Supplementary Figure 9: α-Synuclein oligomers generated in vitro in the presence of dopamine become internalized and are toxic to primary neurons.

Hippocampal neurons at 7 days in vitro were exposed to 0.5 or 1 μM α-synuclein oligomer that had been prepared in the presence of dopamine (Olig), or 1 μM monomer (Mon), 1 μM dopamine that was incubated in parallel to oligomers but without α-synuclein (DA), or an equivalent dose of PBS. (a) Two weeks post-treatment with 1 μM Olig or PBS, cells were labeled with human α-synuclein antibodies in a two-stage protocol. Cells were live-incubated with Syn204 antibody to label extracellular α-synuclein. Following fixation and permeabilization, the cells were incubated with LB509 to label total α-synuclein. Confocal imaging showed puncta that were labeled by LB509 but not Syn204, indicating that exogenous oligomers had been internalized. These puncta were localized to neurites. Scale bar, 10 μm. (n = 3 independent cultures per group). (b-c) At two weeks post-treatments, cell viability was assayed using Calcein AM and propidium iodide (PI) dyes. The number of viable cells, defined as Calcein AM positive and PI negative, was significantly reduced by exposure to 1 μM Olig but was not affected by Mon or DA controls. Treatment with 0.5 μM Olig was insufficient to induce toxicity, indicating that the effect is dependent on dose. Scale bar, 100 μm. (n = 4 independent cultures per group except n = 3 independent cultures for PBS, n = 3 independent cultures for DA, and n = 5 independent cultures for 1 μM Olig, P_{Olig 1 μM versus PBS} = 0.0285, P_{Olig 1 μM versus DA} = 0.0308, P_{Olig 1 μM versus Mon} = 0.0098, F_(4,14) = 6.385; one-way ANOVA with Tukey’s correction for multiple comparisons). Box plots show median, 25th and 75th percentiles, minimum and maximum values. *P < 0.05, **P < 0.01.