Fig. 2: aSyn PFF seeds are predominantly internalized via the endo-lysosomal route and are processed both in the endolysosomes and the cytosol of the neurons.

a, b aSyn KO neurons were treated for up to 72 h with WT fluorescently labeled PFF⁴⁸⁸. The internalization and the truncation of the seeds were evaluated by confocal imaging. a One hour after addition to the KO neurons, we observed that most of the intracellular PFF⁴⁸⁸ were co-stained by an antibody raised against the extremity of aSyn C-terminal domain (epitope: 134–138, yellow arrows). C-terminal truncation of the seeds over time was confirmed by the loss of detection of the seeds by the C-terminal aSyn antibody (134–138, red; green arrows). b The internalization of the seeds via the endo-lysosomal pathway was confirmed by the detection of the fluorescently labeled PFF⁴⁸⁸ seeds in LAMP1-positive (late endosome, red) compartments over time. a, b Neurons were counterstained with MAP2 antibody, and the nucleus with DAPI stain. Scale bars = 10 μm. c Cathepsin B activity was measured in KO neurons treated with 70 nM of WT PFF seeds for up to 48 h. Control neurons were treated with Tris buffer. The graphs represent the mean ± SD of 3 independent experiments. The level of Cathepsin B activity is expressed as a fold change relative to Tris. **p < 0.001, ***p < 0.0001 (ANOVA followed by Tukey HSD post hoc test, Tris vs. PFF-treated neurons). d Truncation of aSyn PFF in the cytosol is confirmed by microinjection. The diagram on the left-hand side shows the experimental approach used to microinject WT PFF⁴⁸⁸ in KO neurons. Cells were fixed after 24 h and immunostained using the N-terminus antibody (aSyn 1–20) or C-terminus antibody (aSyn 134–138). Confocal imaging showed that WT PFF⁴⁸⁸ were detected by the N-terminus antibody (yellow arrows, merge), but not the C-terminus antibody (green arrows, merge). Neurons were counterstained with MAP2 antibody, and the nucleus with DAPI stain. Scale bars = 40 μm.