Human brain tissue
For immunohistochemical analysis, formalin-fixed, paraffin-embedded 6-μm-thick sections from the frontal lobe of the cerebral cortex, pons, cerebellum, and putamen of patients with MSA, Parkinson’s disease (PD)/diffuse Lewy body disease (DLB), and non-neurodegenerative disease controls were deparaffinized and immunostained (see Additional file 1: Table S1 and Additional file 2: Fig. S1 for detailed description of the clinical information and studied regions in each case). Cases with more than 1-month duration of premortal ventilation or a history of sustained generalized seizures were excluded from our analysis. Analysis of oligodendroglial cell populations and p-α-syn pathology in the frontal cortex as well as that of the white matter in the corresponding frontal lobe were performed in nine MSA cases, nine PD/DLB cases, and six controls (Fig. 1; Additional file 3: Fig. S2A–E). BCAS1 and phosphorylated α-syn staining of the putamen, pons, and cerebellum was conducted in four MSA cases and two controls (Additional file 3: Fig. S2A). For the detection of cytoplasmic inclusions using immunofluorescence staining, cases with a cortical α-syn-pathology score of “stage 2 (moderate)” or greater (MSA; N = 4, PD/DLB; N = 7), which is the criteria used for the diagnosis of DLB, were used to compare the emergence of α-syn-pathology in BCAS1(+) cells (Fig. 2; Additional file 3: Fig. S2F–I) [14]. The detection of such inclusions in BCAS1(+) cells in the pons was investigated in one MSA case (case 10; Additional file 1: Table S1; Additional file 3: Fig. S2J). All procedures involving the use of human materials were performed in accordance with the ethical guidelines set by Kyoto University.
Immunohistochemistry
After heat-induced antigen retrieval with citrate buffer (pH 6.0), human brain sections were incubated overnight at 4 °C with primary antibodies against phosphorylated α-syn (1:1000, Abcam, ab51253), BCAS1 (1:1000, Thermo Fisher, PA5–20904), MBP (1:1000, Thermo Fisher, MA1–10837), and NeuN (1:1000, Merck Millipore, ABN78). For visualization with secondary antibodies, Histofine (Nichirei Bioscience) was used for diaminobenzidine staining. In addition, Kluver–Barrera staining of each section was used to distinguish the cortical areas from white matter. For MBP- and phosphorylated α-syn-immunoreactive area quantification and total BCAS1(+) and NeuN(+) cell count, average values per each 1.57 mm2 visual field were calculated using Image J analysis, with 10–14 images captured from each case. Late-stage BCAS1(+) cells were defined morphologically as BCAS1(+) cells with four or more processes and included cells with myelinating morphology. Early BCAS1(+) cells were defined as those with three or less processes.
Double-staining of DAB and alkaline phosphatase (AP)
For double staining of DAB and AP, the following primary antibodies were used: NaBC1, 1:1000, Santa Cruz, sc-136,342; BCAS1, 1:1000, Thermo Fisher, PA5–20904; NG2, 1:50, Millipore, AB5320; and APC, 1:50, OP80, CalBio. Antigen retrieval was performed by autoclaving for 20 min at 120 °C using the Histofine deparaffinizing antigen retrieval buffer, pH 6 (Cat No. 415281; Nichirei, Tokyo, Japan) followed by overnight incubation at 4 °C with the first primary antibodies in phosphate-buffered saline (PBS) containing 3% bovine serum albumin. The samples were then washed three times with 0.01 M PBS with Triton X (PBS-T). Subsequently, the sections were incubated with peroxidase-conjugated anti-mouse IgG (Histofine Simple Stain Max-PO (M), Nichirei, Tokyo, Japan) for 30 min at 37 °C. Reaction products were visualized by immersing the slides in DAB solution (DAB Substrate Kit SK 4100, Vector Laboratories, Burlingame, CA, USA) for 3 min. After the sections were washed with deionized water, the second primary antibody was applied and incubated overnight at 4 °C. The sections were then washed three times with PBS-T before incubating with alkaline phosphatase-conjugated anti-rabbit IgG antibody (Histofine Simple Stain AP(R), Nichirei, Tokyo, Japan) for 30 min at 37 °C. The sections were again washed three times with 0.01 M PBS-T. Finally, the sections were visualized using Fast Red Substrate Kit (Cat No. 415261; Nichirei, Tokyo, Japan) according to the manufacturer’s instructions.
Immunofluorescence staining
For human samples, antigen retrieval was performed as described above, and sections were incubated overnight at 4 °C with primary antibodies against phosphorylated α-syn (1:200, Wako, 015–25,191), BCAS1 (1:200, Thermo Fisher, PA5–20904), cleaved caspase-9 (h331) (1:200, Santa Cruz, sc-22,182), MBP (1:200, Thermo Fisher, MA1–10837), and α-syn (1:200, BD Biosciences, 610,787). Samples were subsequently incubated with donkey-derived secondary antibodies (Alexa Fluor 488, 594, and 647, 1:200, Thermo Fisher) for 1 h at room temperature. Sections were then covered with VECTASHIELD mounting medium (Vector Laboratories, H-1200) with DAPI. For thioflavin S assessment, sections were incubated with 20 μM thioflavin S (Sigma Aldrich, T1892) in distilled water for 20 min at room temperature before mounting. Images were obtained using an Olympus Fluoview FV1000 confocal microscope (Olympus), with image analysis and 3-D surface reconstruction performed by FV10-ASW software (Olympus). Sections were imaged at 0.46 μm in the z dimension. For the quantitative evaluation of α-syn immunoreactivity within the BCAS1(+) cells, a total of 60–120 cells, which included 5–10 late-stage BCAS1(+) cells per each case, were analyzed three-dimensionally.
For the immunofluorescence analysis of primary rat cell and slice cultures, samples were fixed with 4% PFA for 15 min. After a 10-min incubation with PBS/0.1%Tween, the samples were incubated overnight at 4 °C with primary antibodies against BCAS1 (1:200, Bioss antibodies, bs-11462R), rat and human α-syn (1:200, BD Biosciences, 610,787), human α-syn (1:200, Thermo Fisher Scientific, 180,215), PDGFRα (1:200, R&D systems, AF1062), MBP (1:200, Thermo Fisher, MA1–10837), NG2 (1:200, Millipore, AB5320), CC1 (APC) (Calbiochem, OP80), Olig2 (1:200, R&D, AF2418), and cleaved caspase-9 (Asp330) (1:200, Affinity, AF5244). Visualization with secondary antibodies and thioflavin S staining were conducted as described above. The intracellular immunoreactivity of α-syn and its colocalization with the LysoTracker probe were confirmed by confocal microscopy as described above. The quantitative assessment of intracellular inclusions in OLG lineage cells was performed with BZ-X710 (Keyence). The average counts of the cells containing thioflavin S-positive inclusions were obtained from four visual fields captured at a 20 × magnification, and compared between PDGFRα(+)/BCAS1(−) and PDGFRα(+)/BCAS1(+) cells as well as between BCAS1(+)/MBP(+) and BCAS1(−)/MBP(+) cells.
Primary OLG lineage cell culture
OPCs were prepared as previously described [8, 10]. Briefly, the cerebral cortices from 1- to 2- day-old Sprague Dawley rats (Shimizu Laboratory Supplies Co., Ltd) were dissected, minced, and digested. Dissociated cells were plated in poly-D-lysine-coated 75 cm2 flasks, and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 20% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin. When the cells became confluent (~ 10 days), flasks were shaken for 1 h on an orbital shaker (220 rpm) at 37 °C to remove microglia. The flasks were then changed to a new medium and shaken overnight (~ 20 h). After a 1 h incubation of the collected medium at 37 °C on non-coated cell culture dishes, the non-adherent cells (OPCs) were re-plated at a density of 20,000 cells/cm2 in Neurobasal medium containing 2 mM glutamine, 1% penicillin/streptomycin, 10 ng/mL PDGF-AA, 10 ng/mL FGF-2, and 2% B27 supplement onto poly-DL-ornithine-coated plates. To differentiate the OPCs into mature OLGs, the culture medium was replaced with DMEM containing 1% penicillin/streptomycin, 10 ng/mL CNTF, 15 nM T3, and 2% B27 supplement. For chronological assessment of each cell marker’s expression during differentiation, the cells were fixed or collected at different time points indicated by the day from differentiation induction and then further analyzed.
Preparation of recombinant human α-syn pre-formed fibrils (PFFs)
The purification of recombinant human α-syn was conducted in accordance with previously established method [8, 12, 20]. Briefly, human wild-type α-syn cDNA was cloned into the bacterial expression vector pRK172. Transformations and selection were performed using E. coli BL-21 (DE3) competent cells (BioDynamics) and ampicillin (100 μg/mL) in Luria-Bertani media. Following the overnight incubation of the transformed cells in Luria-Bertani media containing ampicillin (100 μg/mL) at 37 °C, the culture was incubated for another 5 h after a 300-fold dilution and then induced with 1 mM isopropyl-β-D-thiogalactopyranoside for 5 h at 37 °C. Bacterial pellets were then resuspended in high-salt buffer (1 M Tris-HCl, pH 7.5, and 1 mM EDTA), heated to 100 °C for 5 min, and centrifuged at 15,000 rpm for 15 min. The supernatants were subjected to chromatography on a Q-Sepharose fast-flow column (GE healthcare) with a gradient of 0 to 0.5 M NaCl in Tris buffer. Resulting proteins were dialyzed overnight against 50 mM Tris-HCl, 150 mM KCl, and pH 7.5 and centrifuged at 55,000 rpm at 4 °C for 20 min. The removal of endotoxin was performed with EndoTrap HD (800,053, Hyglos), and the concentration of lipopolysaccharide was confirmed to be less than < 0.035 EU/μg αS protein using the LAL endotoxin assay kit (L00350C, GenScript). For PFF generation, proteins were incubated with constant agitation at 37 °C for 3–7 days.
Application of α-syn PFFs to primary oligodendroglial cell culture
To observe intracellular inclusions in OLG lineage cells (Fig. 3, Fig. 4a, Additional file 5 Fig. S4A), α-syn PFFs were diluted in PBS at 1 μM, sonicated several times (60 s in total), and diluted in media. Protein concentrations were determined using the bicinchoninic acid protein assay (Thermo Fisher), with bovine serum albumin as the standard. To evaluate the cell viability and the maturation of differentiating OLG lineage cells exposed to pathological α-syn (Fig. 4b–f), 3 μM α-syn PFFs was added to the culture medium at different time points (day 0–1 or day 3–4 from differentiation induction) and incubated for 24 h. After incubating with α-syn PFFs, cells were washed with DMEM containing 1% penicillin/streptomycin once to remove residual α-syn PFFs. The cells were then incubated with α-syn-free differentiation medium until day 8, at which point they were subject to the WST assay and immunoblot analysis.
Protein labeling and time-lapse imaging
After sonication, α-syn PFFs (2 mg/mL) were labeled with the Alexa Fluor 594 protein labeling kit (Thermo Fisher, A10239) according to the manufacturer’s instructions. Time-lapse imaging was performed with a BZ-X710 camera (Keyence) equipped with an incubator (37 °C, 5% CO2), and images were acquired at defined positions every 10 min. The images were then converted to mp4 files. Immediately after time-lapse imaging, cells were fixed and subjected to immunostaining to validate the maturation status of the imaged cells (Additional file 5: Fig. S4B, C).
Slice culture
Rat cortical slices were prepared from Sprague Dawley rats (Shimizu Laboratory Supplies Co., Ltd) on the 5th day after birth. The brains were removed and immediately submerged in cold HBSS with 0.6% D-glucose and 1% penicillin/streptomycin. Coronal slices from the cortices (100 μm) were cut using a vibrating-blade microtome (Neo-LinearSlicer, D.S.K). Slices were placed onto a 0.4 μm culture plate insert (Millicell-CM, Merck Millipore) and incubated for 24 h in six-well culture dishes containing 3 μM α-syn PFFs in 1.5 mL of DMEM containing 2% B27 supplement and 1% penicillin/streptomycin.
Lysotracker
For the use of the LysoTracker (Life technologies) probes, samples were incubated with probe-containing medium (50 nM) for 30 to 60 min before fixation. Immunostaining was conducted as described above.
WST assay
Cell viability was assessed by the WST reduction assay kit (Cell Counting Kit-8, Dojindo). Briefly, the cells were incubated in a 10% WST solution for 1 h at 37 °C. The absorbance of the culture medium was measured at a wavelength of 450 nm and a reference wavelength of 630 nm.
Immunoblot analysis
For immunoblot analysis, cells were rinsed twice with PBS and collected into sample buffer containing 50% Tris-Glycine SDS buffer (Novex), 45% RIPA buffer (20 mM HEPES-KOH pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Nonidet-P40, and 1% sodium deoxycholate), 5% 2-mercaptomethanol (Nacalai tesque), 1% phosphatase inhibitor (Nacalai tesque), and 1% protease inhibitor (Nacalai tesque). Subsequently, the samples were heated at 95 °C for 5 min, and each sample was loaded onto a 5–20% polyacrylamide gel (Wako). After electrophoresis and the transfer of the gels onto PVDF membranes (Merck Millipore), the membranes were fixed with 4%PFA for 30 min and blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat dry milk for 30–60 min at room temperature. The membranes were then incubated overnight at 4 °C with primary antibodies for MBP (1:500, Thermo Fisher Scientific, MA1–10837), BCAS1 (1:1000, Bioss antibodies, bs-11462R), α-syn (1:1000, BD Biosciences, 610,787), PDGFRα (1:500, Santa Cruz, sc-338), or anti-β-actin (1:10000, Sigma Aldrich, A5441). This was followed by a 60 min incubation with the appropriate secondary antibodies (Novus, NB7574 and NB7160) at room temperature, with visualization by enhanced chemiluminescence (Nacalai tesque). The density of each band was quantified using Image J software.
Quantitative real-time PCR
For quantitative real-time PCR, RNA was extracted from the cells with RLT lysis buffer (QIAGEN) according to the manufacturer’s instructions. RNA concentration was measured using a NanoDrop 1000 spectrometer (Thermo Scientific). cDNA was generated using reverse transcription and the PrimeScript RT reagent kit (TaKaRa). The amount of cDNA was quantified with real-time PCR using the LightCycler 480 SYBR Green I Master mix (Roche) and a Roche LightCycler 480 system. The primer sets used in this study were as follows: Pdgfrα, forward: CTAATTCACATTCGGGAAGGTTG, reverse: GGACGATGGGCGACTAGAC, Bcas1, forward: AGGGAAGGACATAGTGGACAGC, reverse: TTCTCGGCGTCCGTGTCTT, Mbp, forward: ACACACAAGAACTACCCACTACGG, reverse: AGCTAAATCTGCTGAGGGACAG, Snca, forward: CAACAGTGGCTGAGAAGACC, reverse: GAAGGCATTTCATAAGCCTC.
Statistical analysis
All quantitative data were analyzed using Prism 6.0 (Graphpad). The statistical significance was evaluated using Mann-Whitney’s U test for two-group comparisons or one-way non-parametric ANOVA (Kruskal-Wallis test) for multiple-group comparisons. Data are expressed as the mean ± S.D. Spearman rank correlation coefficient analysis was used for the correlation analysis of phosphorylated α-syn-immunoreactive areas and late-stage BCAS1(+) cell counts.