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 are structurally similar to human E46K-mutated and MSA-amplified α-syn fibrils

Chemical structure and binding curves of ThT (J), Nile Red (K), and FSB (L) to mouse and human sonicated α-syn fibrils (average radii: 16.68±1.44 nm and 15.14±4.02 nm, respectively). Data points indicate means from three independent experiments and error bars are S.E.M.  ****p<0.0001 and ***p<0.01 from 2-tailed t-tests.

Fig. 2 Distinct β-fold stacking arrangements in mouse α-syn fibrils contribute to low tensile strength and resilience

(C) Representation of proposed model of fibril 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 six rungs. Error bars represent S.D. from 100 independent simulations. (D) Group comparison of fibril breakage under sonication conditions shown as the percent of size population of 10-100 nm (e.g., short fibrils) at 0, 2, and 30 minutes, measured by DLS. Error bars indicate S.E.M of three independent experiments with 30 acquisition measurements corresponding to each experiment. (E) Filter-trap slot-blot analysis of sonicated fibrils (i.e., PFFs) exposed to different concentrations of guanidinium chloride (GuHCl), and then remaining fibrils detected with the fibril-selective antibody MJFR14-6-4-2. Error bars indicate S.E.M from three independent experiments. (F) DLS analysis of PFFs after incubation with GuHCL. Error bars indicate S.E.M of three independent experiments with 10 acquisition measurements for each biological sample. Curves in (E) and (F) show asymmetric sigmoidal models with a goodness of fit >0.96, and  **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 lysosome damage in macrophages

(B) Internalized Alexa-647-PFFs (%area) inside cells and (C) % of LAMP1-positive vesicles positive for PFFs. Each data point represents the means of cells analyzed from at least eight images from three independent experiments. (D) ELISA analysis of the extracellular IL-6 and (E) CCL5 from MDM cultures treated with PFFs (1 μg/mL) for 3 and 24 hours. Each data point represents the mean from two technical replicates from four independent experiments. (F) % of Gal3-positive vesicles calculated per mm² of cell surface area in PFF-treated MDM cultures after 24 hours of incubation and (G) % of Gal3-positive vesicles also positive for DQ-PFFs after 48 hours of PFF incubation. Data points show the mean values from cells imaged across three independent experiments with at least eight images analyzed per group. (H) Representative images of Gal3 immunostaining after 24-hours treatment with Alexa-647- or DQ- labeled PFFs. Orthogonal views of sequential z-stacks are shown. Side left image = x,y plane, side right image = y,z plane; top image = x,z plane. 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) or from Tukey’s post-hoc test after ANOVA (panels D,E).

Fig. 4. Mouse and human α-syn fibril uptake in neurons is similar and clathrin-dependent

(A) Time-dependent dynamics of pHrodo-labeled mouse or human PFF (1 µg/mL) internalization over 24 hours into human-PAC-wt-SNCA+/+/Snca-/- hippocampal primary neuronal cultures at DIV7, with normalized pHrodo-channel intensity to DAPI counts at the indicated time point. Each dot represents mean values from four images each from three independent neuronal cultures. (D)  Alexa-568 intensity in dopaminergic neurons and (E) intensity exclusively in the perinuclear area 8 hours post PFF incubation. Each dot represents the mean value of one image with at least 20 images collected from three independent experiments. (F) Calculated uptake of pHrodo-labeled α-syn PFFs at 24 hours in the presence of endocytosis inhibitors with signals normalized to vehicle only controls. Each dot in panel F represents the mean value of four images evaluated per condition from three independent experiments. Error bars for each group analysis represent S.E.M., and ns is 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 assessed relative to the number of neurons in the corresponding cultures cultured from human-PAC-wt-SNCA+/+/Snca-/- hippocampal primary neuron culture treated with 1 µg/mL of α-syn PFFs for 14 days, or equivalent amounts of monomer as indicated, and stained against pS129-α-syn, Tau and NeuN. (B) To ensure the specificity of pS129-α-syn signals, control groups show the lack of signal in neurons cultured from Snca-/- mice following 14 days of incubation with α-syn PFFs or monomer as shown. (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 a group in the graphs represents the mean of signal from an individual litter with two technical replicates per litter and at least 25 images analyzed for each replicate, with 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. Mouse PFF induced α-syn pathology spreads through the mouse brain to seed human α-syn pathology more efficiently than human α-syn PFFs

(B) Group analysis of α-syn pathology propagation ratio to contralateral side in piriform cortex, (C) thalamus, and (D) dorsal striatum, quantified as proportion of pSyn neuronal inclusion spread between Ipsi and Contr. areas. (E) Analysis of pS129-α-syn pathology near the injection site within the ipsilateral dorsal striatum. Each data point (n=5 per group) in group analysis plots represents the mean of the signal from 20-25 sections from an individual animal, and error bars indicate 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 into mouse PFFs leads to the generation of mouse-like fibrils.

(A) Representative aggregation assays showing mouse and human PFF-templated aggregation with human α-syn monomer, with (B) the calculated lag phase. Data points represent normalized ThT fluorescence from three independent experiments with error bars indicating S.E.M. (C) Representative filter-trap slot-blot membranes stained with MJFR14-6-4-2 α-syn aggregate-specific antibodies for the detection of aggregated α-syn in corroboration of aggregation kinetics without amyloid dyes.  (E) Group analysis of ThT binding (fluorescence units, F.U.) from chimeric or homogenous-sequence extracted, monomer-free, sonicated α-syn fibril (PFF) products, as well as (F) Nile Red binding. Each data point in panels E and F represents a mean from an individual experiment measured in duplicate with three independent experiments.Error bars indicate S.E.M.,  **p<0.01, from a 2-tailed t-test, and ***p<0.001 from Tukey’s post-hoc test after ANOVA.

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. Mouse α-syn PFFs are highly susceptible to sarkosyl denaturation.

(a) Filter-trap slot-blot analysis of sonicated fibrils (PFFs) exposed to different concentrations of sarkosyl (left) and the remaining fibrils detected with the fibril-selective antibody MJFR14-6-4-2 in a dose-response curve (middle), with group analysis between two PFF variants at 1% Sarkosyl compared (right). Error bars indicate S.E.M from three independent experiments. (b) DLS analysis of sonicated fibrils (PFFs) exposed to sarkosyl concentrations in a dose-response curve. Error bars indicate S.E.M of three independent experiments with 10 acquisition measurements for each biological sample.  *p<0.05 from unpaired 2-tailed t-tests.

fig. S11. Evaluation of α-syn aggregation ELISA using control recombinant mouse and human α-syn fibril PFFs.

Standard curves generated from mouse and human PFFs in a pan-α-syn aggregate-specific ELISA. The ELISA approach was utilized to quantify the level of aggregates present in lysates derived from the neuronal cultures. The standard curve, along with the indicated goodness of fit and corresponding r² values, provides reliable measures at physiological ranges. Each data point represents the mean from three technical replicates from two independent experiments with errors bars indicating S.E.M.

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. S12. Elevated p-S129-α-syn levels in iPSC-derived dopaminergic neurons following treatment with mouse α-syn PFFs

(b) Levels of pS129-α-syn in the β-III-tubulin area in iPSC-derived DA neurons after 7 days of treatment with 10 μg/mL of PFFs or control. (c) The quantity of pS129-α-syn 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. 

fig. S13. Evaluation of kinetic properties in aggregation assays with cross-seeded chimeric fibrils show that the chimeric fibrils largely replicate the functional properties of their parental seeds, despite different amino acid sequences between the fibril preparations.

(a) Representative RT-QuIC (real-time quaking induced)  assays of normalized relative fluorescence values (RFUs) with mouse monomer templating on different PFF seeds in the creation of new fibrils. The lack of formation of spontaneous fibrils (gray color, no PFFs added “no template”) indicate that spontaneous aggregation is not occurring under the given aggregation conditions within the specified timeframes. Line graphs show corresponding lag in amplification (in hours, or time to initial fluorescence threshold) of the different reactions combined with different PFF concentrations added and analyzed to a linear regression model. Data points represent normalized ThT fluorescence to their maximal fluorescence in the individual reactions, accounting for differential ThT binding to different fibril structures. (b) Comparable reactions as above but with human instead of mouse monomer templating with the indicated PFF seed.