Materials

Rabbit anti-phospho-synuclein (S129) antibody (1:1000) and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (1:500), were obtained from Abcam (ab51253, Abcam) and Abcam (ab7260, Abcam), respectively. CSB was purchased from Tocris (0846, Tocris). Bafilomycin A1 was purchased from Calbiochem (196000, Calbiochem).

Animals

Adult (6 months old) male and female PrP-A53T mice [G2-3 line, Tg(Prnp-SNCA*A53T)23Mkle/J, C57BL6/J strain] and their wild-type littermates (male and female) were used. All experimental procedures described below were performed in accordance with Seoul National University (Republic of Korea) Institutional guidelines for the care and use of experimental animals (IACUC number: SNU-190721-1-10).

Cell culture

Cell lines stably expressing each half of the split Venus fluorescent system (V1S and SV2) for testing cell-to-cell transmission of α-synuclein were generated as described previously [18]. V1S and SV2 cells were subcultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; SH30243.01, Hyclone) supplemented with 10% fetal bovine serum (FBS; SH30919.03, Hyclone) and 100 units ml−1 penicillin/streptomycin (GIB-15070-063, Gibco), additionally containing 200 μg ml−1 G418 (11811-031, Invitrogen). Cultures were maintained at 37 °C in a humidified 5% CO2 atmosphere, and the culture medium was replaced every 2 days.

Human neuroblastoma SH-SY5Y cells (CRL-2266, ATCC) were cultured and maintained at 37 °C in a humidified 5% CO2 atmosphere with media exchange every 2 days. Cells were differentiated by culturing in growth medium (DMEM + 10% FBS + 100 units ml−1 penicillin/streptomycin) containing 50 μM retinoic acid (R2625, Sigma Aldrich).

High-content screening of drugs in an α-synuclein BiFC cell model

The effects of drugs on α-synuclein propagation were determined using a dual cell bimolecular fluorescence complementation (BiFC) method employing α-synuclein–conjugated split Venus fragments, which are expressed in separate cell lines (V1S and SV2 cells) and after cell-to-cell transfer, reassemble to form a fluorescent protein upon interactions of the conjugated protein [18]. V1S and SV2 cells were first cultured for 6 passages and then seeded onto a flat-bottom, 96-well black plate (655090, Greiner Bio-One) at 8 × 104 cells per well. After 24 h, cells were incubated with 10 μM CSB at 37 °C for 48 h. CSB-treated cells were then incubated at 37 °C for 10 min with 10 μg/ml Hoechst 33342 (H1399, Invitrogen) to stain nuclei and with Topro-3 iodide (T3605, Invitrogen), diluted 1:1000, to detect dead cells. Topro-3 is only permeable in cells undergoing necrosis or apoptosis. Cells were washed once with DMEM, after which new medium without phenol red was added. Venus BiFC fluorescence in live cells was measured by imaging with an automated high-content screening reader (In Cell Analyzer 2200; GE Healthcare), and images were analyzed using the In Cell Developer Toolbox software. The concentration dependence of CSB-mediated modulation of α-synuclein cell-to-cell transmission was analyzed by incubating 1000 cells with different concentrations (1 nM, 10 nM, 0.1 μM, 1 μM and 10 μM) of CSB at 37 °C in a humidified CO2 environment. Three independent experiments were performed.

Cell viability test

Cell viability was assessed by seeding differentiated SH-SY5Y cells overexpressing α-synuclein on 6-well plates and then adding CSB (1 nM, 10 nM, 0.1 μM, 1 μM, 10 μM) to each well and incubating at 37 °C for 2 days. After collecting cells by trypsinization, 2 μl of propidium iodide, which binds to DNA and is commonly used to detect dead cells, was added to 18 μl of cell suspension and the fluorescence of the resulting samples was measured using a LUNA cell counter (Logos Biosystems).

Preparation of recombinant α-synuclein fibrils

Human α-synuclein was expressed in Escherichia coli BL21 DE3, and α-synuclein fibrils were prepared as described [19]. Briefly, human α-synuclein protein was induced by adding IPTG (isopropyl β-d-1-thiogalactopyranoside) to a final concentration of 0.1 mM and incubating cells for 3 h at 37 °C. The cells were centrifuged and resuspended in 20 mM sodium phosphate buffer (pH 7.4), then sonicated, boiled, and centrifuged at 10,000×g for 10 min at 4 °C. The supernatant was subjected to anion-exchange chromatography and Superdex-200 gel filtration column chromatography, after which fractions containing purified α-synuclein were immediately dialyzed and lyophilized. α-Synuclein polymerization reactions were performed using 200 μM α-synuclein monomers after filtration of the monomers by using a 100,000 MWCO (molecular weight cut-off) filters. α-synuclein was incubated with CSB or a vehicle control solution at 37 °C for 9 days with constant shaking at 1050 rpm in a Thermomixer C (5382000015, Eppendorf).

Thioflavin-T binding assay

Amyloid fibril formation was assayed using thioflavin T (ThT), which binds to β-sheet–rich structures in the presence of amyloid fibrils. Specifically, 40 μl of 10 μM recombinant α-synuclein was added to 50 μl of a 10 μM Thio-T solution in glycine–NaOH (pH 8.5). After incubating for 5 min at room temperature, fluorescence was measured at 450 nm (excitation)/490 nm (emission) using a microplate reader (Synergy Neo, BioTek).

Circular dichroism

For evaluation of the secondary structure of α-synuclein protein incubated with CSB, samples were diluted in PBS to 0.5 mg/ml and analyzed using a circular dichroism detector (Chirascan Plus).

Transmission electron microscopy (TEM)

Samples of α-synuclein with CSB (0.1, 1, or 10 μM) or vehicle (DMSO) were applied to 200 mesh carbon-coated copper grids, then negative-stained by placing 10 μl of a 2% uranyl acetate solution on the grid for 5 min. Samples on grids were visualized using a JEM-1400 transmission electron microscope (JEOL).

NMR spectroscopy

Expression and purification of 15N-labeled α-synuclein protein was performed as described previously [20]. For NMR experiments, 15N-labeled α-synuclein was dialyzed against buffer containing 100 mM NaCl, 50 mM HEPES, and 0.02% NaN3 (pH 7.4). 1H-15N heteronuclear single-quantum coherence (HSQC) experiments were recorded at 15 °C on a 600 MHz Bruker NMR spectrometer using the Bruker pulse program, hsqcetf3gpsi. 1H-15N HSQC experiments were performed using α-synuclein (80 μM) alone and in the presence of CSB at α-synuclein:CSB ratios of 1:0.1, 1:0.2, 1:0.5, 1:0.8, 1:1, 1:2 and 1:5. The combined 1H/15N chemical shift perturbation was calculated according to [(δH)2 + (δN/5)2]/21/2, where δH and δN are the chemical shift values of 1H and 15N respectively. In addition, NMR signal intensities were fitted assuming a simple two-state exchange model, and the dissociation constant (Kd) was derived from a global fit to the intensity decay curves of selected residues (residues 1–9, 12, 14–20) according to the following relationship:

$$\left( }}} \right) = I_ \left[ + x + K_ } \right) - \sqrt + x + K_ } \right)^ - 4P_ x} }} }}} \right],$$

where I is the intensity value along the titration, I0 is the intensity value of the free state, P0 is the total amount of protein, Kd is the dissociation constant, and x is the concentration of α-synuclein (in μM) along the titration. Errors were estimated by evaluating the standard deviation of the intensity according to the following:

$$\sigma_ = \left( }}} \right)\sqrt } \right)^}} + \left( }} }}} \right)^ } ,$$

where σI and σI0 are the standard deviations of the noise in the spectra.

Animal treatment

Transgenic mice (Tg) expressing A53T human α-synuclein under control of the cellular prion protein (PrP) promoter were used. Briefly, adult (6-month old) Tg mice and wild-type littermate controls were injected intraperitoneally with either CSB (20 mg/kg/day) or PBS once a week for 3 months.

Immunohistochemistry

Adult mice were deeply anaesthetized with ketamine:Rompun (3.5:1; 2.5 μl g−1) and perfused with PBS followed by 4% paraformaldehyde (PFA; P6418, Sigma-Aldrich). Excised brains were post-fixed overnight in 4% PFA at 4 °C. Coronal section (40 μm) were cut with a vibratome, rinsed three times in PBS and then once with 3% H2O2 (Sigma-Aldrich) to quench endogenous peroxidase. Sections were washed with PBST (0.1% Triton X-100 in PBS) three times and incubated with blocking solution (4% bovine serum albumin in PBST) for 1 h at room temperature. Sections were incubated overnight in a mixture of rabbit anti-phospho-synuclein (S129) (ab51253, Abcam) and rabbit GFAP (ab7260, Abcam) primary antibodies, diluted 1:1,000 and 1:500, respectively, in blocking solution. The next day, sections were incubated with species-appropriate horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h and 30 min at room temperature and then washed three times. Following incubation with an avidin–biotin complex (Vectastain ABC kit; PK6200, Vector Laboratories), immunocomplexes were visualized using 3,3′-diaminobenzidine (DAB; D5637, Sigma-Aldrich) with H2O2. Sections were mounted on gelatin-coated slides using Canada balsam (C1795, Sigma-Aldrich).

Open field test

Locomotor activity in an open field (40 × 40 cm) was monitored in 9-month old mice using a video tracking system (EthoVision XT14; Noldus, Netherlands) that detects the center of gravity of the mouse in the arena. Spontaneous horizontal activity (total distance travelled) in the center and peripheral zone were measured for 10 min.

Forelimb grip strength test

The forelimb grip strength test provides a measure of the neuromuscular activity of mice. In this test, the maximal force of the forelimbs of mice hanging on a metal grid surface was measured twice. Mean forelimb grip strength values were normalized to mouse body weight.

Balance beam test

Motor balance and coordination were assessed by monitoring walking of familial Parkinson’s disease model mice across a beam apparatus (length, 1 m; width, 2 cm; height, 50 cm). Mice were habituated to a black box (finish point) for 2 min and trained to walk on the beam, after which they were placed at one end of the beam and the time to reach the black box at the other end and the number of slips were recorded.

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 7.04, SPSS and ImageJ software. All data are presented as means ± SEM. The significance of differences among means was assessed using a one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test or two-way ANOVA with Tukey’s post-hoc test. p-values < 0.05 were considered significant; individual p-values (*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001) are shown in figure legends.