The GT1-1 cells were obtained from the laboratory of Professor Krister Kristensson, Department of Neuroscience, Karolinska Institutet Stockholm, Sweden, following an MTA agreement with Dr. Pamela Mellon of the University of California, San Diego, USA. This line is often used in prion biology and it is established from gonadotropin hormone-releasing neurons immortalized by genetically targeted tumorigenesis in transgenic mice . The SH-SY5Y cell line is a thrice-cloned sub-line of SK-N-SH cells, which were originally established from a bone marrow biopsy of a neuroblastoma patient with sympathetic adrenergic ganglial origin . GT1 cells were seeded in 10-cm plates containing 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) culture media, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. SH-SY5Y normal cells were cultivated in 10-cm plates, containing 10 mL of minimal EMEM: Ham F12 (1:1) culture media, supplemented with 15% FBS, 1% non-essential amino acids, 0.5% L-glutamax, 1% penicillin-streptomycin and 1% G-418 (only for SH-SY5Y wild-type cells transfected for overexpressing α-syn). The cells were grown at 37°C in 5% CO2 to 95% confluence for 1 week before splitting at 1:10 for further cultivation.
The cytotoxic effect of α-syn fibrils was assessed by measuring cellular redox activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Briefly, 100,000 SH-SY5Y and GT1 cells/well were cultured in a 96-well microtiter plate and treated with α-syn oligomers, short fibrils and long fibrils (suspended by vortexing). Following a 24-h incubation, the cytotoxic effect was assessed by measuring cellular redox activity, following the manufacturer’s (Sigma) instructions.
Expression and purification of recombinant human α-syn
Expression and purification of recombinant human α-syn were performed in accordance with the method previously described . Briefly, the human α-syn gene was cloned and expressed in pET11a vector using BL21 (DE3) E. coli strain. Expression of α-syn was obtained by growing cells in Luria-Bertani broth medium with 100 mg/mL ampicillin at 37°C until an O.D.600 of about 0.6, followed by induction with 0.6 mM isopropyl β-D-thiogalactoside (IPTG) for 5 hours. The protein was purified according to the method of Huang et al..
α-syn amyloid preparations
All solutions were sterilized by filtration through a 0.22-μm filter prior to each assay run. Reactions were prepared in a 96-well black plate (BD Falcon), and each well contained 200 μL of reaction solution [1.5 mg/mL recombinant human α-syn, 100 mM NaCl, 10 μM Thioflavin T (ThT) in 20 mM Tris–HCl pH 7.4]. Each sample analysis was performed in fifteen replicates. Each well contained one 3-mm glass bead (Sigma). The plate was covered with sealing tape (Fisher Scientific), incubated at 37°C under continuous shaking, and read on SpectraMax M5 fluorescence plate reader (Molecular Devices) by top fluorescence reading every 5 min at excitation of 444 nm and emission of 485 nm.
AFM analysis was performed in accordance with the method previously described . Three to five μL of fibril solution was deposited onto a freshly cleaved piece of mica and left to adhere for 30 min. Samples were then washed with distilled water and blow-dried under a flow of nitrogen. Images were collected at a line scan rate of 0.5-2 Hz in ambient conditions. The AFM free oscillation amplitudes ranged from 25 nm to 40 nm, with characteristic set points ranging from 75% to 90% of these free oscillation amplitudes. AFM data were analyzed with Gwyddion (gwyddion.net) and SPIPTM (http://www.imagemet.com) software.
α-syn amyloid solution for cell infection
The α-syn amyloid solution was transferred from the 96-well plate into a 1.5 mL Eppendorf tube and collected under sterile conditions. The solution was ultracentrifuged at 100,000 g for 30 min at 4°C (Beckman Coulter). Pellets were resuspended in 1X PBS and then sonicated (Branson 2510) for 5 min prior to adding to the cultured cell plate.
α-syn fibril infection in cell lines
Three hundred μg of α-syn amyloids was added to GT1 and SH-SY5Y cell plates (10 cm-plate) and exposed in the cell culture media for 7 days before the next splitting and media change. Cells were split and maintained for six additional passages. Cell lysates were collected at each passage for Western blotting and immunofluorescence studies. For immunofluorescence of amyloids internalization, cells were cultured in 12-well plates with coverslips; 30 μg of α-syn amyloids was added to the cell culture media and incubated for 7 days.
Detection of α-syn aggregates in infected cells and analysis by Western blotting
The total protein content of samples was measured by bicinchoninic acid assay (BCA) (Pierce). Fifty μg of cell lysates protein was used and 5X loading buffer was added in a 1:5 ratio. The samples were boiled for 5 min at 100°C, loaded onto a 10% Tris-Glycine SDS-PAGE gel, and transferred overnight onto Immobilon P PVDF membranes (Millipore). Membranes were blocked by 5% nonfat milk, incubated with 0.4 μg/mL rabbit polyclonal anti-α-syn antibody (Santa Cruz) followed by incubation with goat anti-rabbit IgG F(ab)2 fragment conjugated with horseradish peroxidase. Blots were developed with the enhanced chemiluminescent system (ECL, Amersham Biosciences) and visualized on Hyperfilm (Amersham Biosciences). To analyze α-syn aggregation in cell samples, cells were scraped in TBS buffer containing 1% Triton X100, protease cocktail inhibitors and phosphatase inhibitor. After sonication (Sonicrep 150) with 10 amplitude microns for 3 times (30 seconds of sonication and 30 seconds intermediate stop) cells were centrifuged at 2000 RPM for 5 min before the protein was quantified. Fifty μg of sample was centrifuged at 100,000 g for 30 min. Supernatant (in term S-TX) was collected and the pellets were resuspended in 2% SDS. Samples in 2% SDS were centrifuged, supernatant (S) and pellet (P) were collected. All the fractions were added to loading buffer 5X in ratio 1:5 and prepared similarly for Western blotting.
Reverse transcribed–polymerase chain reaction
Total RNA was extracted from cultured cells using the Trizol® Reagent (Life Technologies) extraction method. From 1 μg of total RNA, cDNA was synthesized using 200 units MoMuLV-reverse transcriptase (SuperScrip™ III RT; Life Technologies), oligo (dT) primers and 1 μl 10 mM dNTP Mix in a final volume of 13 μl. Ten microliters of the 25-fold diluted solution was subjected to quantitative PCR analysis. The following primer pairs were used for α-syn: Hu_Syn_FW 5′-ATGGATGTATTCATGAAA-3′, Hu_Syn REV 5′-TTAGGCTTCAGGTTCGTA-3′. PCR amplification was conducted using the Phusion® High-Fidelity DNA Polymerase protocol, under the following conditions: initial denaturation at 98°C for 30s, 10s at 98°C, 30s at 45°C and 30s at 72°C for 25 cycles, followed by 10 min at 72°C. After amplification, 10 μl aliquots were electrophoresed in 1.5% agarose gel, followed by photographic recording of the gel stained with ethidium bromide.
Immunocytochemistry and ThS staining of α-syn fibril-infected cells
Cells on coverslips were washed with PBS and fixed with 4% paraformaldehyde, then washed twice, 15 min/time with PBS and blocked in blocking buffer [5% normal goat serum (NGS) in PBS + 0.3% Triton] for 1 hour. For Thioflavin S (ThS) staining, fixed cells were incubated with 0.025% of the fluorophore (Sigma) for 8 min and washed three times with 80% ethanol for 5 min each time, prior to the antibody incubations. Fluorescence immunocytochemistry was performed using the primary and secondary antibodies listed in Additional file 6: Table S2. Primary antibodies were made up in 1% blocking buffer and PBS. After incubation, the cells were washed 5 times for 5 min/time with PBS; secondary antibodies were incubated in 1% blocking buffer and PBS for 45 min. Finally, cells were washed 5 times, 5 min/time with PBS, and counterstained with DAPI to reveal nuclei, then mounted in Vectashield Mounting Medium. Cell coverslips were stored at 4°C for confocal fluorescence microscopy. The fluorescence was measured in at least 10 randomly chosen observation fields for each experimental condition using Leica SP5 confocal laser-scanning microscope. The increase in fluorescence was measured calculating CORRECTED TOTAL CELL FLUORESCENCE (CTCF) using the formula: CTCF = Integrated density – (Area of selected cell X Mean fluorescence of Background readings) using the ImageJ 1.47 Software (NIH). Quantification experiments were carried out independently at least three times; more than 150 cells were counted for each condition. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software (San Diego, CA). Data are shown as mean ± standard deviation (SD).
Immunofluorescence microscopy analysis of phosphorylated aggregates
All dopaminergic neuroblastoma SH-SY5Y cells were grown on ibidi dishes (Biovalley) for microscopy. After washing in PBS, cells were fixed using 4% (w/v) paraformaldehyde (Sigma) in PBS for 15 min and permeabilized with 0.01% Triton X-100 (Sigma) in PBS for 3 min, washed and blocked in 2% Bovine serum albumin (Sigma) in PBS for 20 minutes. Cells were immunostained using anti-α-syn (Santa Cruz Biotechnology, INC) and anti-phospho S129 α-syn (Abcam) followed by secondary antibody coupled to Alexa 488 (Invitrogen). Cells were mounted in Aqua Poly/Mount (Polysciences) and pictures were acquired using white field Axiovision microscope (Zeiss) with a 63x objective. Quantification experiments were carried out independently at least three times; more than 150 cells were counted for each condition. Individual differences were assessed using individual student’s t-tests in GraphPad Prism software (San Diego, CA). Data are shown as mean ± standard error of the mean (SEM).