Antibodies and reagents
Antibodies against cav-1 (610407) and α-syn (610786) were purchased from BD Biosciences (Franklin Lakes, NJ). Antibodies against human α-syn (ab138501) and cav-1 (ab17052) were purchased from Abcam (Cambridge, MA). Antibodies against mCherry (ab167453) were purchased from Abcam. Antibody against pCav-1 (3251S) was purchased from Cell signaling Technology (Danvers, MA). Antibody against GAPDH (SC-32233) was procured from Santa Cruz Biotechnology (Santa Cruz, CA). Rhodamine-conjugated transferrin (T23365) and boron-dipyrromethene (BODIPY) FL C5-LacCer (B34402) were purchased from Molecular Probes (Leiden, The Netherlands). Saracatinib (11497) was procured from Cayman Chemical (Ann Arbor, MI). SKI-1 (c-Src inhibitor 1, S2075), retinoic acid (RA) (r2625), and bafilomycin A1 (B1793) were obtained from Sigma (St. Louis, MO). Antibody for pSer 129 α-syn (015-25191) was purchased from Wako (Richmond, VA).
Immunohistochemistry
C57BL/6 mice (Orient Bio, Korea) and M83 mice overexpressing A53T human α-syn under the control of mouse prion protein promoter (B6;C3-Tg(Prnp-SNCA*A53T)83Vle/J, The Jackson Laboratory) at indicated age were perfused, and their brains were fixed with 4% paraformaldehyde for 2 days. Frozen sections were cut at 35 μm in the coronal plane. The sections were stained using the free-floating method, adhering to the slide after staining. For 3,3’-diaminobenzidine (DAB) staining, the samples were twice rinsed with phosphate-buffered saline (PBS) containing 0.2% Triton X-100 (PBST), treated with 3% H2O2 for 5 min, and rinsed with PBST. After blocking non-specific binding by incubating with 1% bovine serum albumin (BSA) in PBST, the sections were incubated overnight at 4℃ with cav-1 antibody. Sections were rinsed three times with PBST, incubated with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and visualized using VECTASTATIN avidin-biotin complex (ABC kit) and DAB solution (0.05% DAB and 0.003% H2O2 in 0.1 M phosphate buffer). Next, the sections were mounted on slides and examined under a bright-field microscope (Olympus Optical, BX51, Tokyo, Japan). Images were captured using the PictureFrames Application 2.3 software.
Plasmids
Plasmids for WT cav-1 and WT cav-1-EGFP were constructed using PCR. The plasmid for Y14A cav-1 was constructed using the Quick-Change site-directed mutagenesis kit (Stratagene, La Jolla, CA). Each construct was subcloned into pCDH-EF1 for generating lentiviral vectors. All plasmids were verified via DNA sequencing and prepared using the Maxi prep Kit (Qiagen, Valencia, CA).
Cell culture
SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS). Primary cortical neurons were cultured from Sprague-Dawley rat embryos or human A53T α-syn overexpressing transgenic (TG) mice at embryonic day 18, maintained in neurobasal medium (Invitrogen, Carlsbad, CA) with L-glutamine and B-27 supplement (Invitrogen, Carlsbad, CA).
Generation of stable cell lines
WT cav-1, Y14A cav-1, WT cav-1-EGFP, and Y14A cav-1-EGFP overexpressing (OE) SH-SY5Y cells were prepared using lentiviral constructs and selected using puromycin, respectively. A53T α-syn-EGFP (A53T-E) and A53T α-syn-mCherry (A53T-M) were prepared as described previously [24]. WT cav-1/A53T-E and Y14A cav-1/A53T-E OE SH-SY5Y cells were prepared using lentiviral transfection of A53T-E in WT cav-1, Y14A cav-1 OE SH-SY5Y cells and selected using FACSAria III, respectively.
Western blot
Cells were washed with PBS and lysed in ice-cold RIPA buffer (50 mM Tris-HCl [pH7.4], 150 mM NaCl, 0.25% sodium deoxycholate and 1% Triton X-100) containing protease inhibitor cocktail (Calbiochem, Germany) and phosphatase inhibitor cocktail (GenDEPOT, Baker, TX). After brief sonication, the lysates were incubated on ice for 20 min and centrifugated at 15,700 g for 30 min at 4℃. After centrifugation, the supernatant was collected. The protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Bio-Rad, CA, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The PVDF membrane was blocked with 1% BSA in Tris-buffered saline. After 1 h blocking, the membrane was immunoblotted with appropriate antibodies and visualized using an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA).
Dual Chamber assay
The dual chamber assay was performed as described previously [24, 25]. WT α-syn or A53T-M OE SH-SY5Y cells as donor cells were differentiated by treatment with 50 μM RA for 5 days. Next, SH-SY5Y cells or primary cortical neurons cultured on coverslips in a 12-well plate as recipient cells were cocultured with differentiated WT α-syn or A53T-M OE SH-SY5Y cells cultured on the insert for 24 h. The recipient cells were prepared for staining with anti-α-syn or anti-mCherry antibody. To measure the amount of internalized α-syn, five random fields were selected and intensities of more than 100 cells were analyzed using the MetaMorph software (Molecular Devices).
Coculture assay
The coculture assay was performed as described previously [24, 25]. Briefly, A53T-E, WT cav-1/A53T-E or Y14A cav-1/A53T-E and A53T-M OE SH-SY5Y cells were cocultured in a 1:1 ratio for 5 days in the presence of 50 μM RA. After 5 days of coculture, the cells were then subcultured on coverslips in a 12-well plate. The cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed again with PBS, stained with DAPI for 10 min, and washed again with PBS before analysis. Then, the samples were observed under a confocal microscope. To quantify double fluorescence-labeled puncta, five random fields were selected and more than 100 cells containing double fluorescence-labeled puncta were counted manually. The number of A53T-E OE SH-SY5Y cells containing double fluorescence labeled puncta and the number of A53T-M OE SH-SY5Y cells containing double fluorescence-labeled puncta were counted separately and expressed as percentages of total cells analyzed.
Microfluidic chamber assay
Microfluidic chamber assay was performed as described previously with slight modification [26]. Briefly, triple compartment microfluidic devices (TCND1000) were obtained from Xona Microfluidic, LLC (Temecula, CA, USA). Glass coverslips were prepared and affixed to the microfluidic device according to the manufacturers’ instructions. Approximately 100,000 A53T TG cortical neurons (TP18) were plated per chamber. At 7 DIV, 0.5 μg of α-syn fibrils were added into chamber (C) 1, with lentiviruses for EGFP-only, WT cav-1-EGFP or Y14A cav-1-EGFP expression added into C2. To control the direction of flow, a 50 μl difference in media volume was maintained between C1 and C2, and C2 and C3, according to the manufacturers’ instructions. Neurons were fixed at 7 days after treatment with α-syn fibrils using 4% paraformaldehyde in PBS. Devices were then removed, and immunofluorescence assay was performed.
Confocal microscopy
Cells cultured on poly-D-lysine coated coverslips were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. The fixed cells were then washed with PBS and incubated with PBS containing 0.1% Triton X-100 for 10 min at room temperature. After washing several times with PBS, cells were blocked with PBS containing 1% BSA for 1 h at room temperature, and then incubated overnight with indicated antibodies at 4°C. The samples were then stained with fluorescence conjugated secondary antibodies for 1 h, mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA), and observed under a confocal microscope (Zeiss, Germany).
Statistical analysis
All values are expressed as the mean ± SEM. Statistical significance was evaluated using Graphpad Prism (La Jolla, CA).