Figure S1.

Characterization of α-syn fibrils and its transfer between microglia, related to Figures 1, 2, and 3

(A-B) Characterization of α-syn fibrils used throughout this study. Electron micrographs of α-syn fibrils stained by Uranyl acetate, before (A) and after (B) fragmentation.

(C) Sedimentation coefficient of fragmented fibrils determined by analytical ultracentrifugation. The sedimentation velocity measurement shows a distribution centered at 110 Svedberg. The sedimentation coefficient is compatible with a molecular species of 16,000 kDa corresponding to ∼1,100 monomers of 14.46 kDa.

(D) Proteolytic profile of fibrillar α-syn. Time course of fibrillar α-syn (100 μM) degradation by proteinase K (0.38 μg/mL) analyzed by SDS-PAGE after Coomassie blue staining.

(E) All α-syn preparations were confirmed to have an endotoxin concentration below 0.02 endotoxin units/μg (EU/μg). n = 7 independent α-syn preparations.

(F) Quantification of the percentage of α-syn monomers containing microglia (left) and the relative individual uptake index per cell (middle) after exposure to ATTO488-labeled α-syn monomers, 2 μM; n = 3 independent experiments per group. Diagram representing the α-syn monomers uptake as measured by FACS (right).

(G) Representative immunostaining showing the internalization of ATTO488-labeled α-syn monomers into CD11b-labeled microglia.

(H) Representative time-lapse recordings demonstrating that α-syn fibrils are transferred from one microglia to another via thin cellular membrane connections.

(I) Representative time-lapse recording demonstrating that α-syn fibrils are transferred from overloaded microglia to naive microglia via cellular connections.

(J) Representative particle tracking of aggregates transferred from donors to acceptors as shown in (I) (upper panels). Quantification of the directionality of transferred particles. D = donors, A = acceptors. A total of 37 particle transfer events were analyzed.

(K) Quantification of particles that underwent transfer from α-syn-containing microglia toward naive cells for their size, traveling distance, total particle transfer time, and particle transfer velocity. n = 33 individual particles.

(L) Quantification of the number of individual cell neighbors and proportion of cells involved in a network before and after α-syn fibrils uptake. Network formation was analyzed using a CellProfiler script, identifying individual cells and measuring the number of adjacent cells. A total of at least 205 cells per condition were analyzed. n = 5-6 individual experiments.

Graphs in F are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Graphs in K present individual particles and the mean. Graphs in L were analyzed by t test analysis. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗p < 0.05 compared to 0 min.

Scale bars: 100 nm (A-B), 20 μm (G, I), 10 μm (H).