Extended Data Fig. 10: Ectosome-mediated propagation of phosphorylated α-synuclein in GBA1-N370S neurons.

a-b, Maximum intensity projections of GBA1-N370S and control neurons incubated with large vesicle fraction derived from GBA1-N370S + /- PFF for 14 days and stained with β3-tubulin and pS129-α-synuclein. Quantifications of the amount of intracellular pS129-α-synuclein normalized to the neuron area in GBA1-N370S and control neurons treated with different centrifugation fractions after 14 days of incubation. n = 3 independent experiments; 6 cells/experiment. Mean ± SEM, One-way ANOVA with uncorrected Fisher’s LSD. c, Schematic representation of the multi-step propagation of pathogenic α-synuclein through large vesicles/ectosomes. d-e, Quantifications of the amount of intracellular pS129-α-synuclein normalized to the neuron area in GBA1-N370S and control neurons treated with large vesicle fraction from multi-step process depicted in (C) after 7 days of incubation. n = 4 independent experiments; 8 cells/experiment. Mean ± SEM; One-way ANOVA with uncorrected Fisher’s LSD. Max intensity projections of GBA1-N370S and control neurons incubated with the second-generation large vesicle fractions derived from GBA1-N370S. f-h, Size distribution of attached β3-tubulin-positive vesicles from GBA1-N370S + /- GW4869 and corresponding isogenic control neurons. n = 4 independent experiments; a total of 138, 199 and 234 attached vesicles for isogenic control and GBA1-N370S + /- GW4869 were analyzed; bars show mean ± SEM. i, Average size of vesicle diameter of GBA1-N370S + /- GW4869. n = 4 independent experiments; bars show mean ± SEM; Two-tailed Mann-Whitney test. j, Cytotoxicity assessment of GW4869 on GBA1-N370S and isogenic control neurons by MTT assay. n = 4 independent experiments; 3 technical replicates per experiment; bars show mean ± SEM; One-way ANOVA, with Dunn’s multiple correction test. k, Percentage of Iba1 positive microglia neighboring the tdTomato-expressing neuron containing tdTomato puncta. n = 5 animals; 6 images per n; mean ± SEM; Two-tailed unpaired t-test. l, Maximum intensity projection showing a cortical neuron expressing tdTomato after saline or CBE administration and immunostained for Iba1 and DAPI. Boxed area, maximum intensity projection, single slice magnification and orthogonal views (YZ; scale bar 2 µm) showing tdTomato-positive puncta internalized by neighboring microglia stained with Iba1. Graphs are depicted as superplots where biological replicates are shown in large shapes, and where applicable technical replicates are shown as small shapes.