Fig. 4: Oligomer formation and cytotoxicity of dopanized f-αSyn.
From: DOPAnization of tyrosine in α-synuclein by tyrosine hydroxylase leads to the formation of oligomers
a Negatively stained TEM images of FLAG-tagged αSyn (f-αSyn). Control (upper) and TH-treated (lower) f-αSyn were incubated for 18 h before staining with 2% uranyl acetate. Control f-αSyn formed large fibril clusters, whereas dopanized f-αSyn tended to form a separated oligomeric conformation. High-magnification images are shown on the right side of each panel. Scale bars: 200 nm (left) and 50 nm (right). b WB analysis of the sucrose gradient fractionation of f-αSyn. Before (0 h) or after (18 h) incubation, control (TH−) and dopanized (TH+) f-αSyn were separated by 5–30% sucrose gradient centrifugation. Twelve 1-mL fractions were examined by WB using an anti-αSyn antibody. The corresponding sucrose concentrations are indicated at the top of the panels. After 18 h of incubation, control f-αSyn formed aggregates with large masses, whereas dopanized f-αSyn retained a smaller mass. c SEC of dopanized f-αSyn oligomers. The chromatogram of SEC using monomeric (upper) or TH-treated f-αSyn after 18 h of incubation (lower) showed that dopanized f-αSyn formed oligomers up to 15-mers. d Detection of f-αSyn uptake in PC12 cells. Control (upper) and TH-treated (lower) f-αSyn after 18 h of incubation were added to PC12 cells for 24 h and immunostained with anti-FLAG antibody (green) and Y136DOPAmab (magenta). Rectangle-surrounding areas were enlarged on the right side. Black arrowheads indicate control f-αSyn taken up into cells, and white arrowheads point to dopanized f-αSyn (yellow). Scale bar: 20 µm. e Real-time quantification of cell death in PC12 cells. The numbers of dead cells were counted using IncuCyte Cytotox Green Dye, resulting in a significant increase in cytotoxicity by treatment with dopanized f-αSyn (TH+) compared with control f-αSyn (Cont). Data are presented as mean ± SEM. Sample size: n = 3 well/treatment. P-values were calculated using two-tailed unpaired t-test between Cont and TH + groups (29 h *p = 0.044; 33 h *p = 0.027; 37 h *p = 0.018; 41 h *p = 0.011; 45 h **p = 0.007). None indicates the sample without any αSyn treatment. Data are representative of two independent experiments with similar results (a, b, d).
