Supplementary Figure 7: Biochemical and structural characterization of α-synuclein oligomers resulting from incubation with dopamine in vitro.

(a) Incubation of recombinant wild-type human α-synuclein under standard aggregation conditions (400 μM, 37°C, 1400 rpm) resulted in Thioflavin T (ThioT)-positive fibril formation, whereas ThioT signal was abolished in the presence of equimolar dopamine (DA). rfu, relative fluorescence units. The data are presented as mean ± s.e.m. (n = 3 independent experiments per group, P_{-DA day 4 versus +DA day 4} = 0.0045, P_{-DA day 6 versus +DA day 6} = 0.003, F_(1,4) = 8.901; repeated measures two-way ANOVA with Bonferroni’s correction for multiple comparisons). (b) α-Synuclein fibrils exhibited characteristic β-sheet secondary structure by circular dichroism, while α-synuclein species resulting from interaction with dopamine maintained random coil structure on day 5 of incubation. Representative traces are shown. (n = 3 independent experiments per group). (c) Sedimentation analysis and coomassie blue staining revealed that over the 6-day incubation, in the absence of dopamine, soluble α-synuclein (supernatant) was lost while insoluble α-synuclein (pellet) accumulated. In the presence of dopamine, α-synuclein was detected as multiple soluble oligomer species. Similar to ex vivo oligomers extracted from A53T TH-RREE mice, in vitro generated oligomers were immunoreactive to Syn505 (N-terminal) and LB509 (C-terminal) α-synuclein antibodies. (n = 4 independent experiments per group). (d) Immunoelectron microscopy on α-synuclein from day 4 of incubation with dopamine confirmed both Syn505 and LB509 labeling indicating that dopamine-induced oligomers generated in vitro share common epitopes and potentially similar conformations to oligomers resulting from dopamine elevation in vivo. Scale bar, 20 nm. (n = 3 independent experiments per group). **P < 0.01.