All procedures were performed in accordance to the institutional committee for animal care of the School of Medicine, University of Sao Paulo (#0659/08).
Methodology employed for cell culture was a modification of the previously described protocol . Briefly, neonatal Lewis rats had their brains dissected and the areas containing hippocampus, locus coeruleus and substantia nigra were excised. After dissection, blood and epithelial cells were removed in sterile cold solution consisting of NaCl 120 mM, KCl 5 mM, KH2PO4 1.2 mM, MgSO4 1.2 mM, NaHCO3 25 mM, glucose 13 mM, pH 7.22. Subsequently, the tissues were cut into small pieces using a scissors and incubated with 0.05% trypsin (Gibco) at 37°C for 40 minutes in a water bath kept under agitation. Then, trypsin inhibitor (0.006%, Gibco) was added and the cells were mechanically dissociated using a Pasteur pipette, after the total decoupling the cell solution was centrifuged at 300 g for 5 minutes. The supernatant was discarded and cells were resuspended in Neurobasal A medium (Gibco) supplemented with Glutamax (Gibco) 0.25 mM, B27 (Gibco) 2%, L-Glutamine (Sigma) 0.25 mM and Gentamicin (Gibco) 40 mg/l.
Cells were plated into either 8-well glass slides or 35 mm petri dishes (Nunc), at the concentration of 1800 cels/mm2. Plates were treated the day before with poly-D-lysine 10 μg/ml (Sigma), and with fetal bovine serum 10% (Gibco) for 2 hours before plating the cells to facilitate adhesion. Cultures were kept in a humidified incubator with 5% CO2 at 37°C for nine days. Culture medium was changed three hours after plating the cells and every three days of cultivation.
Cell culture characterization
Cell cultures were washed in PBS, fixed in 50% methanol and 50% acetone for 10 minutes at -20°C, permeabilized with PBS containing 0.2% Triton for 30 minutes at room temperature. Unspecific binding sites were blocked with PBS containing 2% NGS (Vector Laboratories), 0.2% Triton and 4% bovine serum albumin (BSA, Sigma) for 30 minutes at room temperature.
Cells from substantia nigra and locus coeruleus were incubated with mouse polyclonal antibody against tyrosine hydroxylase (1/3000, Sigma) for 24 hours at 4°C, followed by incubation with anti-mouse immunoglobulin conjugated to FITC (Jackson, 1/120) for 45 minutes at room temperature protected from light. Hippocampal cultures were subjected to MAP2 immunolabeling (1/1000, Sigma) also at 4°C overnight followed by incubation with FITC-conjugated secondary antibody in order to identify the neurons present in cultures.
The slides were mounted with mounting medium containing DAPI (4',6-diamidino-2-phenylindole, Vector Laboratories) to visualize cell nuclei. Immunolabeled cells were analyzed using a fluorescence microscope (Zeiss) equipped with appropriated filters using a 40× lens. Quantification was done by comparing images taken of 16 fields of culture plate using filters to visualize the label generated by FITC and DAPI. Cell culture characterization was repeated twice.
Exposure to Rotenone
Rotenone (Sigma, USA) was prepared with DMSO (Sigma, USA) (stock solution of 1 mM) and diluted in culture medium applied to cell cultures from hippocampus, locus coeruleus and substantia nigra in concentrations of 0.5, 1, 10 and 25 nM for 48 hours. Control groups were exposed to less than 0.01% DMSO diluted in culture medium. Cells were then subjected to trypan blue staining in order to identify cell death; to immunocytochemistry for identification of protein aggregates containing hyperphosphorylated tau, alpha-synuclein and beta-amyloid; and protein extraction for western blot experiments.
Analysis of cell death
After exposure to rotenone, 10 μl of trypan blue stain solution (Gibco) which stains in blue the cytoplasm of cells with damaged plasma membrane, were added to the culture medium of cells. Immediately after the addition of trypan blue, the cells were examined under a microscope (Olympus) using an objective of 40× (400× magnification) and photographed to detect stained cells.
Identification of protein aggregates through immunocytochemistry
Cell cultures were fixed as described above and incubated for 24 hours at 4°C with either a mouse polyclonal antiserum against alpha-synuclein (Abcam, 4D6, Ab1903), or rabbit polyclonal antiserum against hyperphosphorylated tau (Sigma, Ser 199/202, T6819) or beta amyloid peptide (Abcam, Ab14220), the three antibodies were diluted 1/1000 in PBS containing 0.3% Triton X-100 (Sigma) and 0.5% BSA (Sigma). Cells were washed in PBS and incubated with biotinylated goat anti-rabbit or anti-mouse immunoglobulin both diluted 1/200 (Vector, USA) for 2 hours at room temperature. Cells were washed in PBS and incubated with an avidin-biotin peroxidase complex (both diluted 1/120, Vectastain, Vector) for 2 hours. Immunoreactivity was visualized after 10 minutes of reaction with 3-3'-diaminobenzidine tetrahydrochloride (DAB, Sigma) as a chromogen and H2O2 (0.01%, v/v, Sigma).
The occupied area (μm2) of beta amyloid peptide immunoreactivity in the hippocampus cultures was calculated by means of a KS 400 image analyzer (Kontron, Zeiss, Germany) linked to a CCD 72 camera (Dage; MTI, Michigan City, Ind, USA) mounted on a Zeiss microscope (40× objective). Nine randomly chosen fields were considered for the quantification. The procedures have been described in detail elsewhere .
Western blot analysis of protein aggregation
Cultured cells were homogenized in PBS, pH 7.4, containing 1% NP40, 0.5% sodium deoxycholate, 1%SDS, 1 mM EDTA, 1 mM EGTA and 1% protease inhibitor cocktail (Sigma). After centrifugation at 14000 rpm for 20 minutes, the resulting supernatant was fractionated by SDS-PAGE (10 μg of protein/lane) using a 12% tris-HCl gel at 100V for 1 h. Proteins were transferred to nitrocellulose membrane for 1 h at 100V.
Blots were incubated in blocking solution containing 5% milk/TBS-T during 1 h at room temperature followed by incubation with primary antibodies against alpha-synuclein (Abcam, 4D6, Ab1903) or hyperphosphorylated tau (Sigma, Ser 199/202, T6819) both were diluted 1/1000 in solution containing 3% milk/TBS-T, overnight at 4°C.
Horseradish peroxidase-conjugated secondary antibody incubations were performed at room temperature for 1 h with antibody anti-mouse 1/6000 (Amersham) or anti-rabbit 1/10000 (Amersham).
Development was done after 5-minute incubation with enhanced chemiluminescence reagent (Millipore) and exposure of membranes to ECL sensitive films (Hyperfilm ECL, Amersham Biosciences). After development, blots were incubated with anti-beta-actin antibody 1/1000 (Santa cruz, C4, sc-47778) during 1 h at room temperature, followed by horseradish peroxidase conjugated secondary antibody anti-mouse (Amersham) diluted 1/6000 for 1 hour also at room temperature and developed as previously described.
Density normalization was done by dividing the density of the bands relative to proteins of interest by beta-actin value. Films were quantified by optical densitometry using a system of image analysis (Imaging Research Inc., Canada, model M4/SK/ALU).
All the analyses were made using protein samples from control and treated cells that were fractionated in a same gel and transferred to a single membrane which was incubated with the antibody solution.
The presence of alpha-synuclein isoforms was confirmed in neonate and adult (6 months old) Wistar and Lewis rats. This was performed because the isoform of 14kDa did not appear in cultured cells from Lewis newborn rats. To this end, rats were euthanized and had their brains excised instantly to protein extraction of substantia nigra which was subjected to the same western blot method described previously for cell culture. In addition to the antibody from Abcam, an immunoglobulin against alfa-synuclein from Santa Cruz Biotechnology (1/500; cat. 7011R) was employed to confirm the pattern of isoforms present in neonates and adults. An assay of adsorption was performed by adding specific blocking peptide (1/100, Santa Cruz; cat. 7011P) to the solution containing the antibodies to confirm their specificity.
All the antibodies used in the present study were tested for specificity. In the case of immunocytochemistry it was done by incubating the sections with the secondary antibody only, and for western blot the control of specificity was tested in the presence of the blocking peptide. Furthermore, the antibodies are commercially available, their specificity are warranted by the manufacturer as well as they have been tested by other authors [19–21].
Results were analyzed by unpaired Student's T test accessed through GraphPad Prism (GraphPad Software Inc., version 4.00, CA). A p-value ≤ 0.05 was considered to indicate statistically significant differences. Data are expressed as mean ± standard deviation (SD).