α synuclein

Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0 °C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500 μl, 5 μl aprotinin, PMSF 50 μl, Na₃VO₄ 100 μl and NaF 20 μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4 °C (13,200g for 20 min). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE [45 (link)]. The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), α-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.

All mice were deeply anesthetized by intraperitoneal injection of 4% avertin for the brain extraction. After perfusion with 0.9% NaCl, mice were sacrificed by cervical dislocation and extracted brains were fixed in 4% paraformaldehyde (Biosesang, Korea). After 24 h of fixation, brains were immersed in 30% sucrose for 2 days. For immunofluorescence staining, 35 µm-thick frozen sections were incubated with 6E10 (Biolegend, USA, Catalog# SIG-39320, 1:200), 4G8 (Biolegend, USA, Catalog# SIG-39220, 1:200), AT8 (Invitrogen, USA, Catalog# MN1020, 1:200), or α-synuclein (BD Transduction Laboratories, USA, Catalog# 610786, 1:250) antibody diluted in 5% horse serum (Gibco, USA), then with Alexa555-conjugated secondary antibody (1:200 in PBS). Each stained section was incubated in 500 µM of Thioflavin S (ThS, Sigma-Aldrich, USA) dissolved in 50% ethanol for 7 min for ThS double-staining. Hoechst 33342 (10 µg/mL, Sigma-Aldrich, USA) was used to observe nuclear morphology. All images were taken using a fluorescence microscope (Leica DM2500, Germany). The number and area of plaques detected by ThS and 6E10 were quantified using Image J software.

Cells were homogenized in lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.1 mM EDTA, 30 mM NaF, 3 mM orthovanadate, 0.5 mM DTT, 2 mM sodium pyrophosphate, 0.5 mM PMSF, 2 μg/mL leupeptin, 2 μg/mL antipain) and incubated on ice for 10 min. After addition of NP-40 (0.5 %), samples were vigorously mixed and centrifuged for 30 s at 12,000 × g, and the resulting supernatant was collected. Protein concentration in cell lysates was determined using a protein assay kit (Bio-Rad). The immunoblotting was performed as described previously [26 (link)]. The primary antibodies used were the following: α-synuclein (1:500, BD Transduction Laboratories), phospho-MAPK42/44 (1:2000, Cell Signalling Technology), α-tubulin (1:2000, Santa Cruz Biotechnology) and β-actin (1:2000, Sigma). The HRP-conjugated secondary antibodies (1:5000, Sigma) were applied and blots were developed using an ECL kit (Millipore).

The following antibodies were used for Western blot analysis: α‐synuclein (BD Transduction Laboratories, Clone 42/α‐synuclein (RUO), 610787; dilution 1:500); β‐actin (Sigma, A5316; dilution 1:20,000); Flag‐tag (Sigma, F3165; dilution 1:5,000); and Horseradish peroxidase (HRP)‐conjugated secondary (goat anti‐mouse IgG, Jackson Laboratory, 115‐035‐146; goat anti‐rabbit IgG, Jackson Laboratory, 111‐035‐144; 1:5,000 dilution). For FACS analysis, anti‐NeuN antibody was used (EMD Millipore, ABN78; 1:500). For chromatin immunoprecipitation experiments, the following primary antibodies were used: (Normal mouse IgG, Millipore, 12‐371; H3K4me3, Abcam, ab8580; H3K27me3, Active motif, 39155; H3K27ac, Abcam, ab4729). Anti‐Cas9 antibody (Takara, 632607; 1:1,000) and anti‐GFP antibody (Fisher Scientific, MS1315P0; 1:5,000) were used for immunoprecipitation experiments. For immunocytochemistry, TUJ1 (Neuromics, MO15013; 1:500), TH (Santa Cruz, SC‐25269; 1:200) and Cas9 (Takara, 632607; 1:500) antibodies were used.

The slices, including OE, OB, STR, and SNc, were rinsed three times with 0.01 M PBS, dewaxed at 65℃ in a drying cabinet, and rinsed again. And the slices were incubated with 3% H₂O₂ for 30 min, blocked with 10% goat serum in PBS, and then incubated with primary antibodies against α-synuclein (BD Transduction Laboratories, 1:200) and TH (Sigma, 1:500) overnight at 4℃, respectively. The slices were then rinsed with PBS and incubated with secondary antibodies conjugated to HRP (Invitrogen Technologies, 1:1000) for 2 h at room temperature. The pieces were subsequently stained with DAB, mounted on gelatin-coated glass slides, and cover-slipped. The pieces were imaged with an Olympus microscope. The images were quantitatively analyzed by Image J software.

The protease inhibitor cocktail, cOmplete™ ULTRA tablets from Roche Diagnostics was used (#5892988001, Roche Diagnostics, Indianapolis, IN, United States). For immunoblotting, the following antibodies were used : total Tau (1:50000 #A0024, Dako, Santa Clara, CA, United States); GFP (1:1000 #3H9, Chromotek Inc., Hauppauge, NY, United States); ɣ-actin (1:10000 #Sc-65635, Santa Cruz, Dallas, TX, United States); Tau-46 (1:500 #ab203179, Abcam, Cambridge, MA, United States); Tau-C3 (1:1000 #AHB0061, Invitrogen, Carlsbad, CA, United States); Caspase3 (1:1000 #9662, CellSignaling); Cleaved Caspase3 (1:1000 #9661, CellSignaling); α-Synuclein (1:1000 #610787, BD Biosciences, Franklin Lakes, NJ, United States). All the secondary antibodies were coupled with HRP from Jackson ImmunoResearch (West Grove, PA, United States). The Flag-Tau, Flag-empty, GFP-VAMP8 and GFP-empty plasmids used for co-transfection of Neuro-2A cells were described previously (Pilliod et al., 2020 (link)). Tau-D421A, Tau-Δ421-441, Flag-TauNT and Flag-TauMBD-CT were generated by mutagenesis from Flag-Tau and Tau-N410A+D421A from pEGFP-C1-4R-Tau by removing the GFP tag (Civic Biosciences limitée, Beloeil, QC, Canada). Myc- α-Synuclein was obtained from Dr. EA Fon. Recombinant Tau protein (0N4R) was obtained from Bio-techne (SP-499, Bio-Techne, Minneapolis, MN, United States).