Pathological analysis of α-synuclein by immunofluorescence staining

Neurons were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS, then blocked in 2% BSA, 5% normal goat serum in PBS-Triton for 30 min. LB509 (1:100) and anti-TH antibodies (1:100; Merck Millipore, 657012) were incubated overnight, washed in PBS-Triton, then AlexaFluor 568-conjugated anti-mouse IgG was used to detect α-syn,whereas AlexaFluor 488-conjugated anti-rabbit was used to detect TH. Next, thioflavin S (Thio S) staining was performed as previously described (Mazzulli et al., 2006). The number of cells with α-syn and Thio S in the cell body [as either a diffuse (type I) or punctated (type II) pattern, as characterized in our previous study; Mazzulli et al., 2016] were scored and normalized to total cells in the field-of-view (calculated by nuclear DAPI stain). Only the Thio S+ cells that colocalized with α-syn were counted. Neuritic pathology was calculated by counting the number of α-syn+ neurites that colocalized with Thio S, and divided by the total number of neurites in the field-of-view. At least three fields of view were used per coverslip, and 100–500 cells counted per condition, per cell line. Images were obtained on a Leica epifluorescence microscope using a 40× objective (DMI3000B). To determine synaptic localization of α-syn, neurons were fixed as described but permeabilized in 0.1% Triton-containing blocking buffer. Anti-synapsin antibodies (ThermoFisher, A6442; 1:50 dilution) with LB509 (1:100 dilution) were detected with AlexaFluor-conjugated secondary antibodies and analyze by confocal microscopy as described previously (Mazzulli et al., 2016). Synapsin/α-syn colocalized puncta were quantified from merged images and expressed as fold-change compared with control conditions. The quantification represents the mean (±SEM) from five different fields-of-view from different culture wells with 100–200 puncta counted per field.

H4 cells were seeded on cover glass at a cell number of 4 × 10⁴/24 wells. Cells were treated with DOX (2 μg/ml) for 5 d to turn off α-syn expression. For 758 analysis, H4 cells were cultured without DOX and treated with compound 758 (5 μm) or DMSO for 5 d. Cells were fixed in 4% PBS-buffered paraformaldehyde for 20 min and washed three times in PBS. Cells were permeabilized with 0.2% saponin (w/v)/PBS for 5 min, then with 0.2% (w/v) glycine/0.2% saponin in PBS, and blocked with 2% bovine serum albumin, 5% normal goat serum (Jackson Immunofluorescence) in 0.2% saponin/PBS for 30 min. Primary antibodies [anti-GCase, gift from Johannes Aerts, University of Leiden, NL; anti-LAMP2, Invitrogen, 51-2210; anti-protein disulfide isomerase (PDI) Abcam, ab11432] were diluted 1:100 in blocking buffer, incubated overnight at 4°C, then washed and incubated with secondary antibodies (AlexaFluor 568-conjugated anti-mouse, and AlexaFluor 488-conjugated anti-rabbit, 1:400 dilution). Cells were mounted on microscope slides using DAPI-Fluoromount G (Southern Biotech) and imaged using a Leica confocal microscope as described previously (Mazzulli et al., 2016). Images were analyzed by ImageJ software using the colocalization plugin Coloc 2. Pearson correlation values of GCase + LAMP2, or GCase + PDI were obtained from six fields-of-view from different culture wells for each condition, and data were expressed as fold-change compared with the control condition. The quantifications represent the mean ± SEM.