C. elegans strains
C. elegans were maintained at room temperature (21 °C) on nematode growth medium (NGM) agar plates with E. coli OP50 as a food source as previously described . The N2 (or Bristol) strain is the canonical wild-type strain used in most C. elegans research and was obtained from Caenorhabditis Genetics Center (CGC; University of Minnesota, St. Paul, MN, USA). The following additional strains were obtained from CGC: BR2823 (by155), BZ555 (egIs1[dat-1p::gfp]), CB102 (e102), CB587 (e587), CB933 (e245), and CB1112 (e1112). Strains containing the following transgenes were obtained from National BioResource Project (NBRP; Japan): tmIs903, tmIs904, tmIs905, tmIs1082, tmIs1083, and tmIs1084. VN305, VN306, and VN307 were obtained by crossing with YT2022 (tzIs3[cre::gfp])  (kindly provided by Dr. Yoshishige Kimura, Kanagawa University of Human Services) with wild-type α-synuclein, A30P α-synuclein, and A53T α-synuclein transgenic animals, respectively (kindly provided by Dr. Takeshi Iwatsubo, University of Tokyo) . VN310 was obtained by backcrossing YT2022 with N2. VN162 was obtained by crossing YT2022 with CB1112. Control GFP animals were obtained by crossing VN310 with BZ555 and used as a control for ectopic protein expression in dopaminergic neurons, since GFP was expressed under the same dat-1 promoter as α-synuclein. VN306 was crossed with BR2823 to produce double mutants. VN306 was crossed with BZ555 to use GFP to track dopaminergic neuron loss over time.
C. elegans locomotion
Animals were synchronized by isolating eggs using alkaline hypochlorite treatment and hatching overnight. Larvae were plated onto NGM agar plates with OP50 and grown for 4 days before recording locomotion. For individual C. elegans recordings, a single adult animal was randomly picked, washed for 1 to 2 min in M9 buffer, and then transferred to a fresh NGM plate without food for imaging. Video images were acquired (2 frames per second) for up to 6 min using a sCMOS camera (pco.edge, PCO) mounted on a stereomicroscope (Olympus MVX10 MacroView) controlled using the μManager software . A total of 40 individual animals were imaged for each strain. For C. elegans population recordings, ten adult animals were randomly removed using an eyelash pick, washed for 1 min in M9 buffer, and then transferred to a fresh NGM plate without food, with a ring of copper sulfate solution (150 mM) around the area of recording (a chemorepellent to keep the animals in the recording frame). Exploratory locomotion was recorded using a CCD camera (GX1920, Allied Vision) which was attached to a dissecting stereomicroscope (Leica M165C, 1.0x PlanApo lens). A Labview program captured video (2 frames per second or 10 frames per second) at 1936 × 1456 resolution. Using ImageJ, the displacement, perimeter, and area of each animal was calculated, from which a circularity value was computed using the following formula: circularity = 4π(A/P2), where A is the area of the animal’s body and P is the perimeter of the animal’s body (both expressed in arbitrary pixel units). We set a cut-off for coiling at a circularity value of 0.6; an animal that had a circularity value > 0.6 was considered to be coiling. Using MATLAB (Mathworks), we calculated various behavioural metrics including the coiler score, which is defined as the percentage of frames with a circularity value > 0.6. Speed was automatically computed from the ImageJ based WrmTrk plugin as the distance travelled by each animal (in pixels) over the amount of time that it was tracked (in seconds) . We determined average speed by calculating the weighted average of each tracked animal’s speed within a single population of animals. On the day of the recording for dopamine or raclopride treatment, adult animals were washed for 1 min in M9 buffer, treated in a solution containing either drug for 1 min, and then placed onto fresh NGM plates without food, with a copper sulfate solution ring around the area of recording. For all other drug treatment experiments, larvae were first grown for 2 days after hatching and then treated on drug plates for 3 days prior to recording, since drug treatment of younger larvae resulted in abnormal development and unhealthy animals. Drug plates were prepared by adding each drug to LB medium with OP50 before spreading onto fresh NGM plates. The solvent used to dissolve each drug was used as vehicle control for the drug treatment experiments (M9 buffer for dopamine, distilled water for raclopride, DMSO for all other drugs). A concentration of 100 μM was used for treatment with rapamycin based on previous reports  and the same concentration was used for treatment with acetaminophen, caffeine, losartan, and rifabutin.
C. elegans lifespan
Synchronized larvae were plated onto NGM plates with food and grown for 2 days. Thirty L4 animals were picked and transferred to a fresh NGM plate containing floxuridine (FUDR; 0.005 mg/mL) with food (day 0). A total of 15 plates were prepared for each C. elegans strain (total N = 450 animals per strain). Animals were scored as alive or dead every 2 days until no alive animals remained on the plate. An animal was marked as dead and removed from the plate if it no longer exhibited movement and did not respond to prodding with an eyelash pick. Censored animals included those C. elegans that could not be found (e.g., burrowed into the agar) or died due to desiccation on the walls of the plate. An individual animal’s lifespan was the age at which it was scored as dead, and mean lifespan was calculated for each plate. Mean lifespan for each C. elegans strain was calculated as the average of the 15 plates.
Enzyme-linked immunosorbent assay (ELISA)
Synchronized larvae were plated onto NGM plates with food and grown for 4 days. Animals were collected, washed, and pelleted in M9 buffer. Animals were resuspended in ice-cold RIPA buffer (BioBasic, RB4478) containing a protease inhibitor cocktail (Roche, 11,836,153,001) (1 mL of buffer per 0.1 g of C. elegans pellet) and then sonicated at 70% power for 2 runs of 10 s each (Qsonica, Q125). Sonicated samples were incubated at 4 °C for 2 h in a shaker and then centrifuged at 17,000 g for 30 min. ELISAs were performed to measure α-synuclein protein levels in the protein lysate (supernatant). Total protein was quantified using a commercial BCA assay (ThermoFisher, 23,227) according to the manufacturer’s instructions. ELISAs were conducted using a commercial human α-synuclein ELISA kit (BioLegend, 844,101) according to the manufacturer’s instructions.
Dopaminergic neuron loss in C. elegans
Control (tzIs3;egIs1) and α-synuclein ([A30P α-synuclein];tzIs3;egIs1) gravid adult animals were synchronized, and eggs were hatched overnight (day 0) on NGM plates without food. Synchronized L1 larvae were transferred to NGM plates with food and grown at room temperature. L4 larval animals were then picked and transferred to fresh NGM plates with food for better synchronization. The animals were transferred to new plates every 48 to 72 h. On day 3 to day 9, anterior deirid (ADE) neurons were scored from 26 to 58 animals in a blinded fashion by GFP fluorescence using a widefield microscope (Zeiss AxioObserver). Any absent or unidentifiable ADE neuron was scored as degenerated.
CRE-GFP reporter of dopaminergic dysfunction in C. elegans
Animals were synchronized by isolating eggs using alkaline hypochlorite treatment and hatching overnight. Larvae were plated onto NGM agar plates with OP50 and grown for 3 to 4 days prior to CRE-GFP analysis. Animals were washed with M9 buffer until clear of bacteria and then resuspended in a small volume of M9 buffer. Animals were mounted on 10% agarose pads and immobilized on the pads with 0.2 to 0.5 μl of 0.1 μm diameter polystyrene microspheres (Polysciences, 00876–15). GFP fluorescence of 4 cholinergic head neurons named SIA (sublateral interneuron) was scored in a blinded fashion using a widefield fluorescence microscope (Zeiss AxioObserver). We recorded the number of animals with at least one GFP positive SIA neuron, as well as the number of GFP positive SIA neurons per animal.
Gene ontology (GO) analysis of C. elegans coiler strains
Yemini et al.  published a database of behavioural phenotypes for 305 C. elegans strains, including the coiler phenotype (reported as coiling frequency and coil time). Ninety-seven genes were associated with C. elegans strains having a statistically significant increase in either coiler frequency or coil time compared to N2 (binned q value of < 0.05). These genes were analysed with PANTHER gene ontology “GO biological process complete” (pantherdb.org/geneListAnalysis.do). The C. elegans genome was used as the reference list. For all GO terms > 100-fold enrichment, we ranked the biological processes by the -log10 of the false discovery rate (FDR).
Immunoprecipitation and immunoblotting
Synchronized animals (4 or 5 days old) were lysed in 0.5X Tris-HCl buffer with 0.5% Triton X-100 at 100 °C for 5 min and then passed through a 23-gauge needle 10 times. The lysate was centrifuged at 10,000 g for 10 min in a 4 °C refrigerated centrifuge, and the protein lysate (supernatant) was transferred to a new tube. Protein concentration was determined with a Bio-Rad Lowry protein assay (Bio-RAD, 5000116) according to the manufacturer’s instructions. For immunoprecipitation, protein lysates were incubated on a rotator at 4 °C overnight with mouse anti-human α-synuclein (Clone 42, BD Laboratories, 610,787; dilution 1:100). Immune complexes were isolated by the addition of washed protein G agarose beads followed by incubation for 4 h at 4 °C. Beads were washed and samples were analysed by immunoblotting and mass spectrometry (see below). For immunoblotting, protein lysates (9.5 μg) or beads from immunoprecipitations were mixed with 6X Laemmli sample buffer, and proteins were then separated on a 10% acrylamide gel by SDS-PAGE. Proteins were transferred to a PVDF membrane using a wet transfer system. The membranes were probed with anti-α-synuclein mouse monoclonal antibodies (Clone 42, BD Laboratories, 610,787; dilution 1:500). Biotinylated goat anti-mouse antibodies (Jackson Immuno Research, 115,065,146; dilution 1:20,000) were used as secondary antibodies with streptavidin-conjugated with horseradish peroxidase (Jackson Immuno Research, 016030084; dilution 1:10,000). Signals were detected with ECL (ThermoFisher, 32,132) and developed on HyBlot CL autoradiography film (DV-E3018).
Mass spectrometry and GO analysis of α-synuclein-interacting C. elegans proteins
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed by SPARC BioCentre Molecular Analysis, The Hospital for Sick Children, Toronto, Canada using samples of proteins co-immunoprecipitated with α-synuclein (described above). One hundred and thirty-one proteins were identified in immunoprecipitants from α-synuclein C. elegans lysates but absent from GFP C. elegans lysates. The genes that encode these proteins were analysed with PANTHER gene ontology “GO biological process complete” (pantherdb.org/geneListAnalysis.do). The C. elegans genome was used as the reference list. Biological processes with FDR < 0.05 were ranked according to fold enrichment.
In silico ranking of drugs predicted to reduce α-synuclein oligomers
IBM Watson for Drug Discovery Predictive Analytics identified ~ 26 million records in Medline that cited either the known or candidate entities, as we previously described . Every single Medline abstract was then converted into a multidimensional vector of the words and phrases contained in the document, relative to the 20,000 most common words and phrases within the English lexicon, using a term frequency–inverse document frequency statistic. A centroid for each known and candidate entity was then generated by averaging the multidimensional vectors of all documents associated with each entity and used to produce a distance matrix comprising a similarity index for every pair of entities. Finally, a graph diffusion algorithm was applied to rank each candidate entity by similarity to the entire known set rank, thus producing a ranked candidate list ordered by predicted semantic similarity to the known set. The model performance was validated using a leave-one-out (LOO) cross-validation in which the ranking process (as described above) was run 15 times, each time with one entity removed from the known set and added to the candidate set. Receiver-Operating Characteristic (ROC) and Precision-Recall curves were generated.
Luciferase protein-fragment complementation and cell viability assays in cell lines
H4 neuroglioma cells (ATCC, HTB-148) were maintained in Dulbecco’s Modified Eagle Medium plus high glucose, L-glutamine, and sodium pyruvate (ThermoFisher, 11995–065) with 10% heat-inactivated fetal bovine serum (ThermoFisher, 16140071) and 1% antibiotic-antimycotic cocktail (ThermoFisher, 15240062) at 37 °C and 5% CO2. DNA expression constructs encoding for full-length human α-synuclein fused to the N-terminal fragment of Gaussia princeps luciferase (syn-luc1), full-length human α-synuclein fused to the C-terminal fragment of Gaussia princeps luciferase (syn-luc2), and full-length Gaussia princeps luciferase were kindly provided by Dr. Pamela McLean, Mayo Clinic Jacksonville . DNA expression constructs encoding for human amyloid-beta peptide Aβ1–42 fused to the N-terminal fragment of Gaussia princeps luciferase (Aβ-luci) and human amyloid-beta peptide Aβ1–42 fused to the C-terminal fragment of Gaussia princeps luciferase (Aβ-ferase) were kindly provided by Dr. Tadafumi Hashimoto, University of Tokyo . A DNA expression construct encoding for full-length human tau protein (2 N, 4R) fused to the C-terminal fragment of Gaussia princeps luciferase (tau-ferase) was kindly provided by Dr. Susanne Wegmann, DZNE Berlin . To generate a construct encoding for full-length human tau protein (2 N, 4R) fused to the N-terminal fragment of Gaussia princeps luciferase (tau-luci), the luciferase fragment from luci-Aβ was PCR amplified using Q5 polymerase (New England Biolabs) and the primers: BspeILUCIf 5′ GTGGGTCCTCCGGAAAGCCCACCGAGAACAACGAAGACTTCAAC 3′ and KpnILUCIr 5’ATCGGATCCGGTACCGATTTAAACGGGCCCTCTAGATTAGCCTATGCCGCCCTG3’. The fragment was cloned into the Bspe I/ Kpn I sites of tau-L2 to produce tau-luci (pAK 348–1) which was sequenced to confirm accuracy and freedom from PCR induced errors. Cells were transiently co-transfected with syn-luc1 and syn-luc2, Aβ-luci and Aβ-ferase, tau-luci and tau-ferase, or transfected with luciferase alone using Superfect transfection reagent (Qiagen, 301305) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were plated on a 96-well microplate at 60,000 cells per well. Twenty-four hours after plating, cells were treated with drugs or vehicle control. Following 24 h of treatment, cells were washed 1 time with PBS and bioluminescence was measured using an automated CLARIOstar plate reader (Mandel, 430–0505). Cell permeable, native coelenterazine was used as the Gaussia luciferase substrate. Lyophilized coelenterazine (Nanolight, 303–500) was reconstituted in NanoFuel Solvent (Nanolight, 399–1), then diluted in PBS to 16.6 μg/mL, and dispensed per well to a final concentration of 20 μM). The bioluminescent signal generated by the luciferase enzyme was assessed at 470 nm over 5 s. Raw luminescence units were normalized to the DMSO vehicle control of each 96-well microplate. To assess cell viability, H4 neuroglioma cells were prepared and treated as described above and, 1 h prior to viability analysis, cells were treated with PrestoBlue viability reagent (ThermoFisher, A13261) according to the manufacturer’s instructions. Fluorescence was automatically measured using a CLARIOstar plate reader (Mandel, 430–0505) and then normalized to the fluorescence measure of the DMSO vehicle control.
Venus YFP protein-fragment complementation assay in primary cortical neurons
Pregnant Sprague Dawley rats (E17) were purchased from Charles River. Embryos were surgically removed, and neurons were harvested in a sterile environment. Neurons were plated on poly-D-lysine coated glass coverslips at a density of 5 × 105 cells per well in Neurobasal-A medium, (Gibco, 10,888,022) supplemented with B27 (2%) (Gibco, 17,504,044), antibiotic-antimycotic (1%) (Gibco, 15,240,062), and Glutamax (1%) (Gibco, 35,050,061). Fifty percent media changes were performed every 3 days. DNA expression constructs encoding for full-length human α-synuclein fused to the N-terminal fragment of Venus YFP (V1S), full-length human α-synuclein fused to the C-terminal fragment of Venus YFP (SV2), and full-length Venus YFP were kindly provided by Dr. Pamela McLean, Mayo Clinic Jacksonville . The following AAV serotype 1/2 vectors were custom ordered from GeneDetect (Auckland, New Zealand): AAV1/2-CBA-V1S-WPRE-BGH-polyA (AAV-V1S), AAV1/2-CBA-SV2-WPRE-BGH-polyA (AAV-SV2), and AAV1/2-CBA-Venus YFP-WPRE-BGH-polyA (AAV-Venus). Two days post isolation, neurons were co-transduced with AAV-V1S and AAV-SV2 or transduced with AAV-Venus alone at a MOI of 3000. AAV-containing media was removed after 72 h and cells were then treated with drugs or DMSO control for 72 h at the following concentrations: acetaminophen (100 μM), caffeine (200 μM), losartan (100 μM), rapamycin (50 nM), rifabutin (20 μM). Concentrations were selected to avoid neuronal toxicity according to previous reports [22,23,24,25,26]. Drug-containing media was removed, and cells were washed 1 time with ice-cold PBS before being fixed with 4% PFA (Sigma) at room temperature for 10 min. Levels of α-synuclein oligomers were measured by YFP fluorescence. To measure total α-synuclein levels, neurons were immunostained with anti-α-synuclein mouse monoclonal antibodies (Syn211, ThermoFisher, 32–8100; dilution 1:500) and goat anti-mouse secondary antibodies conjugated to Alexa Fluor® Plus 555 (ThermoFisher, A32727; dilution 1:500). Images of fixed cells were acquired using a confocal microscope equipped with 405, 488, 555, and 639 nm laser lines (Zeiss LSM880). All images for each biological replicate were taken within the linear range at constant gain and pinhole settings at a resolution of 1024 × 1024 pixels using a 63x/NA1.4 oil objective with the zoom set to 1.2. The imaging medium used was Zeiss Immersol 518F. Fluorescence intensity was analysed automatically using Imaris imaging software (Oxford Instruments) by measuring the mean fluorescence intensity of each individual fluorescent neuron throughout a 3D field of view generated by z-stack image acquisition with the “surfaces” module. This module detects fluorescence above background threshold in each field of view, identifies a cell body and neuronal projections of each cell, and calculates the mean fluorescence intensity of each fluorescent cell within the field of view. The mean fluorescence intensity of 10 fields of view was recorded for each biological replicate and normalized to the mean intensity of the DMSO treated condition. The value generated for each neuron was normalized to the mean of fluorescence intensity of neurons treated with vehicle alone.
For crosslinking experiments, primary cortical neurons were cultured and transduced as above. Eight days post isolation, media was removed, and cells were washed twice in PBS before being scraped and centrifuged at 1200 g for 5 min. Cells were re-suspended in PBS containing 50 μM disuccinimidyl glutarate (DSG; ThermoFisher, 20,593) and incubated at 37 °C for 30 min according to the manufacturer’s instructions. The reaction was quenched by the addition of 50 mM Tris for 15 min. Cells were pelleted again and re-suspended in RIPA buffer and prepared for SDS-PAGE.
Stereotactic surgery and rifabutin treatment of rats
Adult female Sprague-Dawley rats (250–300 g) were purchased from Envigo. The animals were pair-housed in cages with wood bedding and access to food and water ad libitum. The animal colony was maintained in a regular 12-h light/dark cycle. All procedures were approved by the University Health Network Animal Care Committee in accordance with guidelines and regulations set by the Canadian Council on Animal Care.
AAV serotype 1/2 was used to express A53T α-synuclein (AAV-A53T) under the control of the CAG promoter, a hybrid of the chicken beta actin (CBA) promoter fused with the cytomegalovirus (CMV) immediate early enhancer sequence (GeneDetect, Auckland, New Zealand), as previously described . An AAV1/2 vector lacking the A53T α-synuclein open reading frame was used as an empty vector control (AAV-EV). Animals were secured in a stereotactic frame under isoflurane/oxygen anaesthesia (2.5% isoflurane and 1.5 L/min O2) and anafen (5 mg/kg) analgesia. The surgical site was shaved and sterilized with iodine/betadine/isopropanol prior to making a 2-cm incision along the midline. The skull was exposed and a unilateral injection targeting the SN was performed at coordinates AP − 5.2 mm, ML − 2 mm, and DV − 7.5 mm with respect to bregma. For each animal, a total volume of 1.5 μl of AAV-A53T or AAV-EV (5.1 × 1012 genomic particles/ml) plus 0.5 µl of sterile PBS was injected at a rate of 0.5 μl/min using a microinjection pump and 10 μl Hamilton syringe with a 26-gauge needle. At the end of virus injection, the needle remained in place for 5 min before gradual removal.
Rats were randomly assigned to receive 25 mg/kg rifabutin (prepared as 1.5 mg/ml in 5% DMSO in saline) or vehicle control (5% DMSO in saline) daily. Treatments were started 2 days following stereotactic surgery. Rats were weighed and treated each day between 7:00–8:00 am with rifabutin or vehicle by intraperitoneal injection using a 25-gauge needle for 6 weeks.
Collection of rat plasma, cerebrospinal fluid, and brain tissue
Animals were euthanised after 6 weeks of rifabutin or vehicle treatment by cardiac puncture under isoflurane/oxygen anaesthesia, followed by transcardial perfusion with approximately 100 to 200 ml of ice-cold heparinised saline. Blood obtained via cardiac puncture was centrifuged at 4 °C in microtainer tubes (BD, 365974) for 2 min at 10,000 g, and the upper layer plasma was collected, frozen on dry ice, and stored at − 80 °C until use. Cerebrospinal fluid (CSF) was collected using a latex dropper bulb attached to a custom-made glass micropipette (Drummond Scientific, Broomall, PA) inserted into the cisterna magna. Approximately 50 to 100 μl of CSF were transferred into autoclaved vials and centrifuged at 3000 rpm over 3 to 5 s; samples with a detectable red blood cell precipitate were excluded due to blood contamination. CSF samples were frozen on dry ice and stored at − 80 °C until use. Brains were then removed, and tissue anterior to the optic chiasm was snap frozen in dry ice-cooled isopentane. A single 1 mm thick section of the ventral striatum was immediately cut, using a matrix, and frozen on dry ice. These sections were sent to Vanderbilt University Neurochemistry Core (Nashville, TN, USA) for measurements of biogenic amines by high-performance liquid chromatography (HPLC). Approximately 100 mg of frozen brain tissue plus frozen plasma samples from a subset of animals were sent to InterVivo Solutions (Toronto, ON, Canada) for measurements of rifabutin concentrations by LC-MS/MS. Tissue posterior to the optic chiasm, including the posterior striatum and SN, was immersion-fixed in 4% paraformaldehyde in 0.1 M PBS for 2 days at room temperature and cryoprotected at 4 °C in 15% sucrose and then 30% sucrose in 0.1 M PBS solution until the brains sank. For immunostaining, 40 μm thick coronal cryosections were prepared using a sliding microtome (Leica Microsystems Inc.), and 6 series of sections were stored in cryoprotectant (30% glycerol, 30% ethylene glycol, 40% PBS) at − 20 °C until use.
Immunostaining of brain cryosections
Immunostaining for stereology was performed by washing free-floating sections with PBS-T (PBS with 0.1% Tween-20) three times for 10 min each at room temperature. Sections were then immersed in 3% H2O2 for 3 min to quench endogenous peroxidases. Sections were rinsed in PBS-T three times for 5 min each before incubation in blocking solution (2% BSA, 10% normal goat serum in PBS-T) for 1 h at room temperature. After blocking, sections were incubated with rabbit anti-tyrosine hydroxylase (TH) antibodies (ThermoFisher Scientific, AB152; dilution 1:2000) in blocking solution overnight at room temperature. Sections were washed in TBS-T (TBS with 0.1% Tween-20) before incubation with alkaline phosphatase-conjugated goat anti-rabbit (H + L) secondary antibodies (Jackson ImmunoResearch, 111–055-144; dilution 1:500) in 2% NGS TBS-T for 2 h at room temperature. Sections were then washed three times for 5 min each in TBS-T before incubation in Vector Blue substrate, prepared by adding 2 drops of reagents 1, 2, and 3 to 5 ml of 100 mM Tris-HCl pH 8.2 (Alkaline Phosphatase Substrate Kit III, Vector Labs, SK-5003). The reaction was stopped by incubation of sections in 100 mM Tris-HCl pH 8.2 before the sections were washed five times for 3 min each in PBS. Sections were mounted onto slides and allowed to air-dry overnight. Slides were dehydrated by incubating for 3 min in ddH2O, then for 1 min each in 70, 95, and 100% EtOH, and finally two times for 3 min each in Histoclear (Harleco, 65,351). Vectamount (Vector Labs, H-5000) was applied prior to coverslip application.
Immunofluorescent staining was performed by washing free-floating sections with PBS-T (0.2% Tween-20 or 0.1% Triton X-100) three times for 5 or 10 min each at room temperature. To detect total α-synuclein, sections were then incubated in blocking solution (2% BSA, 10% normal goat serum in PBS-T) for 1 h at room temperature. After blocking, sections were incubated with rabbit anti-TH antibodies (ThermoFisher Scientific, AB152; dilution 1:1000) and anti-α-synuclein mouse monoclonal antibodies specific for human α-synuclein (Syn211, ThermoFisher, 32–8100; dilution 1:500) in antibody solution (2% normal goat serum in PBS-T) overnight at room temperature. Sections were washed in PBS-T and incubated with secondary fluorescent antibodies in antibody solution for 1 h in the dark at room temperature. Secondary antibodies were Alexa Fluor goat anti-rabbit 594 (Invitrogen, A11037; dilution 1:500) and Alexa Fluor goat anti-mouse 488 (Invitrogen, A11029; dilution 1:500). To detect α-synuclein oligomers, sections were treated with 1 M glycine for 30 min and then incubated in blocking solution (1% BSA, 10% normal goat serum in PBS-T) for 1 h at room temperature. After blocking, sections were incubated with chicken anti-TH antibodies (Abcam, ab76442; dilution 1:1000) and Syn-O2 antibodies (dilution 1:5000) in blocking solution overnight at room temperature. Syn-O2 is a mouse monoclonal antibody which specifically recognizes early soluble oligomers and late fibrils of α-synuclein, and Syn-O2 has a high binding affinity for oligomeric α-synuclein . Sections were washed in PBS and incubated with biotin-conjugated goat anti-mouse secondary antibodies (Jackson ImmunoResearch, 115–065-146; dilution 1:500) in PBS for 1 h at room temperature. Sections were washed in PBS and incubated with Alexa Fluor goat anti-chicken 594 (Invitrogen, A11042; dilution 1:500) and Alexa Fluor 488 streptavidin (Invitrogen, S32354; dilution 1:500) in PBS for 1 h in the dark at room temperature. Sections were washed in PBS, mounted onto glass slides, and allowed to dry. Fluorescence mounting medium (DAKO) was applied, followed by coverslip application. Appropriate targeting of AAV-A53T injection in the SN was based on the findings from immunostaining human α-synuclein; animals were considered mistargeted and thus excluded from analyses if their SN cells were not transduced with AAV-A53T.
Image analysis of brain cryosections
Imaging analyses were performed by a researcher blinded to the drug treatments. Confocal images of immunofluorescent staining were acquired with a Zeiss LSM700 confocal microscope equipped with 405, 488, 555, and 639 nm laser lines. All images were taken within the linear range at constant gain and the pinhole settings at optimal resolution settings determined by the software. The whole midbrain regions were imaged using a 10X objective. Four serial coronal midbrain sections were imaged per animal, separated by 240 μm intervals. Confocal images of immunofluorescent stained midbrain sections were processed using HALO software (Indica Labs). Injected SN was selected as a region of interest (ROI), and dopaminergic neurons were identified by automated detection of TH-labelled cells within this ROI. Dopaminergic neuron densities, which correlate with neuronal counts obtained by conventional stereology as previously described , were estimated using HALO software. Levels of total human α-synuclein (detected by Syn211 staining) or α-synuclein oligomers (detected by Syn-O2 staining) were assessed in this ROI by measuring fluorescence intensity.
Stereology of brain cryosections
The optical fractionator method was used for the unbiased stereological estimation of dopaminergic (i.e., TH-positive) cell counts in the injected and non-injected SN (Stereo Investigator software version 9, MBF Biosciences). The investigator was blinded to the experimental groups. Every sixth section throughout the SN was quantified (7 sections total). The injected or non-injected SN was selected as a ROI bounded at a 10x objective, and counting was performed using a 40x oil-immersion objective. A guard zone thickness of 2 μm was set at the top and bottom of each section. The sampling interval in the X-Y coordinate axis was set as follows: 175 μm × 175 μm counting frame size; 300 μm × 200 μm grid size; 20 μm dissector height. Coefficient of error was calculated according to Gundersen and Jensen , and values < 0.10 were accepted.
Real-time quaking induced conversion (RT-QuIC) assay
A modified version of the α-synuclein RT-QuIC assay described by Barger et al.  was used. Dilution of rat CSF (1:10) was performed with 1x N2 supplement (Thermo Fisher) diluted with PBS. Black 96-well, clear bottom plates were pre-loaded with six 1 mm in diameter silica beads per well (OPS Diagnostics). Lyophilized recombinant human α-synuclein (Sigma) was reconstituted to 1 mg/ml with filtered ddH2O and centrifuged for 10 min at 4 °C through 100 kDa Amicon filters (Millipore). Diluted rat CSF (15 μl) was added to individual wells containing 85 μl of RT-QuIC reaction mixture composed of 40 mM NaPO4 pH 8.0 (Boston BioProducts), 170 mM NaCl, 0.1 mg/ml recombinant α-synuclein, 20 μM ThT (Sigma), and 0.0005% SDS. The plates were sealed with clear plastic film (Thermo Fisher) and incubated at 42 °C in Clariostar plate reader (BMG Labtech) with cycles of 1 min shaking (400 rpm, double orbital) and 1 min rest throughout the indicated incubation time. ThT fluorescence (450 nm excitation and 480 nm emission) was recorded every 45 min for 60 h. Replicate reactions were run in four separate wells for each sample. Positive RT-QuIC reactivity of individual wells was defined as enhanced ThT fluorescence above a predefined threshold at 60 h. This threshold was calculated as the mean background fluorescence of the diluent plus 5 standard deviations, equal to approximately 3200 relative fluorescence units (rfu) in our experiments. A sample was considered positive when at least one of the four replicates displayed positive ThT reactivity above the threshold. For each positive sample, the mean fluorescence intensity from the positive replicates was calculated and plotted against time. For each negative sample, the mean fluorescence intensity from the negative replicates was calculated and plotted against time.
All statistical analyses were conducted using GraphPad Prism 6 or 8. Mean ± SEM with individual replicates are presented when appropriate. The specific statistical tests performed are indicated.