Fig. 3.

Non-toxic overexpression of α-synuclein sensitizes dopaminergic neuroblastoma cells and primary midbrain cultures to Cu toxicity. In A and B, neuroblastoma cells were transduced with Ad-Empty, Ad-α-synuclein or Ad-α-synuclein A53T mutant (A53T) at the indicated MOI for 24 h. Cells were washed, and the expression levels of WT or A53T α-synuclein were measured by WB after 48 h of transduction (A), while cell survival was evaluated by Calcein retention (B). In C and D, cell death induced by CuCl₂ treatment (48 h, 0.75 mM in C) was determined in neuroblastoma cells previously transduced with WT or A53T α-synuclein using PI uptake (C) or Calcein retention assay (D). In C, cell death is represented in two-dimensional 5% probability contour plots of PI fluorescence vs cell size (forward scatter) to depict cells with both compromised plasma membrane integrity (PI uptake) and decreased cell size. %s in contour plots represent the number of viable cells (PI- and normal cell size, broken line regions). Data in B and D represent means ± SE of n = 3. In E, primary midbrain cultures transduced, or not, with WT α-synuclein (MOI = 10, 72 h) were incubated in fresh media with or without CuSO₄ (10 μM) for 24 h. Cells were stained with antibodies specific for MAP2 and TH and scored for dopaminergic cell viability. The data are presented as the mean ± SE of n = 7. Two-way ANOVA, Holm-Sidak post hoc test, ^ap < 0.05 vs Empty without CuCl₂ or CuSO₄ supplementation; ^bp < 0.05 vs 0 mM CuCl₂ within the corresponding category of α-synuclein or A53T; ^cp < 0.05 vs Empty within the corresponding [CuCl₂] tested.