The hereditary mutation G51D unlocks a distinct fibril strain transmissible to wild-type α-synuclein.

Purification of recombinant WT and G51D α-syn Preparation of full-length

WT and G51D α-syn follows the similar protocol described previously 11 . Briefly, the genes encoding WT and G51D α-syn were inserted into the pET22 vector. G51D point mutation gene was generated based WT α-syn gene by PCR kit (TransGen Biotech, Cat. # AP101-11). Primer sequences used in this work are listed in Supplementary Table 1 . α-Syn was co-expressed with yeast N-acetyltransferase complex B in BL21 (DE3) cells (TransGen Biotech, Cat. # CD601-02) to obtain the N-terminally acetylated α-syn protein 40 . Cells were harvested after protein expression at 37 °C for 4 h with 1 mM isopropyl-1-thio- d -galactopyranoside (IPTG). Then, cells were lysed by sonication in 50 mM Tris–HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA. The supernatant was obtained by centrifugation at 15,000 × g for 25 min. Then the supernatant was processed by boiling at 100 °C for 10 min, streptomycin treatment (20 mg/ml), pH adjustment to 3.5 using 2 M HCl, and dialysis 50 mM Tris–HCl (pH 8.0) overnight in turn. Anion exchange column (GE Healthcare, 17-5156-01) and Superdex 75 (GE Healthcare, 28-9893-33) were then used to purify α-syn protein in high purity. For the anion exchange column, buffer (50 mM Tris–HCl, 1 M NaCl, pH 8.0) was used to elute protein with a gradient (0–60%). Finally, an online EASY-nL-LC 1000 coupled with an Orbitrap Q-Exactive HF mass spectrometer was used to validate that the α-syn protein is indeed acetylated. Preparation of WT 1a and G51D α-syn fibril strains Recombinant WT (100 μM, in 50 mM Tris, pH 7.5, 150 mM KCl, 0.05% NaN 3 buffer) and G51D (100 μM, in 50 mM phosphate buffer, pH 7.0, 50 mM NaCl, 0.05% NaN 3 buffer) were shaking at 37 °C, 900 rpm in ThermoMixer (Eppendorf) for 7 days, respectively. α-Syn preformed fibril seeds (PFFs) were obtained by sonication with 20% power for 15 times (1 s per time, 1 s per interval) on ice by JY92-IIN sonicator. Then, 100 μM WT and G51D α-syn monomer were incubated in the presence of α-syn PFFs (0.5 mol%, concentration by monomer equivalent) at 900 rpm, 37 °C for a week. The residual soluble α-syn in the supernatant was removed after pelleting the fibrils. The pellets were suspended with buffer to 100 μM (equivalent to monomer concentration). The fibril samples were further used for NS-TEM, AFM, cryo-EM sample preparation, PK assay, sonication, and primary neuron treatment.

ThT kinetic assay

Seeding of WT and G51D monomer by α-syn PFFs was conducted by using ThT assay. 50 μM α-syn WT (in 50 mM Tris, pH 7.5, 150 mM KCl, 0.05% NaN 3 ) monomer was incubated with the WT 1a α-syn PFFs and G51D α-syn PFFs (5 mol%, equivalent to monomer concentration) with 10 μM ThT in the reaction mixture, separately. Similarly, 50 μM α-syn G51D (in 50 mM phosphate buffer, pH 7.0, 50 mM NaCl, 0.05% NaN 3 ) monomer was incubated with the G51D (5 mol%, equivalent to monomer concentration) with 10 μM ThT in the reaction mixture. A Fluoroskan Ascent microplate reader (Thermo Scientific) was used to test reactions performed in a 384-well optical plate (Thermo Scientific) in triplicate, with 440 nm excitation wave-length and 485 nm emission wave-length, a bottom read. Graphing was performed with GraphPad Prism 6. The data shown in each ThT experiment are mean ± s.d., n = 5 independent samples. A co-aggregation experiment of α-syn WT and G51D was conducted by using ThT assay. 100 μM α-syn WT, 100 μM G51D monomer or mixed monomer (50 μM WT + 50 μM G51D) was incubated with 10 μM ThT in the reaction mixture in PB buffer (50 mM phosphate buffer, pH 7.0, 50 mM NaCl, 0.05% NaN 3 ) or Tris Buffer (50 mM Tris, pH 7.5, 150 mM KCl, 0.05% NaN 3 ). A Fluoroskan Ascent microplate reader (Thermo Scientific) was used to test reactions performed in a 384-well optical plate (Thermo Scientific) in triplicates, with 440 nm excitation wavelength and 485 nm emission wavelength, a bottom read. Graphing was performed with GraphPad Prism 6. The data shown in each ThT experiment are mean ± s.d., n = 3 independent samples. The sample from the ThT assay was further characterized by NS-TEM. 50 μl aqueous solution of each sample from the ThT assay was pelleted by centrifugation (14,462 × g , 25 °C, 45 min). SDS-loading buffer was added into 45 μl supernatant and boiled for 10 min. The pellet was washed by phosphate-buffered saline (PBS) and dissolved in 45 μl buffer, then sonicated for 5 min and boiled for 30 min. Lastly, the solution was boiled in the SDS-loading buffer for 10 min. The supernatant samples and dissolved pellet samples were loaded on 4–20% Bis–Tris gels (GenScript), respectively. The gels were stained by Coomassie brilliant blue and images were acquired and analyzed with Image Lab 3.0 (Bio-Rad).

Atomic force microscopy

Fibril samples were mounted on mica for 3 min, rinsed gently with water, and dried with nitrogen flow. Images were captured by Nanoscope V Multimode 8 (Bruker) with SNL-10 probes (a constant of 0.35 N m −1 ) on ScanAsyst air mode. Images were acquired with a 1.5 Hz rate at 512 × 512 pixels and analyzed on the Nanoscope software.

Negative-staining transmission electron microscopy

Samples were prepared by loading 5 μl of fibril solution onto a glow-discharged 200 mesh carbon support film (Zhongjingkeyi Technology Co., Ltd., Beijing). The samples were held for 45 s and washed with double-distilled water followed by 3% uranyl acetate. The grid was then stained with 3% uranyl acetate for 45 s and allowed to dry in air. The samples were imaged by a Tecnai T12 microscope (FEI). PK digestion of α-syn PFFs WT 1a or G51D α-syn PFFs were prepared by sonication at 20% power for 15 times (1 s per time, 1 s per interval) on ice by JY92-IIN sonicator. α-Syn PFFs (3 mg ml −1 , 25 μl, in PBS, pH 7.4) were incubated with proteinase K (final concentration 0.5 and 1.5 μg ml −1 , Invitrogen) at 37 °C for 10, 30, 60 and 200 min. 1 mM PMSF was added to samples to stop the reaction. Then samples were boiled with an SDS-loading buffer for 15 min and loaded on 4–20% Bis–Tris gels (GenScript). The gels were stained by Coomassie brilliant blue and images were recorded and analyzed with Image Lab 3.0 (Bio-Rad). Graphing was performed with GraphPad Prism 6. The data shown in are mean ± s.d., n = 3 independent samples. Characterization of fibril fragmentation 20 μM WT 1a or G51D α-syn fibrils were sonicated with 20% power 2 times and 15 times on ice by JY92-IIN sonicator. And 5 μM WT 1a or G51D α-syn PFFs (under the corresponding condition) were characterized by NS-TEM.

Cell viability assay

SH-SY5Y cells were cultured to test the cytotoxicity of G51D and WT1a PFFs with a CCK-8 kit. SH-SY5Y cells cultured in a 96-well plate were treated with α-syn PFFs at final concentrations of 0.01, 0.1, and 1 μM for 24 h. Then cell viability was tested with the CCK-8 kit following the manufacturer’s protocol. Briefly, CCK-8 solution (10 μl/well) was added to each well. The absorbance of the plate was measured at 450 nm after incubating for 30 min. The data were analyzed with GraphPad Prism 6. Primary neuronal culture experiments Primary cortical neurons were dissected from the cortex of embryonic day (E) 16–E18 Sprague Dawley rats (Shanghai SIPPR BK Laboratory Animals Ltd, China) embryos as previously described 41 . In brief, primary neurons were seeded onto coverslips previously coated with poly-D-lysine (PDL) in 24-well plates (150,000 cells/coverslip). At 8-day in vitro (DIV), neurons were treated with PBS and 100 nM (final concentration) WT 1a or G51D α-syn PFFs, and collected for immunofluorescence at 10/14-day post-treatment. The primary antibodies used in the assay were the phospho-α-synuclein (S129) antibody (Abcam, cat. no. ab51253) and the MAP2 antibody (Abcam, cat. no. ab5392). The antibodies were diluted at a ratio of 1:1000. The intensity of confocal images was analyzed by Image J 2.0.0. The secondary antibodies included goat anti-rabbit IgG Alexa Fluor 568 (Abcam, cat. no. ab175471) and goat anti-chicken Alexa Fluor 488 (Thermo Fisher, cat. no. A-11039). All rat experiments were performed followed the protocols approved by the Animal Care Committee of the Interdisciplinary Research Center on Biology and Chemistry (IRCBC), Chinese Academy of Sciences (CAS). There are three samples in each group. Purity characterization of G51D fibril To characterize the purity of α-syn fibrils, we dissolved the fibrils with buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100, 2% SDS) and performed SDS–PAGE. The gels were stained by Coomassie brilliant blue and images were acquired and analyzed with Image Lab 3.0 (Bio-Rad). In addition, the fibrils were also detected by western blot. The monomer and fibrils were immunoblotted by anti-α-synuclein antibodies: 2642S (Cell signaling) and ab138501 (Abcam), respectively. The antibodies were diluted at a ratio of 1:1000. MALDI-TOF MS A droplet of 1 μl solution containing G51D monomer or dissolved fibrils was mixed with 1 μl of the sinapinic acid matrix (10 mg of sinapinic acid per ml, a 70:30 water/acetonitrile (ACN) with 0.1% trifluoroacetic acid). Deposit 1 μl of final mix onto a MALDI stainless steel target and allow to air dry at room temperature. A 5800 MALDI-TOF/TOF mass spectrometer (AB SCIEX, Framingham, MA, USA) in linear positive mode with a mass range from m / z 2500 to 20,000 was used for MALDI-TOF MS measurements and analyses. LC–MS/MS analysis Excise bands of dissolved fibril samples loaded on 4–20% Bis–Tris gels with a clean scalpel and cuts gel bands into cubes. Gel pieces were then destained with ammonium bicarbonate/ACN (1:1, vol/vol), shrink with neat ACN, and then saturated gel pieces with trypsin. After digestion with trypsin by incubating samples overnight at 37 °C, extract peptide digestion products by incubating samples with extraction buffer (1:2 (vol/vol) 5% formic acid (FA)/ACN for 15 min at 37 °C in a shaker. After centrifugation at 16,000 × g and 4 °C for 15 min, the clear supernatants were collected, dry down in a vacuum centrifuge, and then resuspended in 0.1% (vol/vol) FA for further LC–MS/MS analysis. The peptide mixture was analyzed using an Orbitrap Fusion mass spectrometer coupled to an online EASY-nL-LC 1000 system. Mobile phase A consisted of 0.1% FA, 2% ACN, and 98% H 2 O, and mobile phase B consisted of 0.1% FA, 2% H 2 O, and 98% ACN. A 60 min gradient (mobile phase B: 3% at 0 min, 8% at 5 min, 20% at 46 min, 30% at 54 min, 95% at 55 min, and 95% at 60 min) was used at a static flow rate of 300 nL/min. The data were acquired in a data-dependent (top 20) mode. High-energy collisional dissociation (HCD) was used to fragment the precursor peptides, and the resulting fragment ions were measured in the ion trap analyzer.

Cryo-EM data collection

A solution containing G51D or WT 51cs fibril samples was applied to glow-discharged holey carbon Cu Quantifoil grids (R2/1, 300 mesh) and then plunge-frozen in liquid ethane after blotting with filter paper using Vitrobot Mark IV(FEI). Cryo-EM micrographs with a defocus from −1 to −2 μm were collected on a Gatan K3 direct detector in super-resolution mode on a Titan Krios transmission electron microscope (FEI) operated at 300 kV. 32 movies were recorded per micrograph was a record with a pixel size of 1.06 Å pixel −1 using a dose of 55 e − Å −2 . Automated cryo-EM data collection was performed by Serial EM software 42 . Imaging processing, reconstruction, and model building MotionCorr2 43 was used to correct beam-induced motion of movie frames with dose weighting while CTFFIND4.1.8 44 was used to estimate the contrast transfer function. All filaments were picked manually using the manual picking method of RELION3.0 45 . All subsequent steps of helical reconstruction were carried out using RELION 3.0. G51D dataset 28,989 filaments were manually picked from 1869 micrographs. G51D segments were first extracted using 1024-pixel box size with an inter-box distance of 109 Å and used for subsequent reference-free 2D classification with a decreasing in-plane angular sampling rate from 12° to 1° and a T = 2 regularization parameter to estimate the fibril pitch and helical parameters. Filaments containing selected segments with 1024-pixel box size were then re-extracted using 288-pixel box size with an inter-box distance of 31 Å and particles comprising an entire helical crossover were selected for the following 3D classification. An initial 3D reference was de novo generated using selected particles after 2D classification by relion_helix_inimodel2d and the initial 3D model low-pass filtered to 60 Å was then applied further as a reference map to perform 3D classification. Local optimization of helical twist and rise was performed while β-strands perpendicular to the helical axis was clearly separated. The symmetry of psedo-2 1 was applied and several rounds of 3D classification with K = 3 and K = 1 were used to gain segments belonging to the same conformation and optimize twist parameters. Optimized parameters and selected segments were applied for high-resolution gold-standard refinement. Post-processing with a soft-edge solvent mask in 30% central Z length was performed to sharpen the refined maps. The final overall resolution estimate was calculated to be 2.96 Å based on the 0.143 Fourier shell correlation cutoff. The model was built into the central region of the sharpened density map using E46K α-syn structure ((PDB entry code 6L4S) as an initial model in COOT 46 . A three-layer model was generated and refined by the real_space_refine program in PHENIX 47 , 48 . WT 51cs dataset 12,428 filaments were manually picked from 725 micrographs. WT 51cs segments were first extracted using 1200-pixel box size with an inter-box distance of 127 Å and used for several iterations of reference-free 2D classification. Filaments containing selected segments were then re-extracted using a 686-pixel box size with an inter-box distance of 73 Å and used for subsequent reference-free 2D classification. An initial 3D reference was de novo generated by relion_helix_inimodel2d using selected particles comprising an entire helical crossover and the initial 3D model low-pass filtered to 60 Å was then applied further as a reference map to perform de novo 3D classification. Local optimization of helical twist and rise was performed while β-strands perpendicular to the helical axis was clearly separated. Several rounds of 3D classification with K = 3 and K = 1 were then performed. WT wts dataset 7903 filaments were manually picked from 1056 micrographs and first extracted with a 1024-pixel box size with an inter-box distance of 109 Å and used for several iterations of reference-free 2D classification. Filaments containing selected segments were then re-extracted using a 686-pixel box size with an inter-box distance of 73 Å. Similarly, with G51D and WT 51cs dataset, an initial 3D reference was de novo generated by relion_helix_inimodel2d using selected particles comprising an entire helical crossover, and the initial 3D model low-pass filtered to 60 Å was then applied further as a reference map to perform de novo 3D classification. Several rounds of 3D classification with K = 3 and K = 1 were then performed. Reporting summary Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper.

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