Fig. 4: Preventing aSyn cleavage at 114 does not impact the seeding capacity of PFF in primary neurons.

aSyn KO neurons were treated for 14 h (a) or up to 21 days (b), or aSyn WT neurons were treated for 10 days (c) with PFF^WT, PFF^E114A, PFF^D115A, or with PFF^1–114. Neurons were lysed at the indicated time, and the insoluble fractions were analyzed by WB. The total aSyn (SYN-1 antibody) levels were estimated by measuring the WB band intensity normalized to the relative protein levels of actin. A double red asterisk indicates the 15 kDa species (full-length), and a single red asterisk indicates the C-terminal truncated species running at 12 kDa. The purple arrows indicate the intermediate aSyn-truncated fragments. d–e Newly formed inclusions were detected using pS129 antibody (81a) in WT neurons after 10 days of treatment. Neurons were counterstained with MAP2 antibody, and the nucleus was counterstained with DAPI staining. d Representative confocal images. Scale bar = 5 μm. e Quantification of images acquired by a high-throughput wide-field cell imaging system. For each independent experiment, triplicate wells were acquired per condition, and nine fields of view were imaged for each well. Each experiment was reproduced at least 3 times independently. Images were then analyzed using Cell profile software to identify and quantify the level of LB-like inclusions (stained with pS129 antibody, 81a clone) formed in neurons (MAP2-positive cells). The graphs (a, c, e) represent the mean ± SD of three independent experiments. a, c aSyn species were quantified by densitometry, normalized to actin levels, and expressed as fold change relative to PFF^WT-treated neurons. e pS129 level was expressed as fold change relative to PFF^WT-treated neurons. a, c, e *p < 0.01, **p < 0.001, ***p < 0.0001 (ANOVA followed by Tukey HSD post hoc test, Tris vs. PFF-treated neurons). ^##p < 0.005 (ANOVA followed by Tukey HSD post hoc test, PFF^WT vs. mutants PFF-treated neurons). All the original uncropped and unprocessed WB scans are available in Supplementary Fig. 17.