Data from: Mouse α-synuclein fibrils are structurally and functionally distinct from human fibrils associated with Lewy body diseases

Tabular raw data corresponding to figure sets used in the study. Fig. 1 Mouse α-syn fibrils exhibit structural similarities with human E46K-mutated and MSA-amplified α-syn fibrils, yet show distinct characteristics from WT human α-syn fibrils Overlay of one layer of mouse α-syn (8uie, green) and (g) recombinant human (6sst, magenta), (h) recombinant E46K (6ufr, yellow), and (i) MSA-amplified (7ncg, gray) structures with visualized T53 and N87 amino acid schematics and indicated aligned total RMSD values. The structures were aligned based on residues 54-66 selected after an initial global fit with the lowest RMSD values. Chemical structure and binding curves of ThT (i), nile red (j), and FSB (k) to mouse and human sonicated α-syn fibrils (radii: 16.68±1.44 nm and 15.14±4.02 nm, respectively, Fig. S3) and monomeric equivalents. Data points indicate the mean from three independent experiments and presented error bars are S.E.M. ****p<0.0001 and ***p<0.01 from unpaired 2-tailed t-tests. Fig. 2 Distinct β-fold stacking arrangements in mouse α-syn fibrils contribute to low tensile strength and resilience Cross-β-structure of five protofilament rungs (left) and schematic representation of stacking arrangement (right) with estimated coordinates against the assigned baseline position (i) as the horizontal plane (37-57 amino acids) aligned to primary peptide sequence for mouse, 8uie (a) and human, 6sst (b) cryo-EM models. (c) Representation of proposed model of β-sheet fragmentation for tensile strength estimation used to simulate the MMGBSA energy of α-syn fibrils rupture and group analysis of MMGBSA energy required to disrupt a stack of 6 protofilaments. Error bars represent SD of 100 independent simulations. (d) Group comparison of fibril breakage under sonication conditions shown as the percent of size population of 10-100 nm at 0, 2, 30 minutes measured by DLS approach. Error bars indicate S.E.M of three independent experiments with 30 acquisition measurements for each biological sample. Filter-trap slot-blot analysis of sonicated fibrils (PFFs) exposed to SDS (e), sarkosyl (f), guanidine chloride (g) concentrations detected by α-syn aggregate-specific antibody MJFR14-6-4-2 with a group analysis of the level of mouse and human fibrils detected after exposure to 0.1% SDS, 1% of sarkosyl and 2M GuHCl. Error bars in panels e, f, g indicate S.E.M from three independent experiments . *p<0.05, **p<0.01, **p<0.001, ****p < 0.0001 from unpaired 2-tailed t-tests. Fig. 3 Mouse α-syn fibrils fail to elicit robust cytokine and damage responses in macrophages (b) Number of individual Alexa-647-PFFs captured in cell area per mm². Each data point represents the mean analysis of one individual cell from three independent experiments with at least nine images analyzed per group. (c) Percentage of LAMP1-positive vesicles containing α-syn PFFs in MDM cultures treated with mouse or human PFFs. Eight images from three independent experiments were quantified for each condition, with each dot representing results from one image. ELISA quantification of the extracellular soluble IL-6 (d) or CCL5 (e) concentration in MDM cultures treated with PFFs for 3 and 24 hours. Each data point represents a signal from two technical replicates from three independent experiments. (f) Percentage of Gal3-positive vesicles calculated per mm² of cell surface area in PFF-treated MDM cultures after 48 hours of incubation. Each data point represents the mean of one individual cell, from three independent experiments with at least eight images analyzed per group. (g) Percentage of DQ-PFFs in Gal3-positive vesicles in proportion to the overall DQ-fibril count after 48 hours of incubation. Each dot represents the mean analysis of one individual cell, from three independent experiments with at least eight images analyzed per group. The scale bar is 5 um.Error bars represent S.E.M and ***p<0.001, *p<0.05 and ns for not significant from unpaired 2-tailed t-tests (panels b, c, f, g) and ANOVA with Tukey’s multiple comparisons test (panels d, e). Fig. 4. Mouse α-syn fibril uptake in neurons is clathrin-dependent and similar to human α-syn fibril uptake (a) Time-dependent dynamics of pHrodo-labeled mouse or human PFF internalization in human-PAC-wt-SNCA+/+/Snca-/- hippocampal primary neuron culture at DIV7 measured as normalized pHrodo-channel intensity to DAPI count. Each dot represents the mean value of three independent neuronal cultures with four images analyzed for each replicate. Group analysis of total Alexa-568 intensity in neuronal cells (d) and Alexa-568 signal in perinuclear area (e) after 8 hours of incubation with labeled α-syn PFFs. Each dot represents the mean value of one image with at least 20 images collected from three independent experiments. (f) Uptake of pHrodo-labeled α-syn PFFs at 24 hours in primary hippocampal cultures previously treated with endocytosis inhibitors shown as normalized pHrodo intensity in relation to untreated internalization rate. Each dot in each column graph represents the mean value of four images per condition from three independent experiments. Error bars for each group analysis represent S.E.M and **p<0.01, *p<0.05 and ns for not significant from 2-tailed t-tests. Fig. 5. Mouse α-syn fibrils pathology propagation is more efficient than human α-syn in primary neurons. (a) Levels of pS129-α-syn signal relative to the number of neurons in corresponding cultures (left). Control groups include mouse (ms) and human (hum) α-syn monomer treatments. (b) Group analysis of pS129-α-syn signal per neuron in Snca-/- cultures following 14 days of exposure with α-syn PFFs or monomer (ms and hum) equivalents. (d) Abundance of distinct pS129-α-syn signals in cell bodies or neuritic morphology in neuronal cells treated with 1 µg/mL of mouse or human α-syn PFFs. (e) Proportion of pS129-α-syn occupancy in cell body and neurites in primary hippocampal cultures incubated with mouse or human α-syn PFFs for 14 days. (f) ELISA quantification of α-syn aggregate levels in cell lysates from human-PAC-wt-SNCA+/+/Snca-/- or Snca-/- neuronal cultures treated with fibril PFFs or monomeric protein for 14 days. (g) Group analysis of NeuN-positive nuclei abundance normalized to DAPI count. Each data point in group analysis plots represents the mean of signal from an individual litter with two replicates per litter and at least 25 images analyzed for each replicate and error bars indicating S.E.M. Significance was determined by 2-tailed t-tests; **p<0.001, ****p < 0.0001, ns for not significant. Fig. 6. Progressive α-syn pathological spread differs between mouse and human α-syn PFFs Group analysis of α-syn pathology propagation ratio to contralateral side in piriform cortex (b), thalamus (c) and dorsal striatum (d) quantified as proportion of pSyn neuronal inclusion spread between ipsi and contra areas.(e) Quantitative analysis of pSyn neuronal inclusions per mm2 in striatum in mouse and human PFF cohorts. Each data point (n=5 per group) in group analysis plots represents the mean of the signal from 20-25 sections analyzed for each replicate and error bars indicating S.E.M. Significance was determined by 2-tailed t-tests; *p<0.05, ns for not significant Fig. 7. Recruitment of human α-syn monomer in mouse PFF-nucleated amplification leads to the formation of fibrils exhibiting distinct amyloid dye-binding profiles. (a) Representative RT-QuIC assay showing mouse and human PFF-templated aggregation with human α-syn monomer, including the indicated lag phase. Data points represent normalized ThT fluorescence from three independent experiments with error bars indicating S.E.M. (b) Group analysis of the duration of the lag phase in mouse and human PFF-templated aggregation. Dots represent a mean measured in duplicates from three independent experiments. (c) Representative filter-trap slot-blot analysis of dye-free aggregation of human α-syn monomer, with or without fibril PFFs, detected with MJFR14-6-4-2 α-syn aggregate-specific antibodies. (d) Representative images of Alexa-647-labeled mouse or human α-syn PFF-templated aggregation with human α-syn monomer, collected after 48 hours of incubation. The collected reactions were supplemented with 1 µM ThT (shown in green), with Alexa-647-conjugated PFFs depicted in magenta. The scale bar is 0.5 µm. Group analysis of ThT (e) and nile red (f) binding to extracted, monomer-free, sonicated α-syn fibrils (PFFs) performed in various combinations of mouse/human PFFs and their monomeric equivalents. Each data point in e and f represents a mean from an individual sample measured in duplicates from three independent experiments with error bars indicating S.E.M. *p<0.05, **p<0.01, **p<0.001, ****p < 0.0001 from 2-tailed t-tests. Fig. S2. High resolution estimation of the cryo-EM map of mouse and human recombinant α-syn fibrils Fourier shell correlation (FSC) resolution estimation and validation for the 3D reconstruction of the cryo-EM collected images of procured mouse fibrils generated by Duke (a) and EPFL (b) research groups. (c) FSC estimation plot of human α-syn fibrils collected and generated at Duke University. Fig. S7. Generation and validation of mouse and human α-syn PFFs (c) Group analysis of the size population proportions of sonicated mouse and human α-syn preparations, (e) representative average radii of human α-syn,(f) UV absorbance spectra and (g) coefficient extinction adjusted concentration. Each data point or S.E.M in panel c, e and g are extracted from a single acquisition from three independent experiments with ten measurements analyzed per group. Each dot in panel f is the mean of two technical replicates from three independent batches. Significance was assessed via 2-tailed t-tests with ns for not significant. Fig. S9. Evaluation of α-syn ELISA using control recombinant mouse and human α-syn fibril PFFs Standard curve generated from mouse and human PFFs in a pan-α-syn aggregate ELISA. The ELISA was utilized to quantify the level of aggregates present in the cell lysates. The standard curve, along with the indicated goodness of fit and corresponding r² values, provides a reliable measure for interpreting the results. Each data point represents the mean from three technical replicates from two independent experiments with errors bars indicating S.E.M. Fig. S11. Elevated p-S129-α-syn in iPSC-derived dopaminergic neurons followed the treatment with mouse α-syn PFFs (c) The quantity of pS129 puncta relative to the β-III-tubulin area measured in each image and compared across conditions. (d) Proportion of abundance of pS129-α-syn puncta localized in cell bodies and neurites in group analysis between mouse and human α-syn PFF treatments. Each data point in c and d represent the mean value of the images from one well (n=4) and errors bars indicate S.E.M with **p<0.01 from 2-tailed t-tests.