Figure 8

Toxicity of intracellularly overexpressed WT and H50 mutant aSyn. A) H4 neuroglioma cells were transiently transfected with mock, WT, and H50 mutant aSyn, and cell damage was assessed by using MTS cell viability (left) and ToxiLight assays (right). For quantification, measured values were normalized to the mean of WT samples. The background levels of viability and toxicity of mock transfected cells are indicated as dashed lines. Both assays show a slight increase in general cell damage in cells transfected with either H50Q or H50R aSyn. Significance was determined by a one way ANOVA Tukey’s Multiple Comparison Test for randomized block experiments. n = 8 (ToxiLight) and n = 12 (MTS assay). Error bars = SEM. B) The induction of apoptosis by overexpressed H50 mutant aSyn was assessed by ICC using antibodies against aSyn (green) and activated Caspase 3 (aCasp3, red). The number of apoptotic, (aCasp3+ aSyn+, arrow) out of total aSyn overexpressing cells (aSyn+) was counted. The proportion of apoptotic cells in H50Q/R aSyn overexpressing cells was significantly higher than in WT aSyn overexpressing cells. For quantification, measured values were normalized to the mean of WT samples. Significance was determined by a one way ANOVA Tukey’s Multiple Comparison Test for randomized block experiments. n = 6. Error bars = SEM. C) H4 cells transfected with mock, WT, and H50 mutant aSyn were treated with H₂O₂ 24 h after transfection for another 24 h followed by MTS viability assay. The viability of treated cells was compared to the viability of the corresponding untreated cells (vehicle, dashed line). H₂O₂ treatment induces a more pronounced reduction of the viability of H50Q/R overexpressing cells than of WT overexpressing cells. Significance was determined by two way ANOVA Bonferroni Multiple Comparisons. n ≥ 3. Error bars = SEM.