Fig. 4

Overexpression of hα-syn induces the formation of α-syn aggregates in the substantia nigra 3-months post-AAV delivery. (A-E) Dot blot analysis and quantification of 100 ng of protein prepared from the insoluble fraction of the SN of control, mTurq, and hα-syn mice (n = 6 mice per experimental condition). Recombinant monomeric α-syn (Mono) and PFF were used as controls. Membranes were evaluated for protein expression using the following antibodies: (A) Syn1/BD Lab, (B) α-syn-FL140, (C) α-syn LB509, (D) pS129 (Wako), (E) 4B1, and (F) Syn-O2. The data are presented as the means ± s.e.m. (n = 3). One-way ANOVA followed by Tukey’s multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 versus Mono; ## p ≤ 0.01, #### p ≤ 0.0001 versus PFF; $$$ p ≤ 0.001, $$$$ p ≤ 0.0001 versus hα-syn. (G) Representative RT-QuIC analysis illustrating the kinetics of recombinant α-syn aggregation in the presence of protein from the insoluble fraction of the SN of mTurq, and hα-syn mice (n = 6 mice per experimental condition). Monomeric α-syn (Mono) and PFF were used as controls. The average ThT fluorescence intensity was plotted against time. (H) Filter retardation assay and quantification of α-syn protein levels (α-syn-FL140) showing the accumulation of α-syn aggregates in the insoluble fractions of hα-syn and recombinant α-syn PFFs after RT-QuIC. The data are presented as the means ± s.e.m. (n = 3). One-way ANOVA followed by Tukey’s multiple comparisons test; * p ≤ 0.05, ** p ≤ 0.01. Mono: α-syn monomer; PFF: pre-formed fibrils; RT-QuIC, real-time quaking-induced conversion; ThT, thioflavin T. Antibody detection: Syn1/BD Lab and α-syn-FL140: mouse and human α-syn; α-syn LB509: human α-syn; pS129: phosphorylated α-syn at S129; 4B1: Non pS129 α-syn aggregates; Syn-O2: α-syn aggregates. Full dot blots and filter retardation membranes are shown in Suppl. Figure 8