Fig. 1: C-terminal truncation of exogenous PFFs is an early event during the seeding process.

a Seeding model in primary hippocampal neurons. 70 nM of mouse PFF were added to neurons at DIV 5 (days in vitro). Control neurons were treated with Tris buffer (Tris) used to prepare PFF. After 4 days of treatment, positive pS129-aSyn aggregates were detected in the extension of the neurons. After 7 days of treatment, the aggregates appeared in the cytosol of the neurons. The number of LB-like inclusions increased over time, as shown at 10 days of treatment. Scale bars = 40 and 5 μm. ICC analysis of the LB-like inclusions formed 10 days after adding mouse PFF to aSyn KO neurons (b) or WT neurons (c–e). Aggregates were detected using pS129 (MJF-R13) in combination with total aSyn (c, SYN-1), ubiquitin (ub, d), or p62 (e) antibodies. Neurons were counterstained with microtubule-associated protein (MAP2) antibody, and the nucleus was counterstained with DAPI staining. Scale bars = 5 μm. f WB analyses of the insoluble fraction of PFF-treated WT neurons treated with 70 nM of PFF for D1, D2, D4, D7, and D10. Control neurons were treated with Tris. After sequential extractions of the soluble and insoluble fractions, cell lysates were analyzed by immunoblotting. Total aSyn, pS129, and actin were detected by SYN-1, pS129 (MJF-R13), and actin antibodies, respectively. WB band intensities of total aSyn (15 kDa, indicated by a double red asterisk; 12 kDa indicated by a single red asterisk or HMW) or pS129-aSyn were quantified by densitometry, normalized to actin levels, and expressed as fold change relative to D1. Purple arrows indicate the intermediate aSyn-truncated fragments. The graphs represent the mean ± SD of 3 independent experiments. *p < 0.01, ***p < 0.0001 (ANOVA followed by Tukey HSD post hoc test, Tris vs. PFF-treated neurons) and ^#p < 0.01, ^##p < 0.001 (ANOVA followed by Tukey HSD post hoc test, PFF-treated neurons D1 vs. other time point). g Epitope mapping of antibodies raised against the NAC, N-terminal, or C-terminal domains of aSyn. h N-terminal antibodies raised against residues 1–5 or residues 1–20 could detect full-length (15 kDa, indicated by a double red asterisk) or truncated (~12 kDa, indicated by a single red asterisk) aSyn in the insoluble fraction of WT neurons treated with 70 nM of PFF up to D10. Only the LASH-EGT 1–20 was able to detect the HMW at 25, 35, and 40 kDa. i Mapping of the C-terminal cleaved product using antibodies raised against the NAC and the C-terminal domains of aSyn. Immunoblots of insoluble fractions of WT neurons treated with aSyn PFF up to D10 showed that the fragment 1–114 generated in these neurons was well recognized by the NAC antibodies [(FL-140; 61–95) and (SYN-1; 91–99)] and a C-terminal antibody raised against the residues 108–120. However, it was not recognized by antibodies raised against peptides bearing residues after 116 in the C-terminal domain [(ab6162; 116–131); (ab131508; 134–138) and (ab52168; 131–135)]. j WB analyses of the insoluble fractions of aSyn KO primary neurons treated with 70 nM of mouse PFF for up to 48 h using SYN-1 and pS129 (MJF-R13) antibodies. WB band intensities of total aSyn (15 kDa, indicated by a double red asterisk) and C-terminally cleaved aSyn species (12 kDa, indicated by a single red asterisk) were quantified by densitometry, normalized to actin levels, and expressed as fold change relative to 1 h (for the 15 kDa species, *p < 0.0001; ANOVA followed by Tukey HSD post hoc test) or 48 h (for the 12 kDa species, ^###p < 0.0001; ANOVA followed by Tukey HSD post hoc test) after PFF addition. Purple arrows indicate intermediate aSyn-truncated fragments. Graphs represent the mean ± SD from three independent experiments. k, l Insoluble fractions of aSyn KO primary neurons treated with 70 nM of mouse PFF for 4 or 14 h were separated on a 16% Tricine gel. After Coomassie staining, two bands at ~15 (indicated by a black dashed box) and 12 kDa (indicated by a purple dashed box) were extracted from 16% Tricine gels (See Supplementary Fig. 4). Isolated bands were selected based on the size of the proteolytic fragments observed by WB (f) and subjected to proteolytic digestion followed by LC-MS/MS analysis. Proteomic analysis revealed that aSyn fragments generated in KO neurons transduced with PFF originate from C-terminal truncation, rather than N-terminal cleavage, of the PFF seeds. The diagram in (i) shows the different aSyn fragments generated upon C-terminal truncation and their relative position in a WB. Three fragments (1–135, 1–129, and 1–119) were detected in the upper band, and one main fragment (1–114) was found in the lower band. Original uncropped and unprocessed WB scans are provided in Supplementary Fig. 17. In (h–i), multiplexed detection (LiCor technology) allowed simultaneous antibody probing and multiple detections from the same membrane; thus, some immunoblots share the same actin control. See Supplementary Fig. 17 for a detailed explanation.