2.1 Recombinant protein preparation
For the production of recombinant aSyn protein from E.coli we followed established procedures [8, 9]. For purification of recombinant aSyn, the protein was first filtered through a 0.22 µm syringe filter and then applied to Hi Trap Q HP anion exchange column (GE Healthcare Life Sciences) equilibrated with 25 mm Tris, pH 7.6. aSyn was eluted by running a linear gradient of elution buffer (3M NaCl in 25 mm Tris, pH 7.6) up to 30 % elution buffer. aSyn-enriched fractions (analysed by SDS-PAGE and Coomassie staining/destaining) were collected, combined and concentrated with 3 kDa molecular weight cut-off Amicon Ultra Centrifuge filters according to Manufacturer`s instructions. Proteins were further purified by Superdex 75 column (GE Healthcare Life Sciences) equilibrated with 25 mM Tris, pH 7.6, 100 mm NaCl. Pure Fractions corresponding to monomeric state (as determined by Native Gel, Coomassie staining/destaining) were combined, concentrated at a final concentration of 5 mg/ml, and stored at -80 ºC until further use. PFFs were prepared from the monomers according to standard protocols .
2.2. Generation of the inducible stable cell line for aSyn oligomerization based on Bimolecular Fluorescence Complementation (BiFC)
Naïve HEK293 cells were first transfected with the pTet-Off (Clontech) plasmid that express the regulatory protein tTA (tetracycline-controlled transactivator, tTA), which contain a neomycin-resistance gene for the selection. Selection with G418 (500µg/ml; Millipore) was used for the selection of resistant colonies. Inducibility of resistant colonies was determined after transient transfection with the firefly luciferase vector pTRE-Tight-Luc, in presence or absence of Doxycycline (Dox, 2 µg/ml; Sigma-Aldrich). A clonal HEK293-pTet-Off cell line was selected and used for the generation of the different pTRE stable cell lines (pTRE-Tight-VN-aSyn and pTRE-Tight-aSyn-VC). Transient co-transfection for the pTRE-Tight-VN-aSyn and pTRE-Tight-aSyn-VC vectors used to verify the formation of the BiFC oligomers. The VN and VC aSyn constructs were generated from initial plasmids containing the VN and VC fused aSyn proteins, digested with the SnaBI/XbaI enzymes and cloned into the pTRE-Tight vector (Clontech) digested before with SmaI/XbaI. The positive clones were verified by DNA sequencing. Subsequently, the VN and VC fused aSyn vectors were co-transfected with a linear selection marker for Hygromycin (Clontech). Selection was performed with 50 µg/ml Hygromycin (Roche). Resistant clones were picked and aSyn was examined by immunoblotting in the presence or absence of Dox (2 µg/mL added for more than 5 days) using a specific antibody against aSyn (anti-aSyn Syn-1 mouse, BD Transduction Laboratories).
2.3. Generation of HEK293-aSynEGFP stable cell line
HEK293 were transfected with a plasmid encoding human WT aSyn fused to EGFP, at the C-terminus, driven by the cytomegalovirus (CMV) promoter. The plasmid contained a selection marker for the antibiotic geneticin (G418) which was used for the selection of the stable transformants. Protein expression was confirmed by western blot analysis and fluorescence microscopy. A clonal HEK293-aSynEGFP cell line was selected and used for subsequent experiments.
2.4. Construction of lentivirus vectors
Full-length human aSyn was PCR amplified and sub cloned into a 3rd-generation lentiviral plasmid with hUbC-driven EGFP, where the original GFP cassette has been replaced by the aSyn gene including the Kozak sequence. pFUGW vector containing only the GFP cassette was used for control experiments (Addgene) .
2.5. Cell culture and cell treatment
HEK293 cells were maintained in DMEM media supplemented with 10% Fetal Bovine Serum Gold (FBS) (PAA) and 1% Penicillin-Streptomycin (PAN). The cells were grown at 37°C in an atmosphere of 5% CO2. For the seeding experiments, aSyn monomers and PFFs were diluted in PBS, fragmented by sonication in case of PFFs  and then added to the cells at final concentration of 100 nM. Control cells were exposed to vehicle only (PBS). Cells were further incubated for 48 h.
2.6. Primary cultures of cortical neurons
Primary cortical neurons were prepared from embryonic day E16-E17 mouse brains. Briefly, after dissection, the tissue was washed three times with Hank's Balanced Salt Solution (HBSS; Gibco) and digested by Trypsin (Gibco) for 15 minute at 37°C, followed by addition of 100 µl DNase I (10 mg/ml; Roche) and 100 µl FBS (Life Technologies), mixed by inverting and centrifuge at 500 rpm for 5 minutes. After centrifuge trypsin solution was removed and replaced with 1ml of FBS. Cortices then dissociated by mechanical trituration with glass pasteur pipettes. Dissociated cells were transferred in fresh culture medium (Neurobasal medium, Gibco), containing 2% B27 supplement (Gibco), 0.5 mM L-glutamine (200Mm Gibco), and 1% penicillin/streptomycin (Gibco) and plated in 24- or 6-well plates previously coated with Poly-L-ornithin (0,1mg/ml, Sigma-Aldrich) at a density of 250,000 cells/mL. Cultures at DIV5 were treated with recombinant aSyn (monomers and PFFs; final concentration of 100nM) and long-term incubated until DIV25. No further media changes or addition of recombinant protein was made until the end of the culture. After mild trypsinization with 0.5% Trypsin-EDTA for 5 minutes to remove the excess of unbound material, cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes at RT. Fixed cultures were immunostained and subsequently analyzed. In addition, cells were lysed following the Triton soluble/insoluble biochemical assay described below. To verify specificity of effects in our study, two batches of PFFs were used from two different labs. Protein concentration was estimated by the Bradford assay. Primary neuronal cultures were infected at DIV5 with the lentivirus pFUGW coding for WT aSyn or GFP.
2.7. SDS-PAGE and immunoblotting
After treatment, cells were washed with PBS and lysed on ice in radio-immunoprecipitation assay buffer (RIPA) (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% Sodium-Dodecyl-Sulphate (SDS), 1% Nonidet P40, 0.5% Sodium-Deoxycholate, protease inhibitors, Roche). Lysates were centrifuged at 10000 rpm and 4°C for 10 min and post-nuclear supernatants were kept. Protein concentration was determined using the Bradford assay (Bio-Rad). All samples were measured in triplicate. For RIPA samples, equal protein amounts of denatured samples (5 minutes at 95°C in 5x protein sample buffer; 125 mM of 1 M Tris HCl pH 6.8, 4% SDS 0,5% Bromophenol blue, 4 mM EDTA 20% Glycerol 10% β-Mercaptoethanol) were subjected to SDS-PAGE on 12% separating gels with 7% stacking gels, using Tris-Glycine SDS 0.5% running buffer (250 mM Tris, 200 mM Glycine, 1% SDS, pH 8.3). The SDS insoluble fractions were denatured at 42°C for 15 min. The transfer was carried out to 0.45 µm nitrocellulose membranes for 20 min per membrane at constant 25 mA in a semi-dry transfer chamber Trans-Blot® Turbo™ Transfer Solution from Bio-Rad (Bio-Rad). Membranes were blocked in 5% (w/v) skim milk (Fluka, Sigma-Aldrich) dissolved in 1xTBS-Tween (50 mM Tris (hydroxymethyl)-aminomethane (TRIS) supplemented with 0.05% (v/v) Tween-20) for 1 h at RT. Incubation with the primary antibodies (anti-aSyn Syn-1 mouse, BD Transduction Laboratories 1:1000; anti-pS129-α-syn, Rabbit Abcam ; mouse anti-β-actin, 1:10.000, Sigma) was performed overnight at 4°C in 5% Albumin Bovine Fraction V (BSA)/TBS-Tween. Secondary antibodies (anti-mouse and anti-rabbit IgG, 1∶10000 in TBS-Tween) were applied after three times washing in TBS-Tween, for 1 h at room temperature (RT). Membranes were visualized using Fusion Fx (Vilber Lourmat) with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore). Protein levels were quantified using ImageJ and normalized to the β-actin levels.
2.8. Triton soluble/insoluble biochemical analysis
HEK293 cells were treated with aSyn monomers and PFFs for 48 h, harvested with trypsin and washed with ice cold PBS and diluted 1:10 Trypsin. The cell pellet was collected and solubilized in 1% triton buffer (50 mM tris pH 7.6, 150 mM NaCl, 2 mM EDTA, 1% triton, protease and phosphatase inhibitors), followed by 30 min incubation on ice. Cell lysates were then centrifuged at 13.000g for 30 min at 4°C, and the supernatant was collected as Triton soluble fraction. The pellet was washed with ice cold PBS. Insoluble fractions were prepared by resuspending the pellets in 2% SDS soluble buffer (50 mM tris pH 7.6, 150 mM NaCl, 2 mM EDTA, 2% SDS, protease and phosphatase inhibitors), sonicated and centrifuged at 13.000g for 10 min after incubation for 30 min at RT. The collected supernatant represents the SDS soluble fraction. The protein concentration was determined by the Bradford protein assay according to the manufacturer’s instructions, and the samples were then subjected to Western blot analysis. In order to estimate the effect of the levels of aSyn on seeding, we ran all samples in the same blot, or in pairs (HEK293-EGFP with HEK293-aSynEGFP, and HEK293 naive with HEK293-aSynBiFC).
2.9. Filter trap and Dot-blot assay
For the filter trap assay, equal protein amounts of recombinant aSyn monomers and PFFs were adjusted to equal volumes (diluted in PBS) and subsequently subjected to vacuum filtration through a 96-well dot blot apparatus (Bio-Rad) with an acetate cellulose membrane; immunoblotting (IB) was then performed with antibody against total aSyn (Syn-1). As a loading control experiment, the same samples were subjected to vacuum filtration through a 96-well dot blot apparatus with a nitrocellulose membrane followed by immunoblotting (IB) with Syn-1 antibody. Results shown are representative of 3 independent experiments.
2.10. Mitochondrial Fractionation
Mitochondria were isolated as previously described , with some modifications. All buffers were prepared fresh before the isolation and placed in ice at least 15 min before harvesting the cells. Briefly, cells were harvested by trypsinization, pelleted and washed with cold-PBS buffer twice at 600 g for 5 min at RT. After the last wash, PBS was discarded and one volume of the cell pellet of IB 0.1x buffer (NaCl 2.5 mM, MgCl2 0.5 mM, TRIS 3.5 mM pH = 7.8) was added. The cells were resuspended gently and homogenized with 15 strokes using a Heidolph D-91126 Type: RZR1 homogenizer at 1500 rpm. The sample was transferred to Eppendorf 1.5 mL tubes and 1/10 of the initial cell pellet volume of IB 10x Buffer (NaCl 0.25 M, MgCl2 50 mM, TRIS 0.35 M pH = 7.8) was added. A microcentrifuge was used to pellet the debris, unbroken cells and nuclei at 1200 rpm for 3 min at 4°C. The supernatant that contained Mitochondria and Cytoplasmic fraction was collected. An additional centrifuge step was performed at 1200 g for 3 minutes at 4°C to obtain a cleaner fraction and avoid contamination of “heavy material”. The supernatant was collected again. Finally, the collected supernatant was centrifuged at 1500 rpm for 2 min at 4°C. The obtained pellet was the mitochondrial fraction and the supernatant the cytoplasmic fraction, respectively. After collection of the cytoplasmic fraction, the mitochondrial fraction was resuspended in RIPA buffer. Protein concentration was determined by the Bradford protein assay and the samples were then subjected to Western blot analysis.
For ICC, cells were washed with PBS and diluted 1:10 Trypsin, fixed with 4% paraformaldehyde (PFA) for 20 minutes at RT, followed by a permeabilization step with 0.5% Triton X-100 (Sigma-Aldrich) for 20 minutes at RT. After blocking in 1.5% normal goat serum (PAA)/DPBS for 1 h, cells were incubated with primary antibody. Primary antibodies used were: anti-aSyn Syn-1 mouse, BD Transduction Laboratories (1:1000); anti-pS129-α-syn Rabbit Abcam (1:1000), incubated overnight at 4°C and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA) for 2 hours at RT. Nuclei were stained with 4′6′-diamidino-2-phenylindol (DAPI, Sigma-Aldrich) (1∶5000 in DPBS) for 10 minutes. After a final wash, coverslips were mounted by using Mowiol (Sigma Aldrich) and subjected to fluorescence microscopy. Images were analyzed using LAS AF v.2.2.1 (Leica Microsystems) software.
2.12. RT-QuiC assay
Real-time quaking induced conversion (RT-QuiC) was performed as a modification of previously proposed methods for different amyloid proteins [12, 13] and optimized in our lab. In brief, a reaction mixture with the following composition was made: 150 mM NaCl, 1 mM EDTA, 10 µM ThioT, 70 µM SDS, 10 µg/ml of monomeric aSyn. and fibrilar aSyn  at a final concentration of 0,1 µg/ml, 1 µg/ml, 10 µg/ml and 100 µg/ml in PBS buffer. 100 µl of this reaction mixture were pipetted in black 96-well plates (Corning Incorporated, Washington, USA) in triplicates. Additionally, cell lysates were added to the mixture to a final protein concentration of 0,1 µM. Finally, the plates were covered with sealing tape and incubated in a plate reader (41°C, Infinite M200 fluorescence plate reader, Tecan, Hamburg, Germany) for 250 amplification cycles (1 min orbital shaking at 432 rpm; 2 min incubation; measurement of fluorescence intensity at 480 nm). Endpoint fluorescent intensities were normalized by baseline values.
2.13. Statistical analyses
Statistical analyses were performed using the Student's t-test and 2-way ANOVA for comparisons of independent variables. The data are presented as mean ± standard deviations and represents results from at least 3 independent experiments. Differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.