final concentration of LMW α-Syn was 300 µM and that of stress inducers was 150 µM. The aggregation study was initiated in 20 mM Gly-NaOH, pH 7.4 (0.01% sodium azide) at 37°C in Echo Thermo model RT11 rotating mixture (Torrey Pines Scientific, USA), with slight agitation (~ 20 rpm). Aggregation kinetics was monitored by ThT fluorescence and secondary structural changes were studied using circular dichroism (CD) spectroscopy. Amyloid formation was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Circular dichroism spectroscopy (CD) CD spectroscopy was performed with 15 µM protein concentration in 20 mM Gly-NaOH buffer, pH 7.4 using quartz cell (Hellma, Forest Hills, NY) with a path-length of 0.1 cm. Spectra were acquired in the wavelength range of 200-260 nm using JASCO-810 instrument (USA). Three independent sets were performed. Raw spectra were processed and smoothing was done according to the manufacturer’s instructions. All measurements were done at 25°C. Thioflavin T (ThT) fluorescence assay 1 mM ThT was prepared in 20 mM Tris-HCl buffer (pH 8.0, 0.01% sodium azide). 2 µl of 1 mM ThT solution was added to the 10 µM protein solution (final volume 200 µl) in 20 mM Gly-NaOH buffer, pH 7.4, 0.01% sodium azide. ThT fluorescence assay was performed with excitation at 450 nm and emission in 460-500 nm range using Horiba-Jobin Yvon Fluoromax4 (Kyoto, Japan) with excitation and emission slit width of 5 nm each. ThT fluorescence at 480 nm was plotted and the data were fitted to a sigmoidal curve. The lag time (tlag) was calculated using equations 1 and 2 as per the published protocol21 y = y0 + (ymax – y0 )/ (1+ e-k(t-t1/2))------- (1) tlag = t ½ - 2/k ----------(2), where y is the ThT fluorescence at a particular time point, y max is the maximum ThT fluorescence and y0 is the ThT fluorescence at t0. The data described in these assays were obtained from 3 independent experiments. Transmission electron microscopy The end product of aggregation was diluted in distilled water to a final protein concentration of ~ 40 μM and spotted on a carbon coated copper grid (Electron Microscopy Sciences, Fort Washington, 6

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Biochemistry

PA). The sample was blotted off post 5 min of incubation and quick water wash was given. After this, 1% (w/v) aqueous uranyl formate solution was applied to the grid for negative staining and incubated for 2 min. The stain was then gently removed by blotting with a filter paper followed by air drying for 5 min. Transmission electron microscopy (TEM) of the samples was carried out using a PHILIPS CM 200 (Amsterdam, Netherlands) at 200 kV with 6600X magnification. Randomly 10 -12 images were taken for each sample. Two independent experiments were performed. Fibril diameter was analyzed using image J software. Randomly 10 images were quantified and mean diameter was plotted. Atomic force microscopy (AFM) Morphological characterization of different α-Syn species (fibrils and oligomers) were performed using AFM (Asylum Research, USA). For AFM analysis, a small aliquot of α-Syn fibrils and oligomers (isolated from size exclusion chromatography) were taken, diluted to a final concentration of 30 μM and spotted on a freshly cleaved mica sheet and incubated at room temperature for 5 min. Unbound proteins/aggregates were washed with distilled water and dried under vacuum. ASYELEC02 probe from ASYLUM research, made up of silicon having spring constant of 42N/m and frequency of 300KHz, was used. The imaging was performed in tapping mode with silicon cantilever at a scan speed of 1 Hz and around 4-5 different areas were scanned randomly for each sample. Size exclusion chromatography (SEC) Purified α-Syn was dissolved in PBS, pH 7.4, with 0.01% sodium azide as described before in order to get a concentration of 20 mg/ml. The protein solutions were then centrifuged for 30 min at 14000 x g at 4°C using a bench top microcentrifuge (HITACHI, himac CT15RE, JAPAN) to remove any insoluble aggregates. 500 μl of supernatant was loaded on a S200-Superdex gel filtration column attached with AKTA purifier (GE Healthcare). Elution was done isocratically at 4°C in the same buffer at a flow rate of 0.15 ml/min. Oligomers that appeared at void volume ~8 ml were collected for further analysis. Two independent experiments were performed. In order to determine the molecular weight of α-Syn by SEC, we passed two standard protein of known molecular weight through SEC column and standard correlation plot was made and molecular weight of syn monomer was determined. Our data shows that lysozyme from hen egg (mol wt 14.3 kDa) was eluted at 22 ml as single peak. BSA(mol wt 66 kDa) was eluted as monomer at 13.7 ml and 11.8 ml as dimer as consistent with previous report.22 α-Syn oligomers eluted in fraction corresponding to the void volume (∼8 mL) and therefore molecular weight of this fraction cannot be calculated. The other peak of α-Syn was eluted at 15 ml as a 57 kDa protein as reported earlier due to unstructured nature of the protein.23, 24

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MTT assay of fibril formed in presence dopamine and rotenone MTT assay is commonly used to check the cytotoxicity.25 Toxicity of WT and A53E α-Syn fibrils formed in presence of dopamine and rotenone were evaluated using MTT assay. SH-SY5Y cells were seeded in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin at 1×104 cell density in 96 well plate and further grown for 24 hrs at 37°C with 5% CO2. SH-SY5Y cells were then incubated with 25 μM of α-Syn fibrils in 96-well cell culture plate. After 30 hrs of incubation with samples at 37°C in a 5% CO2 humidified environment, viability was measured using MTT assay. In brief, 10 μl of MTT solution (5 mg/ml) was added to each well in a 96-well plate and was incubated for 4 hrs at 37°C. Finally, formazan crystals formed by live cells were dissolved by adding 100 μl/well of solubilization solution containing 50% dimethyl formamide and 20% SDS (pH 4.7) and further incubated for overnight. The absorbance values were then recorded at 560 and 690 nm using a micro plate reader (spectraMax M2e, Molecular Devices). In another set of experiment, the toxicity of WT and A53E α-Syn monomers (25 μM) and oligomers (25 μM) isolated from SEC were examined by MTT assay as per protocol mentioned above. Four independent experiments were performed. Construction of pcDNA3.1 α-Syn expression vector A53E α-Syn mutation was initially created in the bacterially expressing pRK172-WT α-Syn plasmid by site-directed mutagenesis. A53E and WT α-Syn were amplified with primers having NotI and XbaI restriction enzymes sites in it. Primer sequences were as follows: TACTAATGCGGCCGCATGGA-TGTATTCATGAAAG-3’

and

XbaI

NotI α-Syn FP 5’α-Syn

RP:

5’-

TTCAGAGTTCTAGATTAGGCTTCAGGTTCGTAGTC-3'. PCR reaction conditions were set up as mentioned on the Takara Ex Taq polymerase datasheet (Takara, Japan). PCR amplified α-Syn DNA and empty pcDNA 3.1 vector were double digested with NotI and XbaI as per protocol suggested by the manufacturer (Ferments, Vilnius, Lithuania). α-Syn DNA then ligated with cut pcDNA vector using rapid ligation kit (Fermentas, Thermo scientific). Colonies obtained from transformations of ligation mixture were screened by colony PCR and insert release assay. Colony PCR positive and αSyn insert release positive transformants were sequenced using T7 forward primer 5’TAATACGACTCACTATAGGG-3’ and BGH reverse primer 5’-TAGAAGGCACAGTCGAGGC-3’ by Xcelris lab limited (Gujrat, India). Obtained sequences were analyzed by nucleotide sequence alignment tool (NCBI BLAST). Further, the sequence was translated by using ExPASy translate tool (Figure S1). Cell culture and generation of transfected cell lines Human SH-SY5Y cells were grown and maintained in DMEM with 10% FBS (cell clone), 100 U/ml penicillin, and 100 µg/ml streptomycin (complete medium) at 37°C with 5% CO2. For transfection, 8

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Biochemistry

SH-SY5Y cells were seeded at 8×104 cell density in 24 well plates and then grown for 24 hrs at 37°C with 5% CO2. After 24 hrs, cells were transfected with pcDNA vectors (pcDNA vector, pcDNA WT, and A53E α-Syn). One μg of each plasmid construct was used for transfection of cells in each well of 24 well plates. Lipofectamine LTX based transfection was performed as per manufacturers protocol (Invitrogen, USA). Transfected cell lines were initially selected with a high concentration of G418 (800 µg/ml of DMEM). Later, selected cell lines were maintained in 500 µg/ml of G418 containing complete DMEM. Whole cell lysate preparation and western blot of WT and A53E α-Syn overexpressing cells SH-SY5Y cells expressing only vector, WT, and A53E α-Syn were seeded at a density of 3×105 cells in T25 flasks with media (complete DMEM). Cells were grown for 4 days and the cell lysate was prepared by resuspending the cells in PBS with a protease inhibitor cocktail mixture, EDTA-free (190 µl PBS + 10 µl protease inhibitor cocktail). Cells were lysed by sonication (pulse 1 sec, gap 1 sec, amplitude 20%, total cycles 20). Triton X-100 was added to cell lysate at a final concentration of 1% (v/v), incubated on ice for 10 min and centrifuged at 1500 rpm for 2 min at 4°C and total protein lysate was obtained. The resultant supernatant was used as total protein lysate and the cell debris was discarded. The protein concentration of the lysate was determined using Bradford’s assay. An equal amount of samples was boiled with Lamelli sample buffer containing 100 mM DTT for 10 min. Samples were resolved in 12% SDS-PAGE and proteins from the gel were transferred to nitrocellulose membrane by wet transfer method using BioRad transfer apparatus. Transfer was performed at 4°C for 2 hrs at constant current (350 mA). After transfer, the membrane was treated with 0.4% paraformaldehyde (PFA) solution made in PBS for 50 min at room temperature (RT) at slow rocking followed by blocking with 5% blotto (non-fat milk powder) for 1 hr at RT under rocking condition. Two washes (10 min each) were given to the membrane with Tris-buffered saline (50 mM Tris, 150 mM NaCl). Primary antibody was added to the membrane (LB509- 1:1000) and incubated at 4°C for overnight. The membrane was then kept at RT with slow rocking for 1 hr. Primary antibody was removed followed by two washes (8 min each) with TBST (0.01% Tween). The membrane was then incubated with appropriate anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (dilution-1:4000, catalog no-401253, Calbiochem) for 2 hrs at room temperature under constant rocking. Afterward, two washes (8 min each) were given with TBST (0.01% Tween). The membrane was exposed to a chemiluminescent substrate (West Femto, Pierce) in the dark room and signal was captured on X-ray films. Images were processed using Image J software for quantitative analysis. The same membrane was then treated with stripping buffer (2% SDS, 125 mM Tris-HCl pH 6.8, 100mM β-mercaptoethanol) at 55°C for 10 min. Two TBS washes (each wash was of 10 min) were given to membrane. The membrane was then probed against β-tubulin (1:2000) by adding antibody to the membrane and incubating it at 4°C for overnight. Two TBST washes were given to 9

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membrane followed by addition of anti-mouse HRP (1:4000) to the membrane and incubated at RT for 2 hrs under slow rocking. The membrane was then developed with a chemiluminescent substrate. Blots were analyzed using Image J software. Normalized value for α-Syn was obtained by dividing intensity of α-Syn by intensity of β-tubulin for the same lane. Three independent experiments were performed. Measurement of cell viability using α-Syn overexpressing cells SH-SY5Y expressing only vector, WT, and A53E α-Syn were seeded at 1×104 cell density in 96 well plates. Cells were grown for 24 hrs at 37°C with 5% CO2. After 24 hrs, dopamine (500 µM) was added to the cells with media. Incubation was further extended for another 24 hrs. Thereafter, cell death was assessed by MTT assay and Annexin V-FITC and PI binding assay using FACS analysis. Dopamine untreated cells were kept as control. For measuring the toxicity of cells in presence of rotenone, the cells were grown as mentioned above. After 24 hrs, complete media is replaced with media supplemented with 0.5% serum. Rotenone dissolved in the DMSO was added to the cells such that final concentrations of rotenone become 200 nM followed by 24 hrs incubation. The cell death was measured using MTT assay and Annexin V-FITC and PI binding by FACS analysis. Rotenone untreated cells incubated in DMEM with 0.5% serum was kept as a control. Three independent experiments were performed. Measurement of reactive oxygen species (ROS) SH-SY5Y cells expressing only vector, WT and A53E α-Syn were seeded at a density of 3.5×104 cells/well in 24 well plates with coverslips. Cells were treated with dopamine (500 µM) and rotenone (200 nM) as per above-mentioned protocols. To perform ROS measurements, old media was replaced with fresh media and the cells were washed twice with 1 ml PBS. 1 μl dihydroethidium was added from 3 mM stock prepared in DMSO. Cells were incubated in dark for 2 min at RT. Coverslips containing cells were taken out from the 24 well plates and placed on a thin glass slide for imaging. Images were immediately captured by using a confocal microscope (Olympus FV500) and further analyzed by Image J software. Three independent experiments were performed. Measurement of mitochondrial (MT) potential SH-SY5Y cells transfected individually with only vector, WT and A53E α-Syn constructs were seeded at a density of 2×105 cells/well in 12 well plates. Cells were treated with dopamine (500 µM) and rotenone (200 nM) as per above-mentioned protocols. To perform MT potential measurements, cells were trypsinized and collected in tubes with 1 ml complete media. To these cells, 1 ml diluted 1X JC1 dye (JC1 diluted in 1X assay buffer provided by the manufacturer) was added. The positive control was kept by adding 1 µl of valinomycin (1 µg) and 1 ml of diluted 1X JC1 dye to the cells. Cells only with JC1 assay buffer were kept as negative control. Cells were incubated at 37°C in a CO2 10

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Biochemistry

incubator for 20 min. Cells were then pelleted and suspended in 1X JC1 assay buffer. JC1 stained cells were analyzed by flow cytometry. The signal of monomer was recorded in FL1 channel (green), while JC1 aggregates were recorded in FL2 channel (red). Data is represented as % of cell distribution in the green and red region. Three independent experiments were performed. Immunofluorescence study SH-SY5Y cells expressing only vector, WT and A53E α-Syn were used for immunostaining. Cells were fixed with 4% paraformaldehyde for 15 min at 37°C followed by two washes of PBS (each wash for 5 min). Cells were then permeabilized with 0.2% Triton X-100 (prepared in PBS) for 10 min at room temperature. Blocking was done using blocking solution (1% bovine serum albumin, 10% goat serum made in PBS), for 45 min at RT. Cells were stained with mouse anti α-Syn antibody (BD Biosciences, 1:250 dilution) prepared in PBS with 1 % BSA, overnight at 4°C. Three washes were performed with PBST (5 min x 3) and then anti-mouse Alexa 555 (Invitrogen, catalog no. A21424, dilution: 1:400) secondary antibody was added to the cells and incubated for 2 hrs at RT in dark. Three washes were given using PBST (5 min x 3). The nucleus was stained with DAPI (1:1000) for 2 min at RT. After that, cells were washed with PBST (5 min x 3) and mounted on the slides for confocal microscopic studies (Olympus FV 500). Similarly, immunofluorescence study was done for cells that treated with dopamine (500 µM) and rotenone (200 nM). Three independent experiments were performed. To detect α-Syn aggregates, untreated and toxicant treated (dopamine and rotenone) cells of vector, WT and A53E were stained with the phosphoserine 129 α-Syn (p- α-Syn) specific antibody (Abcam catalog no- EP1536Y). Above mentioned protocol was used for immunostaining the cells. p- α-Syn antibody was used at a dilution of 1:500 prepared in PBS with 1 % BSA. Anti-rabbit Alexa 555 antibody (A31572) labeled secondary was used as secondary antibody. Images were acquired on Leica DMi8 fluorescence microscope. Three independent experiments were performed. 30 different images were used for aggregate number quantification. Dot blot analysis SH-SY5Y expressing only vector, WT and A53E α-Syn were seeded at a density of 6×105 cells in T25 flasks. Cells were treated with dopamine (500 µM) and rotenone (200 nM) as per above-mentioned protocols. Whole cell lysates were prepared and protein concentration was quantified using Bradford’s assay. An equal amount of whole cell lysate of each sample was spotted on the nitrocellulose membrane (Immobilon-NC, Millipore) and allowed to air-dry for 10 min. Two washes (2 × 8 min) were performed with PBST (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, and 0.1% Tween) and the membrane was blocked with 5% non-fat milk (Himedia, Mumbai, India) in PBST for 1 hr at RT. The blot was incubated with fibril specific OC antibody (1:1000 11

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dilutions) or oligomer specific A11 antibody (1:1000) for overnight at 4°C followed by three PBST washes. The membrane was then incubated with the anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilutions, cat. no. 401353, Calbiochem). Three washes were performed with TBST and blots were developed by exposing it to a chemiluminescent substrate (Super Signal West Pico, Pierce). β-tubulin was detected from the cell lysates as a loading control. Normalization of intensity values was done by dividing sample intensity by the loading control intensity to obtain a relative value. Three independent sets of experiments were performed. Statistical analysis Statistical significance in the present study was calculated by one-way ANOVA followed by Student-Newman-Keuls Multiple Comparison post hoc test, *P ≤0.05, **P≤0.01, ***P≤0.001; NS P>0.05.

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Results A53E mutation delays aggregation and shows similar cytotoxicity as of WT α-Syn in vitro. Previously our group and few other studies have reported that A53E aggregates slowly as compared to WT α-Syn.14-16 In the present study, we have performed in vitro aggregation kinetics along with cellular toxicity for A53E and WT α-Syn. Aggregation kinetics was performed using 300 µM protein at 37°C, in Echo Thermo model RT11 rotating mixture (Torrey Pines Scientific, USA), with slight agitation (~ 20 rpm). At regular intervals, ThT dye binding assay and CD spectroscopy was performed. Aggregation kinetics showed that A53E mutation leads to longer lag phase (~ 80 hrs), hence it takes longer time (~140 hrs) to form fibrils compared to WT (80 hrs) as revealed by ThT dye binding assay (Figure 1A). At the beginning of aggregation kinetics, both proteins showed a negative minimum near ~198 nm in CD, characteristic of a random coil conformation while at the end of aggregation, β-sheet rich conformation was observed (Figure 1B). Therefore, we find that the secondary structure was not affected by A53E mutation. AFM studies further showed that amyloid fibrils formed by A53E are slightly thinner compared to WT protein (Figure 1C, top panel). Both proteins formed indistinguishable oligomers when freshly dissolved proteins were injected to size exclusion chromatography (SEC) (Figure 1C, bottom panel). Oligomers eluted in fraction corresponding to the void volume (∼8 mL) and while monomer eluted at ∼15 mL fraction. We found the molecular weight of monomer as 57 kDa, as reported earlier.23 SEC profiles of oligomeric and monomeric fractions of both α-Syn WT and A53E mutant proteins were similar (Figure S2). The fibrils and SEC isolated oligomers were further examined for cellular toxicity. The data suggest that oligomers of WT and A53E obtained from SEC showed slightly higher cellular toxicity compared to their fibrils and monomers (Figure 1D). SEC isolated oligomers showed comparable toxicity with that of the oligomers found during aggregation as consistent with our previous report.14 The monomers, fibrils and oligomers by both WT and A53E protein therefore showed no difference in cytotoxicity (Figure 1D). Based on the present data and previous reports14 it can be concluded that A53E mutation delays α-Syn aggregation and its oligomers and fibril toxicities are comparable to WT in vitro. A53E shows dispersed cytosolic localization and is non-toxic to cells It is known that A53E mutation leads to early onset of PD in humans.7 The comparable cytotoxicity of A53E with WT intrigued us to look for the effects of A53E mutant overexpression in model cell line SH-SY5Y. WT and A53E α-Syn were cloned in mammalian pcDNA vector (Figure S1) and transfected to SH-SY5Y cell. The empty vector transfected cells were used as control. The transfected cells were analyzed for protein expression, localization, cytotoxicity and aggregate formation. When analyzed by immunofluorescence studies using α-Syn specific antibody, both cells overexpressing WT and A53E α-Syn showed dispersed staining with maximal localization in the cytosol and to a lesser extent in the nucleus. This indicates that A53E mutation in α-Syn does not affect its localization 13

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(Figure 1E). Consistent with this, previous studies showed that overexpression of α-Syn results in majorly diffuse staining with few punctate appearances in the cytoplasm of neuroblastoma cell lines.26-28 Further, immunofluorescence data suggest that ~80% of the cell population in both WT and A53E α-Syn were found to overexpress the protein (Figure 1F). The western blot analysis of cell lysate revealed that both A53E and WT α-Syn showed ~ five-fold overexpression compared to endogenous α-Syn expressed in SH-SY5Y cells (Figure 1G, 1H). The densitometry analysis further revealed the similar overexpression levels for WT and A53E α-Syn (Figure 1G and 1H) in SH-SY5Y cells. Western blot data further revealed that along with monomeric α-Syn, high amount of SDSresistant dimeric and tetrameric α-Syn were present (Figure 1G, 1H). However, the more intensity of monomeric band in WT and higher intensities of tetrameric band in A53E (Figure 1G) were not consistent among all replicates. To assess the cytotoxicity induced by overexpression of α-Syn, MTT assay 25 was performed. MTT data suggests that mere overexpression

Figure 1. Biophysical and cellular characterization of WT and A53E α-Syn. (A) Aggregation kinetics of WT αSyn and its mutant measured by ThT binding assay showing sigmoidal growth kinetics for both the proteins, n=2. (B) CD spectroscopy showing secondary structural changes from random coil to β-sheet during fibrillation of WT and A53E α-Syn. (C) Morphology of α-Syn fibrils formed at the end of aggregation kinetics (top panel) and oligomers isolated from SEC (bottom panel) examined by AFM, n=2. (D) Comparison of cytotoxicity of WT and A53E α-Syn monomers (25 μM), oligomers (25 μM) and fibrils (25 μM) by MTT assay after 24 hrs of

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Biochemistry

incubation, n= 4, Error bar depicts mean ± s.e.m. (E) Immunostaining of WT and A53E overexpressing cells with α-Syn antibody shows that both WT and A53E α-Syn are located mostly in the cytoplasm. (F) Bar graph depicting the percentage of transfected cells for vector, WT and A53E α-Syn, n= 3, Error bar depicts mean ± s.e.m. (G) Western blot of whole cell lysates obtained from cells transfected with vector, WT and A53E α-Syn. LB509 monoclonal antibody was used to detect α-Syn in the blot. β-tubulin expression was used as loading control. (H) Densitometric analysis of western blots showing ~ four-fold overexpression of α-Syn in transfected cells. After normalization, data has been plotted as relative to vector, n= 3, Error bar depicting mean ± s.e.m. (I) MTT assay for cell viability of transfected cells overexpressing WT and A53E α-Syn. No significant difference in viability was observed across the transfected cells, n= 3, Error bar depicts mean ± s.e.m. (J) Dot blot analysis using amyloid specific OC antibody and oligomer specific A11 antibody of vector, WT and A53E α-Syn overexpressing cells showing negligible amount of oligomers and fibrils in the whole cell lysate, n=2.

of WT and A53E α-Syn does not cause cytotoxicity (Figure 1I). Dot blot analysis was performed to examine the amyloid fibrils (using OC antibody) and oligomers formation (using A11 antibody) by WT and A53E overexpressing cells, which showed negligible immunoreactivity of both OC and A11. This suggests that overexpression of WT and A53E alone in cells does not result in oligomer and fibril formation, in the present experimental conditions (Figure 1J).

Figure 2. Cytotoxicity of WT and A53E α-Syn overexpressing SH-SY5Y cells in presence and absence of various stressors. (A) Dopamine and rotenone treated cells showing enhanced cell death in A53E overexpressing cells compared to the WT and vector controls. Cell death analysis quantified by Annexin V-FITC and PI binding assay using FACS. (B) Bar graph represents percentage cell viability determined by Annexin V-FITC and PI binding assay from FACS, n= 3, Error bars depict mean ± s.e.m.

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A53E overexpression shows increased cytotoxicity in cells compared to WT α-Syn in presence of dopamine and rotenone. Previously, it has been reported that point mutations of α-Syn protein are not sufficient to induce cytotoxicity or PD phenotypes in the model cell lines.12, 29 However, in presence of PD associated oxidative stressors and other environmental toxins, α-Syn familial mutant overexpression in cells gives the biochemical features observed in PD.30,

31

Therefore, we studied the effect of A53E

overexpression in the presence of toxins known to induce PD pathology. Various oxidative stress inducers and environmental toxins were used to study neurotoxicity associated with PD that include dopamine, rotenone, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1,1’-dimethyl-4,4’bipyridinium dichloride (paraquat), and iron.29, 32-34 In this study, we selected dopamine and rotenone as PD associated toxins. In case of dopamine, it has been shown that higher concentration of dopamine (~0.75- 1 mM) is found in cell bodies of dopaminergic neurons29, 35. Furthermore various studies have used ~200 nM and ~500 µM concentration of rotenone and dopamine respectively for cell-based studies29, 36-38. Dopamine induces oxidative stress by generating various dopamine quinones and free radicals in the cells while rotenone interferes functioning of complex I of mitochondria, which leads the production superoxide anion (O2.-) and hydrogen peroxide through dysfunctional mitochondria.39,

40

Moreover, dopamine is known to interact with α-Syn to form adduct which is

suggested to promote the toxic oligomers formation

. Cell death caused by A53E and WT α-Syn

41

overexpression in cells was analyzed in absence and presence of dopamine and rotenone. To analyze cell death quantitatively, we used Annexin V-FITC and PI binding assay followed by FACS analysis. This assay is based on the fact that during apoptosis, phosphatidylserine shuttles from inner leaflet of plasma membranes to the outer leaflet. Annexin specifically binds to this phosphatidylserine of an apoptotic cell.42 PI fluorescence represents the necrotic population of the cells.43

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Biochemistry

Figure 3. Reactive oxygen species generation by WT and A53E α-Syn overexpressing cells using dihydroethidium staining. The data shows up-regulation of ROS in the presence of various stressors in A53E αSyn overexpressing cells compared to WT and vector control. In each image, stained cells (red) indicate ROS positive cells. Under each condition, ~ 500 cells were analyzed and the final data was plotted as percentage of ROS positive cells (right panel), n= 3, Error bar depicting mean ± s.e.m.

The FACS data suggest that cells overexpressing A53E treated with dopamine showed ~79% cell death, majorly in early apoptotic phase (~ 72%); while WT and vector only cells showed ~28% and ~35% cell death, respectively. Rotenone treatment induced ~65% cell death in A53E overexpressing cells, while it was ~30% and ~25% in WT and vector only cells, respectively (Figure 2A, 2B). Since rotenone is known to bind to serum proteins, we performed the toxicity assay using cells grown with media having lesser serum concentration (0.5%) at a specified concentration of rotenone. Untreated and cells treated with media having lesser serum concentration were kept as controls in each experiment, which showed comparable cytotoxicity (~ 13%) in all the samples (Figure 2A, B and Figure S3). Overall, the Annexin V-FITC and PI binding data suggest that A53E overexpression induced cell death upon dopamine and rotenone treatments compared to WT and only vector containing cells. Although we cannot assess the relative cell toxicity in the presence of dopamine and rotenone due to their different concentrations used in this study. However, the study showed higher 17

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toxicity induced by familial A53E mutation compared to WT α-Syn in cells in the presence of dopamine and rotenone. Important to note that majority of A53E mutant overexpressing cell population shifted to early apoptosis after treatment with dopamine and rotenone, indicating induction of programmed cell death cascade in these cells. However, we also observed that only WT or vector expressing cells showed cell death in terms of necrosis. The reason behind this phenomenon is yet unclear and would need further investigation. A53E α-Syn overexpressing cells show higher amounts of reactive oxygen species (ROS) compared to WT in the presence of dopamine and rotenone. Previously it has been shown that production of ROS plays a significant role in cell death associated with PD.39 To study the generation of ROS by cells overexpressing WT and A53E in the presence and absence of dopamine and rotenone, the cells were stained using dihydroethidium. Dihydroethidium passively enters into the cells and upon reaction with superoxide radicals generates 2hydroxyethidium, which intercalates with DNA in the nucleus.44 Therefore, more nuclear staining indicates more ROS generation in cells. The data showed that untreated cells overexpressing A53E and WT α-Syn, as well as only vector-transfected cells, did not show detectable amount of ROS (Figure 3 and Figure S4). In the presence of dopamine, ~ 80% cells of A53E, ~30% cells of WT and vector cells showed ROS generation (Figure 3). In the rotenone treated cells, ~ 60% of the A53E overexpressing cells produced ROS; while ~35% ROS positive cells were observed in WT and vector containing cells (Figure3). The above data suggest that cells overexpressing A53E are more sensitive to dopamine/rotenone and generate more ROS compared to cells overexpressing WT. Mutant A53E overexpressing cells show lower mitochondrial potential compared to WT cells. Mitochondrial dysfunction has been implicated to play a central role in neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, multiple system atrophy.45 It was shown that WT α-Syn overexpression in mice leads to mitochondrial fragmentation and subsequent development of PD phenotypes.10 Moreover, various PD associated toxins such as MPTP, rotenone, paraquat, maneb, and trichloroethylene have been shown to majorly affect mitochondrial functionality,46 which finally leads to apoptosis and cell death.47 To analyze if mitochondrial potential is compromised in WT and A53E expressing cells in the presence of various toxins, mitochondrial potential was measured using JC1 dye. JC1 is a cell permeable dye, which accumulates in the matrix of healthy mitochondria and forms an aggregate known as JC1 aggregate, which emits red fluorescence.48 If mitochondrial potential is lost or low, JC1 aggregate dissociates and comes out from the mitochondria into the cytosol as free JC1 monomers, which emits green fluorescence.48 Cells expressing only vector, WT and A53E α-Syn were treated with dopamine and rotenone followed by staining with JC1 dye for 18

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Biochemistry

FACS analysis. Untreated cells stained with JC1 dye were used as control. . In untreated condition, we observed significant decrease in mitochondrial potential both in WT and A53E mutant compared to vector control. No significant difference in mitochondrial potential was observed between WT and A53E mutant. (Figure 4A, B and Figure S5). Dopamine treatment decreases mitochondrial potential considerably in A53E (~86%) followed by WT (~72%) and then in vector cells (~63%) (Figure 4A, B and Figure S5). Rotenone treatment followed the same trend as seen in dopamine, with vector cells showing ~ 50% decrease in MT potential. For positive control, cells expressing WT, A53E, and vector were treated with valinomycin, which is known to induce the loss of mitochondrial potential (Figure S5, top panel). Overall, the data suggest that cells overexpressing A53E show a significant loss in mitochondrial potential under dopamine and rotenone induced oxidative stress compared to WT.

Figure 4. Mitochondrial potential in cells overexpressing WT and A53E mutant. (A) JC-1 fluorescence based mitochondrial potential of cells treated with dopamine and rotenone were determined by FACS. Untreated cells were used as control. Red and green dots indicate cells with uncompromised and compromised mitochondrial potential respectively. (B) Bar graph represents quantification of percentage of cells showing MT potential (red dots) from three sets of experiments, n= 3, Bar graph depicts mean ± s.e.m.

Cellular aggregation and oligomer formation by WT α-Syn and A53E Previous studies have suggested that soluble oligomers formed during the process of α-Syn aggregation are responsible for neurotoxicity in PD pathogenesis.49, 50 It has been reported that α-Syn aggregates formed in the presence of PD associated toxicants in cells showed classical phenotypes of Lewy bodies.30,

31, 51

Further, rotenone and FeCl2 treatment have been shown to induce α-Syn

aggregates in the COS-7 and BE-M17 cell lines, respectively.31, 51 In this study, α-Syn aggregation was examined in cells via immunostaining with the phosphoserine 129 α-synuclein (p-α-Syn) specific antibody, which detects intracellular phosphorylated α-Syn aggregates. Untreated cells of WT and 19

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A53E showed negligible staining of p-α-Syn in the cytoplasm (Figure 5A, 5D). Further, upon rotenone treatment, immunostaining data suggests punctate staining for phosphorylated α-Syn for WT and A53E expressing cells (Figure 5A). However, the punctate formation was observed more in WT expressing cells compared to A53E α-Syn expressing cells (Figure 5D). Both WT and A53E α-Syn overexpressing viable cells showed lesser number punctate of p-α-Syn under dopamine treatment. Vector cells stained with α-Syn antibody did not show p-α-Syn staining under untreated and treated (dopamine and rotenone) conditions (Figure S6). The data suggests that in these treated conditions, WT α-Syn preferably formed punctate structures compared to A53E. This data is consistent with previous studies which showed more aggregation by WT α-Syn in the cells as compared to A53E mutant.15 To further study the formation of amyloid and oligomers, immunoblot assay was performed using OC and A11 antibody. OC and A11 antibodies were first verified using pure WT α-Syn protein oligomer, monomer and fibrils. Data suggest that A11 only binds to oligomers and OC only bind to fibrils. A11 and OC antibody do not bind to monomeric protein (Figure 5B). In experimental condition, cells were treated with dopamine and rotenone and then the cell lysate was used for dot blot assay using OC and A11 antibody. In the dot blot assay, an equal amount of protein from whole cell lysate was spotted onto the nitrocellulose membrane and then it was probed with A11 and OC antibody. β-tubulin staining done to ensure equal loading of protein. A11 and OC blot intensity was represented after normalization with intensity of β-tubulin. Positive immunoreactivity was observed against OC antibody for cell extracts of both WT and A53E (Figure 5C, 5E). However, OC signal intensity of A53E and WT was comparable. When we performed dot blot using the A11 antibody, we observed that A53E showed positive immunoreactivity in dopamine and rotenone treated conditions (Figure 5C). However, minimal A11 immunoreactivity was observed for WT α-Syn as well as the vector containing cells. Untreated and less serum treated vector, WT and A53E α-Syn overexpressing cells showed very less immunoreactivity to OC and A11 antibody (Figure 5C and Figure S7). This data suggest that A53E in all the treated conditions produces a significantly higher amount of oligomers compared to WT protein, which might cause increased oxidative stress, mitochondrial dysfunction, and cell death. Indeed, previously it has been shown by several studies that α-Syn A11 immunoreactive oligomers are associated with enhanced cytotoxicity in rat model system49, C. elegans and Drosophila melanogaster50 and has also been shown to induce mitochondrial dysfunction in mice.52

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Figure 5. α-Syn aggregation in WT and A53E α-Syn overexpressing cells. (A) Phosphoserine 129-α-Syn immunostaining of dopamine and rotenone treated cells showing more aggregate formation in WT cells (more punctate) as compared to A53E overexpressing cells. Scale bars are 10 µm, n=3. (B) Dot blot assay with oligomer, monomer and fibrils made from purified α-Syn protein showing binding specificity and crossreactivity of A11 and OC antibodies, n=2. (C) Dot blot of dopamine and rotenone treated cells showing more oligomer formation in cells overexpressing A53E compared to WT. Equal amounts of whole cell lysate of WT and A53E expressing cells were spotted on the nitrocellulose membrane and then probed with amyloid specific OC, oligomers specific A11 and β-tubulin antibody, n=3. (D) Bar graph showing average number of p- α-Syn punctate per cell, error bars depict mean ± s.e.m. (E) Bar graph showing densitometry analysis of dot blots. A11 and OC intensity is normalized with intensity of β-tubulin (loading control). Error bar depict mean ± s.e.m.

Rotenone accelerates while dopamine delays the aggregation of WT and A53E α-Syn. To further analyze the effects of these toxicants (dopamine and rotenone) on the aggregation of WT αSyn and A53E in vitro, 300 M of low molecular weight (LMW) form of both proteins were incubated in the presence and absence of these toxicants (150 µM) at 37°C with slow agitation. We have selected toxins:protein (0.5:1) molar ratio based on the previous studies which have used equimolar and sub-equimolar concentration of toxins and protein for studying the aggregation of αSyn in vitro.41, 53 The aggregation was monitored using ThT binding assay and the conformational 21

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transition was monitored by CD spectroscopy. ThT dye generally binds to amyloids and produces a strong fluorescence at 480 nm but it does not bind to the monomers. ThT fluorescence assay for aggregation kinetics showed sigmoidal growth curve for both WT and A53E α-Syn in the presence and absence of rotenone and dopamine with three distinct phases of aggregation (lag, log and stationary phases) (Figure 6A).14 The lag time of aggregation kinetics was further calculated using previously published protocol.21 The data showed that A53E α-Syn alone took longer time (lag time ~84 hrs) (Figure 6A, B) for aggregation compared to WT protein (lag time ~57 hrs) (Figure 6A, B), consistent with previous studies.14 WT in the presence of dopamine and rotenone showed lag time of ~99 hrs, and ~8 hrs, respectively; while in identical conditions, A53E showed a lag time of ~133 and ~33 hrs (Figure 6B), respectively. Therefore, the data suggests that in the presence or absence of toxicants, A53E showed delayed aggregation kinetics compared to WT. The CD data further showed the structural for conversion from random coil to β-sheet, which was in the presence of rotenone and delayed in presence of dopamine for both the proteins (Figure 6C). Overall aggregation kinetics data suggest that rotenone accelerates whereas dopamine delays the aggregation of WT and A53E proteins, which was found to be consistent with previous reports 30, 53.

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Figure 6. In vitro aggregation and amyloid fibril formation by WT and A53E α-Syn.(A) Aggregation kinetics of WT and A53E α-Syn measured by ThT binding assay (Y-axis represents normalized ThT fluorescence), n=2. (B) Lag time for aggregation kinetics of WT and A53E α-Syn in absence and presence of stress inducers, n=2, error bars depict mean ± s.d. (C) CD spectra showing secondary structural changes from random coil to β-sheet during fibrillation for WT and A53E α-Syn in the absence and presence of dopamine and rotenone, n=2. (D) TEM images of fibrils of WT and A53E α-Syn in the absence and presence of dopamine and rotenone at the end of aggregation kinetics showing the appearance of fibrillar morphology, n=2. Scale bar is of 200 nm. Magnified area is shown respective inset.

The morphology of the end products of aggregation kinetics was analyzed by TEM. Both the proteins in absence and presence of rotenone and dopamine showed amyloid fibrils at the end of aggregation (Figure 6D). In presence of rotenone fibrils formed were thinner for both proteins. (Figure 6D and Figure S8). The toxicity of these fibrils was further analyzed using MTT assay (Figure S9). The data 23

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showed A53E fibrils formed in presence of dopamine and rotenone show significant toxicity compared to WT fibril formed under the same condition. Based on present observation, we concluded that along with the increased population of oligomers due to slow aggregation of A53E might be responsible for a more toxic response by A53E mutant compared to WT α-Syn.

Discussion The major pathological hallmark of Parkinson’s disease (PD) is the presence of Lewy bodies (LBs) and Lewy neurites (LNs) in the neurons of the substantia nigra of PD affected brain.4, 5 Based on this observation, it was hypothesized that α-Syn aggregation is the key pathogenic event of PD pathogenesis.54 The SNCA gene duplication/triplication8 and point mutations6, 7 are associated with an autosomal familial form of PD, which further supports the role of α-Syn in PD pathogenesis. Further, the aggregation of α-Syn is strongly linked with PD pathogenesis was convincingly established using animal model overexpressing α-Syn55-57, cell-based

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and in vitro studies.14, 59-61 However, the exact

nature of aggregated species responsible for dopaminergic cell death is not yet established. Recent several studies have suggested that oligomers formed during α-Syn aggregation are more potent neurotoxic species compared to mature fibrils.49, 50 Previously, it was suggested that higher rate of oligomer formation is the shared property of familial α-Syn mutations based on in vitro studies of A30P and A53T mutations.23 Recently three new α-Syn mutations were discovered, which are associated with familial PD, where H50Q mutation accelerated and A53E delayed the α-Syn aggregation.

14, 62, 63

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and the other two mutations, G51D

Both of the slow aggregating new mutants however

did not show faster rate of oligomer formation as shown by A30P.23 The most recent mutation of αSyn associated with familial PD is A53E.7 Our in vitro study showed that structure and toxicity of A53E at monomeric, oligomeric and fibrillar forms are similar as of WT protein (Figure 1B, 1C and 1D). Previous cell-based studies also showed no differences in the toxicity of WT and A53E mutants when overexpressed and studied in the cells.15 We hypothesized that the A53E mutation might increase the susceptibility to various PD associated toxins causing early-onset PD. To study this, we overexpressed WT α-Syn and its A53E mutant in SH-SY5Y cells and toxicity data showed both WT and mutant expression did not cause any cellular toxicity (Figure 1I) with present levels of protein expression. However, we do not negate the toxicity at the different level of α-Syn overexpression. We further studied the effect of PD associated toxins (dopamine and rotenone) on the cells overexpressing WT and A53E α-Syn. Dopamine and rotenone were previously shown to induce PD phenotype either in cell-based studies or in an animal model of PD.29, 32, 64, 65 Dopamine is shown to form adduct with αSyn and promote toxic oligomer formation41; whereas rotenone is known to inhibit the mitochondrial complex 1, induce the ROS generation and cell death66. Interestingly, the addition of dopamine and rotenone increased the toxicity of SH-SY5Y cells overexpressing A53E α-Syn compared to vector 24

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control and WT expressing cells (Figure 2), suggesting only α-Syn overexpression might not play a significant role for causing the cellular toxicity. In this context, dopamine and rotenone have been shown to induce more toxicity in other α-Syn mutants (A30P, A53T and E46K) compared to cells overexpressing WT.64, 65 We further studied the mechanisms of cellular toxicity using these α-Syn overexpressing cells. It has been shown that increased ROS production in cells either due to α-Syn aggregation or other factors plays a significant role for cell death occur in PD.

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We studied the ROS

production by cells expressing WT and A53E in presence and absence of dopamine and rotenone and we observed higher production of ROS in cells expressing A53E compared to WT and vector control cells. This clearly suggests that increased ROS production by A53E overexpression might be associated with increase in cell death (Figure 3). Consistent with this observation, previous studies of cells overexpressing α-Syn or its mutants (A53T, A30P) showed that the addition of dopamine or rotenone elevates ROS production leading to toxicity in cells.67-69 We further studied whether ROS production is associated with dysfunctioning of the mitochondria as impaired mitochondrial potential is also linked to PD pathogenesis.45,70 Cells overexpressing WT and A53E α-Syn showed significantly reduced mitochondrial potential, especially when treated with dopamine and rotenone (Figure 4). Although, without any treatment, cells overexpressing both the proteins showed reduced mitochondrial potential but without significant cell death (Figure 2). This suggests that extent of reduction in mitochondrial potential might be an important factor for cell death. Further, in the presence of dopamine and rotenone, cells overexpressing A53E showed more compromised mitochondrial potential compared to WT (Figure 4). The cellular data therefore suggest that with treatment of dopamine and rotenone, A53E overexpressing cells showed enhanced toxic responses such as ROS generation and compromised MT potential compared to WT, which might cause the higher toxicity in A53E cells. To find the cause of higher toxic response due to A53E mutation, we hypothesized that due to slow aggregation rate, A53E might produce higher amount of oligomers (or higher lifetime of oligomers) compared to WT protein during aggregation. Indeed, in presence of both dopamine and rotenone the A53E mutation showed less amount of phosphorylated α-Syn aggregates (punctate) compared to WT protein (Figure 5A, 5D) irrespective of their similar level of expression (Figure 1). Moreover, we observed more punctate formation in presence of rotenone compared to dopamine for both WT and A53E cells. These differences in staining pattern suggest that rotenone promotes the aggregation of α-Syn 53, 65; whereas dopamine promotes the nonfibrillar aggregates in the cells.30 Indeed, our dot blot assay of A53E cell lysate by fibril17 and oligomers specific antibodies18 showed that irrespective of similar amount of OC immunoreactivity, much higher A11 immunoreactive oligomers were observed when treated with dopamine as compared to rotenone treatment (Figure 5C, 5E). Although for unknown reasons, this difference is not apparent in WT cells. Previously, it has been shown that both dopamine and rotenone interact with α-Syn and modulate the α-Syn aggregation.30, 41, 53 Our in vitro study also showed that rotenone accelerated; whereas dopamine 25

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delayed the aggregation of WT and A53E proteins. However, higher lag time of aggregation (slow) by A53E as compared to WT in all conditions suggest that irrespective of toxins, more oligomers might accumulate for the A53E mutant. Consistent with this ThT kinetics, we also observed less amount of phosphorylated α-Syn aggregates (Figure 5A, 5D) and more A11 positive oligomers (Figure 5C,5E) in presence of both dopamine and rotenone in A53E cells compared to WT protein. However, irrespective of opposite modulation property, how both dopamine and rotenone produce a similar level of OC immunoreactive species in cells (Figure 5C, 5E) is currently unknown. Although dot blot assay showed that both the protein produced similar amount of fibrils, the immunofluorescence study using pSer-129 showed different intensities. Observed discrepancy in amount of fibrils analyzed by pSer129- α-Syn immunostaining and OC dot blot assay could be due different specificity of OC and phospho α-Syn antibody used in this study. OC antibody specifically detects amyloid fibrils of proteins; however phospho-α-Syn antibody specifically detects only phosphorylated (Serine 129) αSyn and might also recognize other forms of phosphorylated (Serine 129) α-Syn. Since different techniques (dot blot analysis and immunofluorescence) have been employed for measuring amyloid formation; the results from these tests might not be directly correlated. Further, the toxicity and amount of oligomers produced in cells do not correlate well as A53E cells in presence of rotenone showed high toxicity, irrespective of low oligomers production compared to dopamine treatment (Figure 2 and Figure 5C, 5E). This indeed suggests that rotenone might play a dual role as a complex 1 inhibitor

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as well as a promoter of α-Syn aggregation and oligomer

formation. Whereas dopamine might promote the higher amount of oligomer formation, which could be the sole source of its toxicity. Further, we also suggest that dopamine/rotenone promotes more oligomers formation in A53E due to its slow aggregation potential where a higher amount of monomers are available (for a longer time) for adduct formation by dopamine or for interacting with rotenone leading to oligomer formation in comparison to WT protein. Therefore, the present study suggests that A53E mutation in the presence of dopamine and rotenone formed higher amount of oligomers, lead to more ROS generation and compromised the mitochondrial potential, which may cause higher cellular toxicity compared to WT α-Syn. This study might help to understand the role A53E mutation in early-onset PD.

Abbreviations α-synuclein (α-Syn); Circular dichroism (CD); Atomic force microscopy (AFM); Alzheimer’s disease (AD); Parkinson’s disease (PD); Size exclusion chromatography (SEC); Reactive oxygen species (ROS), Mitochondria (MT) and Low molecular weight (LMW). Supplementary Material 26

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Supporting Information contains supplementary figures S1 to S9. Acknowledgments The authors wish to acknowledge Central SPM Facility (IRCC, IIT Bombay) for AFM imaging and CRNTS (IRCC, IIT Bombay) for Flow Cytometry facilities and BSBE central facility for confocal microscopy. The authors also would like to thank the ‘Parimal and Pramod Chaudhari Laboratory for Cell Culture’ (BSBE, IIT Bombay) for providing the cell culture facility. We would also like to thank Mrs. Madhura M. Joshi and Mr. Sudesh Roy for their valuable help in flow cytometry analysis and confocal microscopy respectively. We also acknowledge the kind gift of A11 and OC antibodies by Prof. Charles G Glabe, UC Irvine, USA. Conflict of Interest statement. Authors declare no conflict of interest. Funding The work was supported by grants from Department of Biotechnology (DBT), Government of India [BT/PR14344/Med/30/501/2010],Council of Scientific and Industrial Research (CSIR), Government of India [37(1648)/15/EMRII] and Industrial Research & Consultancy Centre (IRCC) Indian Institute of Technology Bombay under scheme [12IRCC001] - Mid stage financial support for TAP/RAP PhD Students. The authors declare no competing financial interest.

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Figure 1. Biophysical and cellular characterization of WT and A53E α-Syn. 130x171mm (300 x 300 DPI)

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Figure 2. Cytotoxicity of WT and A53E α-Syn overexpressing SH-SY5Y cells in presence and absence of various stressors. 635x354mm (150 x 150 DPI)

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Biochemistry

Figure 3. Reactive oxygen species generation by WT and A53E α-Syn overexpressing cells using dihydroethidium staining 301x232mm (300 x 300 DPI)

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Figure 4. Mitochondrial potential in cells overexpressing WT and A53E mutant. 535x287mm (300 x 300 DPI)

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Biochemistry

Figure 5. α-Syn aggregation in WT and A53E α-Syn overexpressing cells. 129x245mm (300 x 300 DPI)

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Figure 6. In vitro aggregation and amyloid fibril formation by WT and A53E α-Syn. 135x232mm (300 x 300 DPI)

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51x37mm (300 x 300 DPI)

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