A mutation (V40G) was introduced into human wild-type α-synuclein (α-synuclein/pDual GC; Agilent Technologies, Santa Clara, CA, USA, #214503) using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies, #200521. The primers used were the following: 5′- GGT GTT CTC TAT GGC GGC TCC AAA ACC AAG − 3′ (sense), 5′- CTT GGT TTT GGA GCC GCC ATA GAG AAC ACC − 3′ (antisense).
Protein purification and fibril preparation
The pDdulGC vector expressing mouse wild-type α-synuclein, human wild-type α-synuclein (WT), or V40G α-synuclein was transformed in Escherichia coli BL21(DE3) (RBC Korea, Seoul, Korea, #RH217). WT-syn and V40G were expressed and purified as previously described 28. For fibrillation, α-synuclein (200 µM in PBS) was incubated at 37°C for 9 days with constant shaking at 1,050 rpm in a ThermoMixer C (Eppendorf, Hamburg, Germany, #5382000015). When used as seeds, the fibrils were sonicated for 1 min (amplitude 30%) before undergoing the fibrillation reaction 28.
Circular Dichroism (CD) spectroscopy
All CD spectra of protein samples (0.5 mg/ml) were recorded using a Chirascan Plus spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK) between 190 and 260 nm in 0.1 mm cells with a step resolution of 1.0 nm, bandwidth of 1.0 nm, and scan speed of 100 nm/min. All spectra were obtained on an average of 10 separate measurements.
Transmission electron microscopy
The aged WT-syn and V40G were adsorbed onto 200-mesh carbon-coated copper grids (Electron Microscopy Sciences, Hatfield, PA, USA, #CF200-Cu), then subjected to add 20 µl of 2% uranyl acetate (Electron Microscopy Sciences, #22400) for negative staining. The prepared grids were observed using a JEM1010 transmission electron microscope (JEOL, Akishima, Tokyo, Japan).
Fluorescent dye binding assay
Recombinant α-synuclein samples were mixed either 50 µl of 10 µM Thio T (Sigma, #T3516) solution in glycine (Fisher Scientific, Hampton, NH, USA, #BP3815)–NaOH (pH 8.5), 40 µl of 0.001% Sybr Green commercial stock solution (#S7563, Invitrogen, Carlsbad, CA, USA), 50 µM X-34 (Sigma, #SML1954), or 100 µl of 50 µM curcumin (Sigma, #C1386). After incubation at room temperature, fluorescence measurements were performed on Synergy NEO plate reader (Biotek, Winooski, VT, USA). Excitation and emission wavelengths were set at 440 nm and 490 nm, respectively, for Thio T, at 485 nm and 520 nm for Sybr Green, at 380 nm and 520 nm for X-34, and at 440 nm and 519 nm for curcumin.
Twenty microliters of human WT and V40G α-synuclein monomer and fibril (200 µM in PBS) were each used for sedimentation assays. Twenty microliters of samples were mixed with 280 µl of DPBS (Gibco, Carlsbad, CA, USA, #A1285601) and placed on top of 30% sucrose, bottom of the tube being 5% sucrose. Samples were centrifuged at 38,000 rpm in a Beckman XL-90K ultracentrifuge using a SW-41Ti rotor (Beckman). Fractions were collected and mixed with Laemmli sample buffer.
Proteinase K (PK) digestion
α-Synuclein samples (5 µM) were incubated with a final concentration of 10 µg/ml PK (Sigma, #P4850) for 1 h 20 min at 37°C. The reactions were stopped by the addition of Laemmli sample buffer, followed by heating at 95°C for 10 min.
Protein misfolding cyclic amplification (PMCA)
PMCA was performed as previously described 28. α-Synuclein monomers were prepared up to a final concentration of 5 µM in conversion buffer (1% Triton X-100, 150 mM NaCl), and 50 µl of aliquot was transferred into PCR tube containing three Teflon beads. Samples were subjected to 48 cycles of 20 s sonication (amplitude 1%) and 29 min 40 s incubation at 37°C for 24 h. Fifteen nanograms of aged WT-syn or V40G were added as exogenous seeds.
Western blotting was performed as previously described with a few modifications 28, 29. In the case of PK digestion experiments, PK-treated samples were loaded onto 16% gels. Primary antibodies and their dilutions were: anti-α-synuclein monoclonal antibody (Syn-1, BD Biosciences, #610787; 1:1500), anti-α-synuclein antibody (LB509, Abcam, MA, USA; ab27766, Abcam, 1:1000), NLRP3 (EPR23094-1, Abcam, ab263899, 1:1000). The membrane was detected with ECL solution (GE Healthcare, # RPN2232). Image detection was performed using an Amersham Imager 600 (GE Healthcare) and Multi Gauge (v.3.0) software (Fujifilm, Akishima, Tokyo, Japan).
Ten-week-old male wild-type C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Three-month-old C57BL/6 mice overexpressing human α-synuclein under the murine Thy1 promoter (mThy1-α-syn tg, Line 61) were used for immune cell infiltration assay 30. Mice were housed and processed according to the standardized conditions at animal facility in the Seoul National University College of Medicine. All mice were maintained in the animal facility for habituation for at least one week before the start of the experiment. All mouse studies were conducted in compliance with the relevant ethics regulations and approved by the Seoul National University Ethics Committee (IACUC SNU-170428).
Stereotaxic injection of WT fibrils and V40G multimers
For intrastriatal injection of WT-syn fibrils and V40G multimers, 10-week-old male C57BL/6 mice were anesthetized with ketamine hydrochloride and xylazine hydrochloride (3.5:1, 2.5µl/g). PBS, WT-syn fibrils or V40G multimers (6 µg) in a volume of 2 µl were stereotaxically injected into the right striatum (anterior/posterior, 1.0 mm; medial/lateral, 1.5 mm; and dorsal/ventral, 3.0 mm) at a speed of 0.5 µl/min using a 30 G needle.
The administration of 2 mg/kg or 40 mg/kg acetylsalicylic acid (Aspirin; Sigma, #A5376) in drinking water was initiated 1 week after intrastriatal injection of PBS, WT-syn, or V40G. Monitoring the amounts of aspirin consumed, once every 2 weeks for 18 weeks, showed a consumption of 4.3–5.2 ml/day, and there was no significant difference in consumption between groups. The concentration of aspirin dissolved in drinking water provided for each animal was given at 12.5 µg/ml (2 mg/kg) or 250 µg/ml (40 mg/kg).
We subjected mice to total activity, motor control, motor strength, sensory spatial memory and emotional behavior tests 1 week prior to sacrifice. Behavioral tests were performed using a computerized video recording and tracking system (Ethovision XT version 14, Noldus, Wageningen, Netherlands).
Open field test
To assess activity, locomotion, and anxiety, we subjected mice to the open field test as previously described 31. The testing room was indirectly illuminated to 15–20 lux. The open field test apparatus was a square arena (40 × 40 × 40 cm) with white Plexiglas walls and floor. We placed the mice individually in the center, allowing them to freely navigate the arena for 10 min while their activity was video recorded.
Motor coordination and balance were assessed with an accelerating rotarod system (Rotamex 5, Columbus Ins., Columbus, OH, USA). Mice were placed on the rotarod spindle (3.0 cm × 9.5 cm), which accelerated from 4 to 35 rpm over 300 s, and the latency to fall was measured. After one practice using this protocol, each mouse was tested twice, and the average latency was taken.
Four-limb hanging test
The four-limb hanging test was used to measure the muscle strength of the four limbs. A grid apparatus (5 × 5 cm) was designed to allow the mice to grab the wire-mesh grid, and then the grid was inverted 180 degrees. The latency for the mice to fall from the grid was measured, with a maximum of 600 s.
Y maze test
To measure the prefrontal cortex- and hippocampus-dependent spatial memory deficits 32, we tested the spontaneous alternation behavior (SAB) and continuous alternation (CA) of mice using a standard protocol 33. The Y maze apparatus consisted of three arms (each 33 cm long) made of white plastic, joined at the center to form a Y shape. We placed the mice individually into one of the arms and allowed them to freely explore the three arms for 5 min while their activity was video recorded.
At 2, 4, 10 or 19 weeks after intrastriatal injection, mice were anaesthetized with ketamine hydrochloride and xylazine hydrochloride (3.5:1, 2.5µl/g) then perfused transcardially with saline followed by ice-cold 4% PFA. Brains were dissected out and fixed in phosphate-buffered 4% PFA for at least 48 h at 4°C for neuropathological analysis. For biochemical analysis, brain samples were dissected and stored at -80°C at 0, 2, 7, 14 days and 5, 19 weeks after intrastriatal injection. Four weeks after intrastriatal injection, the brain samples were dissected into the rhinal cortex before freezing on dry ice and stored at -80°C for RNA analysis,.
Evans blue assay
Two weeks after intrastriatal injection, mice were anaesthetized with ketamine/xylazine mixture, and the cardiac perfusion was performed using 50 ml PBS (pH 7.2) followed by 50 ml of the cocktail containing 1% Evans blue (Sigma–Aldrich) dissolved in 4% PFA. Brains were dissected out and fixed in phosphate-buffered 4% PFA for 4 h, cryoprotected in 30% sucrose overnight at 4°C, and then frozen in OCT medium on dry ice. Twenty-µm-thick brain cryosections were washed with PBST and mounted on fluorescent mounting medium containing DAPI (H1200, Vector Laboratorie, CA, USA). Then, visualized using fluorescence microscope (Olympus IX53) by excitation with 543-nm laser beams and visualized as red fluorescence.
Immunohistochemistry and neuropathological analysis
The procedures for immunohistochemical experiments have been described in detail elsewhere 34. Forty-µm thick free-floating brain sections were reacted with primary antibodies at 4 °C overnight prior to incubation with secondary antibodies (Bio-Rad, #170–6515, #170–6516, #5204 − 2504) diluted 1:200 in PBST, and detected using avidin-biotin-peroxidase complex (ABC Elite kit, Vector Laboratories, Burlingame, CA, USA, #PK6200). Next, 3,3-diaminobenzidine (DAB)-stained sections were imaged using a ZEISS AX10 microscope and an Aperio AT2 microscope. The levels of immunoreactivity against total human α-synuclein (Syn1; BD Biosciences, 1:500), phospho-α-synuclein (pS129; Abcam, #ab59264; Biolegend, CA, USA, #825701, 1:500), Iba-1 (Wako, Osaka, Japan, #019-19741, 1:200), glial fibrillary acidic protein (GFAP; Abcam, #ab7260, 1:500), tyrosine hydroxylase (TH; Abcam, #ab112, 1:2000), IL-1β (Abcam, #ab9722, 1:200), TNF-α (Abcam, #ab6671, 1:200), CD4 (BD Bioscience, #553727, 1:200) and CD8a (BD Bioscience, #553027, 1:200) were determined by optical density analysis using ImageJ (NIH) and corrected against background signal levels. Phospho-α-synuclein-positive cells in each animal were quantified as the percentage of positive cells in a field of view based on cell body recognition using the ImageJ program (NIH).
Immunofluorescence and Thioflavin S staining
Free-floating brain sections were blocked with 4% BSA in PBST and then reacted with primary antibodies phospho-α-synuclein (pS129; Abcam, #ab51253, 1:500), IL-1β (Abcam, #ab9722, 1:200), NeuN (Millipore, #MAB377, 1:500), GFAP (Abcam, #ab10062, 1:500) and Iba-1 (Novus Biologicals, CO, USA, #NB100-1028, 1:200) at 4°C light blocking overnight. The sections were washed with PBST, incubated with fluorescent dye Alexa488-, or Rhodamine red-X-conjugated secondary antibodies (Jackson Immunoresearch Laboratories, PA, USA, #115-545-062, #705-545-147, #111-295-144) diluted 1:200 in PBST, and mounted with fluorescence mounting medium (Vector Laboratories). The phospho-α-synuclein immunoreactive sections were treated with graded EtOH for hydration and incubated in filtered 1% aqueous Thioflavin-S (Sigma, T1892) for 8 min. After washing with 80% and 95% EtOH, coverslip was placed in aqueous mounting medium and slides were dried in the dark overnight. The stained samples were observed under a Carl ZEISS-LSM 700 confocal laser-scanning microscope.
Mouse brain slices were fixed using 2.5% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) at 4°C. Samples were fixed again by using 1% osmium tetraoxide for 30 min at 4°C. The fixed samples were dehydrated using an ethanol series (50%, 60%, 70%, 80%, 90%, and 100% ethanol) for 20 min and were transferred to LR White (Electron Microscopy Science, Hatfield, PA, USA). The samples were impregnated with and embedded in the same resin mixture, sectioned (60-nm-thick sections) with an ultramicrotome (Leica Ultracut UCT; Leica Microsystems, Vienna, Austria), and placed on nickel grids. α-synuclein fibril in the samples was labeled with immunogold by using phospho-α-synuclein antibody (pS129; Abcam, #ab51253, 1:50) and 9- to 11-nm colloidal gold-conjugated goat anti-mouse IgG secondary antibodies (Sigma, St. Louis, MO, USA). The sections were double-stained with 2% uranyl acetate for 10 min and lead citrate for 5 min and were viewed under the transmission electron microscope at 120 kV (Tecnai G2, ThermoFisher, Waltham, MA, USA).
Nigral tissue preparation and neuronal cell counting
Forty-µm thick free-floating brain sections were processed for immunohistochemistry as previously described 35. Rabbit anti-TH antibody (Millipore, Burlington, MA, USA, #AB152; 1:1000) was used as primary antibody prior to incubation in biotinylated goat anti-rabbit secondary antibody solution (Vector laboratories, #BA1000; 1:200). The sections were treated with avidin-biotin-peroxidase (ABC Elite kit, Vector Laboratories) complex. The color reaction was developed by DAB. Sections were then mounted on microscope slides, counterstained with cresyl violet (FD Neurotechnologies) and cover slipped using DPX mounting media (Sigma, #06522). Unbiased stereological counts were performed by an investigator blinded to the experimental groups using the optical fractionator and the Stereo Investigator 2019 software (MBF, version 1.3). The ipsilateral substantia nigra pars compacta was delineated on every 4th midbrain section in the rostro-caudal axis between Bregma − 2.7 mm and − 3.6 mm. Large and densely packed tyrosine hydroxylase-immunoreactive neurons characterized this midbrain region, allowing its delineation using a low-power objective lens (4X UPlanFL N) on an Olympus BX53 microscope equipped with an automated stage (MBF, mac6000) and a Heidenhein Z-axis decoder. The delineation excluded other tyrosine hydroxylase-positive cells in neighboring areas, namely the substantia nigra pars reticulata and pars lateralis (ventral and lateral to the compacta, respectively) and the ventral tegmental area (medial and dorsomedial). Counts were performed at higher magnification (100X UPlanS Apo) using a 1-µm guard zone on the top and bottom of each section. The coefficient of error was calculated according to Gundersen and Jensen 36; all values were < 0.10.
Brain tissue and cell extraction
Samples were homogenized with lysis buffer (1% Triton X-100, 1%(v/v) protease inhibitor cocktail (Sigma-Aldrich, P8340) in PBS. Lysates were incubated on ice for 10 min and centrifuge at 16,000g for 10 min. After collecting supernatant (Triton X-100 soluble fraction), Triton X-100 insoluble fraction was resuspended with 1X Laemmli sample buffer and sonicated briefly.
RNA was extracted using TRIzol (Invitrogen, #15596018) and quantified using an ND-2000 Spectrophotometer (Thermo Fisher Scientific). Libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen, Inc., Greenland, NH, USA) and sequenced with 75-bp single-end reads on a NextSeq 500 (Illumina, Inc., San Diego, CA, USA).
RNA sequencing (RNAseq) data analysis
Analysis of the RNAseq data was performed using TopHat2 (version 2.1.1) and the Cufflinks suite (version 2.1.1). The data were processed and quantified using HTSEq. Differential gene expression analyses were performed using DESeq2 (version 1.24.0) with the Wald test and their respective default filters 37. p < 0.05 was used as the threshold for differentially expressed genes (DEGs). Enrichment analyses of GO terms were analyzed using a Cytoscape plug-in ClueGO based on related terms and statistical significance 38. The enriched Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the DEGs were conducted on DAVID datasets (version 6.8) 39.
Primary microglia culture
Primary microglial cells were obtained from the cerebral cortices of 1-day-old neonatal C57BL/6 mice as previously described 40. Approval for the experiments was granted by the Institutional Animal Care and Use Committee in Seoul National University (SNU-171207-2-5). To induce inflammatory cytokines in microglia cells, microglia cells were treated with 200 nM of aged WT-synuclein or V40G-synuclein for 8 h. To assess inflammasome activation and secretion of inflammatory cytokines, 200 nM of aged WT-synuclein or V40G-synuclein was treated to microglia for 18 h.
Measurement of Cytokine Secretion
Microglial TNF-α and IL-1β secretion were measured in cell supernatants using the Mouse TNF-alpha Quantikine ELISA Kit (SMTA00B, R&D Systems, Minneapolis, MN, USA) and Mouse IL-1 beta/IL-1F2 Quantikine ELISA Kit (SMLB00C, R&D Systems) according to the manufacturer’s protocols.
The procedure for immunofluorescence staining was performed as previously described 41. Cells were incubated with anti-ASC rabbit monoclonal antibody (D2W8U; Cell Signaling Tech, MA, USA, #67824S; 1:100 dilution) diluted in blocking solution. After incubation with fluorescent dye-conjugated secondary antibodies diluted in blocking solution, nuclei were stained with DAPI (D1306, Invitrogen). Images were obtained using Zeiss LSM 700 confocal laser scanning microscope.
Reverse transcription quantitative PCR
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, NRW, Germany, #74106) and reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, #1708891). Target genes were amplified using iTaq Universal SYBR Green Supermix (Bio-Rad, #172–5121) with specific primers. Primer sequences were as follows: mouse TNF-α; 5′- CCT CTT CTC ATT CCT GCT TGT TGG-3′ (forward), 5′- GGT GGT TTG TGA GTG TGA GGG-3′ (reverse), mouse IL-1β; 5′- ATC CCA AGC AAT ACC CAA AGA AGA A-3′ (forward), 5′- GTG AAG TCA ATT ATG TCC TGA CCA C-3′ (reverse), mouse GAPDH; 5′- AGA AGG TGG TGA AGC AGG CAT C-3′ (forward), 5′- CGA AGG TGG AAG AGT GGG AGT TG-3′ (reverse). Relative mRNA levels were calculated according to the 2− ΔΔCT method. All ΔCτ values were normalized to glyceraldehyde-3-phosphate dehydrogenase.
Degradation kinetics of aged WT-synuclein or V40G-synuclein in microglia
Primary mouse microglia cells were treated with either 200 nM of aged WT-synuclein or V40G-synuclein for 30 min at 37°C. After washing with ice-cold PBS, cells were incubated with fresh growth media at 37°C, and harvested at the indicated times.
Cell-to-cell propagation assay
For co-culture with microglia, V1S and SV2 stable cells12 were plated on a poly-D-lysine-coated coverslip. Next day, microglia cells were added to V1S and SV2 coculture. Cells were cultured for 2 additional days. For treatment of recombinant inflammatory cytokines, mixture of V1S and SV2 cells was cultured for 3 days. Recombinant human TNF-α (Prospec, Ness-Ziona, Israel, #CYT223; 50 ng/ml) or IL-1β (Prospec, #CYT208, 50 ng/ml) was treated to V1S and SV2 co-cultured cells at the final concentration of 50 ng/ml for the last 24 h. Cells were fixed in 4% paraformaldehyde in PBS prior to nuclear staining with TOPRO-3 iodide (Invitrogen). Images were acquired by confocal microscopy (Zeiss LSM700, 63X).
All experiments were performed blind-coded and at least in duplicate. Differences were considered significant at p < 0.05 and were calculated using paired, two-tailed Student’s t-tests, one-way ANOVA with Tukey’s post-hoc test, and two-way ANOVA with Bonferroni’s post hoc test using GraphPad Prism 7.04 and 9.0.2 (GraphPad Software Inc., La Jolla, CA, USA). The values in the figures are expressed as the mean ± standard error of the mean (s.e.m.).
Raw sequencing reads for rhinal cortex have been deposited at the National Center for Biotechnology Information under BioProjects PRJNA605306, respectively. The sequences of the mice injected with PBS are deposited in GenBank under accession SRR11060104, SRR11060103 and SRR11060102. The sequences of the mice injected with WT-syn are deposited in GenBank under accession SRR11060101, SRR11060100 and SRR11060099. The sequences of the mice injected with V40G are deposited in GenBank under accession SRR11060098, SRR11060097 and SRR11060096. The Source Data are provided with this paper. Other data are available from the corresponding author upon reasonable request.