rAAV production and preparation:
rAAV 2/8 expressing Dendra2 (16), human WT α-syn with the C-terminal Dendra2 tag and human A53T α-syn with a C-terminal Dendra2 tag, under the control of the hybrid CBA promoter with CMV enhancer were generated as described previously (15, 17). Dendra2, WT-α-syn-Dendra2, A53T-α-syn-Dendra2 were also generated under the control of the human Synapsin promoter and packaged in rAAV 2/8-3Y as reported previously (18).
Organotypic brain slice cultures:
All mouse procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida. BSCs were prepared from postnatal day 8 (p8) B6/C3H F1 mice (Envigo, Indianapolis, IN, USA) as previously described (15, 19). In brief, 350 µm coronal slices containing the cortex or cortex and hippocampus were cut using a McIllwainTM tissue chopper (Mickle Laboratory Engineering Co. Ltd., Surrey, UK) in sterile-filtered Hank’s balanced salt solution (HBSS), calcium, magnesium, no phenol red (Thermo Fisher Scientific), 2 mM ascorbic acid (Sigma Aldrich, St Louis, MO, USA), 39.4 μM ATP (Sigma Aldrich), and 100 units/ml penicillin, 100 µg/ml streptomycin (Thermo Fisher Scientific). Brain slices were collected and plated randomly across wells to avoid any regional effects to contain three slices per semi-porous membrane insert (Millipore, 0.4 µm pore diameter, Thermo Fisher Scientific) in 6-well sterile culture plates. Slices were maintained at 37 °C and 5% CO2 in sterile-filtered culture medium (basal medium eagle (BME, Thermo Fisher Scientific), 26.6 mM HEPES (pH 7.1, Thermo Fisher Scientific), 511 μM ascorbic acid, 1% (v/v) GlutaMAX (Thermo Fisher Scientific), 0.033% (v/v) insulin (Sigma Aldrich), 100 units/ml penicillin, 100 µg/ml streptomycin (Thermo Fisher Scientific) and 25% (v/v) heat-inactivated horse serum (Sigma Aldrich). Slice culture medium was exchanged every 3-4 days for long-term maintenance. rAAVs were applied directly into the culture medium on the first day of culture (DIV 0) at 1-2x1010 VGs of rAAV per well containing 3 BSCs.
Triton X-100 extraction of BSC lysates
BSCs for assessment of insoluble α-syn from three wells (nine slices in total) were harvested into ice-cold PBS and presented as n = 1. BSCs were washed and extraction was performed as previously described (15, 20). In brief, BSCs were lysed in 1% Triton X-100 high-salt (HS) buffer (50 mM Tris–HCl, pH 7.5, 0.75 M NaCl) containing protease and phosphatase inhibitors. Lysates were then centrifuged at 100,000 g for 20 min at 4°C. Supernatants were removed (Triton-soluble fraction), and the pellet was washed in HS buffer with sucrose and centrifuged at 100,000 g for 20 min at 4°C. The pellet was then resuspended in 2% SDS, sonicated, and heated to 100°C for 10 min (Triton-insoluble fraction). Protein concentrations were determined by BCA assay (Thermo Fisher Scientific) before equal amounts of protein were resolved by immunoblotting.
Antibodies
The following antibodies were used for Western blotting and immunohistochemistry. SNL-4, a rabbit polyclonal antibody raised against amino acid sequence 2–12 of human α-syn (21); 94-3A10, a mouse monoclonal antibody which recognizes amino acid sequence 130-140 of human α-syn (21); 81A, a mouse monoclonal antibody specific for α-syn phosphorylated at Ser129 (22); and a rabbit monoclonal antibody specific for α-syn phosphorylated at Ser129 (EP1536Y; Abcam) were used. A rabbit polyclonal antibody for p62/SQSTM1 (18420-1-AP; Proteintech Group Inc, Rosemont, IL, USA) was also used. A mouse monoclonal antibody to β-actin (A5441; Sigma Aldrich), was also used.
SDS-PAGE and Western blotting
15–25 µg protein was separated on 10% (w/v) SDS-PAGE gels (Bio-Rad Laboratories, Hercules, CA, USA) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (15, 23). Membranes were blocked in 0.5% casein for 1 h and then incubated with primary antibodies overnight at 4 oC, washed 3 times with TBS before incubation with fluorophore-conjugated Alexa Fluor 680 anti-mouse IgG (Thermo Fisher Scientific) or IRDye 800 goat anti-rabbit IgG (Li-Cor Biosciences, Lincoln, NE, USA) secondary antibodies for 1 h and washed 3 times with TBS. Protein bands were detected using the multiplex Li-Cor Odyssey Infrared Imaging system (Li-Cor Biosciences).
Immunohistochemistry and Confocal Imaging
BSCs were washed in PBS and then fixed on their inserts in 4% paraformaldehyde (PFA) for 1 h and stained as previously documented (24). Individual BSCs (n=1) were cut out from their membranes after fixation and then used as free-floating sections for subsequent steps. BSCs were permeabilized and blocked for 18 h in 0.5% Triton X-100 in 20% BSA (Sigma Aldrich) at 4oC. BSCs were then incubated overnight at 4 oC with the appropriate primary antibodies in 5% BSA, washed and then incubated with fluorophore-coupled secondary antibodies for 4 h at RT. Slice cultures were washed a further three times before mounting on slides with Fluoromount-G (Southern Biotech, Birmingham, AL, USA) and then imaged using an Olympus FV1200 IX83 confocal laser-scanning microscope (Olympus America Inc, Center Valley, PA, USA). Z-stacks were captured at recommended step-sizes and projected as a maximum projection image using the Olympus Fluoview software.
Long-term Live Imaging and quantification of Dendra2 in BSCs
Images of living BSCs were captured using a Keyence BZ-X700 all-in-one fluorescence microscope (Keyence Corporation of America, Itasca, IL, USA). Dendra2 transduced BSCs were photoconverted using a 2s pulse of blue light through a 20 µm Z-stack at recommended step-sizes with a 20 x objective lens. Images of emitted green and red fluorescence through a 20 µm Z-stack at recommended step-sizes were captured at the relevant time points and then projected onto a full focus image using the BZ-analyzer software (Keyence Corporation of America). Each red and green fluorescent image from each slice at each time point was captured from pre-determined regions. For population analysis, total corrected fluorescence was calculated using integrated density above background integrated density after thresholding for each BSC imaging series using ImageJ (Version 1.51k, National Institutes of Health, Bethesda, MD, USA). For quantification of the cells that showed a lengthened half-life, the number of red cells as a proportion of green cells at DIV 14 was calculated after thresholding.
Expression, purification, assembly, and addition of WT full-length mouse α-syn fibrils:
Recombinant mouse α-syn (residues 1−140) was expressed from pRK172 plasmids containing the cDNA for the SNCA gene and expressed in E.coli BL21 (DE3). Recombinant protein was purified as previously described utilizing size exclusion and Mono Q anion exchange chromatography (25, 26). Proteins were diluted in pH 7.4 sterile PBS, and concentrations were determined by BCA assay. Recombinant mouse α-syn protein was assembled into fibrils by incubation at 37°C at 5 mg/ml in sterile PBS with continuous shaking as previously reported (25, 26). Fibril assembly was monitored with K114 fluorometry (26). Mouse α-syn fibrils were diluted in sterile PBS and underwent bath sonication for 1 h at RT. In experiments where BSCs were seeded, 2μg of mouse α-syn PFFs were applied directly on top of each BSC at the times indicated in schematic diagrams (Fig 4A, Supp Fig 2A).
Thiazin Red staining
BSCs were washed in PBS and then fixed on their inserts with 4% PFA for 1 h. BSCs were stained with 0.05% Thiazin Red as previously described (17). Individual BSCs (n = 1) were cut from their membranes and autofluorescence reagent (Millipore) was applied for 5 min, and then washed in 40% ethanol (EtOH). BSCs were incubated with 0.05% Thiazin Red in ddH2O for 3 min in the dark, and then washed in 50% EtOH, PBS and then ddH2O. BSCs were mounted on slides with Fluoromount-G (Southern Biotech) and then confocal imaged to identify any amyloidogenic β-sheet structures in these sections.
Statistical Analysis
Data were analyzed statistically according to the methods specifically referred to in each figure legend. Data were compared by Student’s two-tailed unpaired T-test, One-way analysis of variance (ANOVA) with post-hoc Tukey’s multiple comparisons test or Two-way ANOVA with post-hoc Sidak’s test (Graphpad Prism 9.0 Software, La Jolla, CA, USA). Half-lives were calculated using non-linear regression analysis of one-phase decay. Differences were considered statistically significant when adjusted p < 0.05. All graphs were generated in GraphPad Prism.