Figure 4: aSyn prevents H₂O₂-induced mitosphere formation.

(a) Mixed cultured aSyn and Ctr H4 cells were treated with 300 μM H₂O₂ for 4 h. Immunofluorescence stainings for aSyn (15G7) and mitochondria (MT) are shown. (b) Quantification of the percentage of aSyn and Ctr H4 cells with mitospheres in a time- and concentration-dependent manner shows that aSyn overexpression substantially prevents mitosphere formation (Two-way ANOVA Bonferroni’s Multiple Comparison Test, n = 3 with >200 cells counted per sample, Ctr vs. aSyn: F = 51.69, p < 0.0001 (time dependence), F = 24.65, p = 0.0001 (concentration dependence)). (c) H4 cells transiently transfected with aSyn, GFP or γSyn were treated with 300 μM H₂O₂ for 4 h. Mitosphere formation was analyzed in transfected (20–30%) vs. non-transfected cells within the same cultures. Only aSyn-transfected cells showed a strong reduction in mitosphere formation (one-way ANOVA Dunnett’s Multiple Comparison Test, n = 3 with >600 cells counted per sample, p = 0.0266, F = 7.051, p = 0.0252 (aSyn vs. GFP), p = 0.0388 (aSyn vs. γSyn), DF = 6; one sample t-tests (two-tailed, theoretical mean = 1), p = 0.0193 (aSyn), p = 0.6621 (GFP), p = 0.0908 (γSyn)). (d–g) Stably aSyn overexpressing H4 cells were treated with 300 μM H₂O₂ for 4 h. (d) DilC1 staining measured via flow cytometry illustrates no differences in the MMP of aSyn H4 cells due to H₂O₂ exposure. Mean cellular fluorescence intensity compared to VC-treated Ctr H4 cells is illustrated (unpaired two-tailed t-test, n = 3, p = 0.0746). (e) H₂O₂ treatment had no influence on MFN1 levels (unpaired two-tailed t-tests, n = 3, p = 0.3627 (1), p = 0.0945 (2), p = 0.3913 (3)) or Drp1 and Fis1 levels (unpaired two-tailed t-tests, n = 6, Drp1: p = 0.4486; Fis1: p = 0.4315) in aSyn H4 cells. H₂O₂ treatment only led to an increase in the shortest OPA1 isoform (unpaired two-tailed t-tests, n = 3, p = 0.2958 (total), p = 0.1346 (1), p = 0.0744 (2), p = 0.3936 (3), p = 0.7992 (4), p = 0.0314 (5)) and (f) did not influence Hsp60, TOM20 and aSyn (15G7) levels (unpaired two-tailed t-tests, n = 6, p = 0.1431 (Hsp60), p = 0.7311 (TOM20), p = 0.3653 (aSyn)) in aSyn H4 cells as quantified via WB. (g) Isolations of crude mitochondrial fractions indicated a marginal increase in aSyn levels in the mitochondrial fractions due to H₂O₂ treatment (300 μM, 4 h), without reaching statistical significance. aSyn levels were normalized to TOM20 or Hsp60 levels (one-sample t-tests, theoretical mean = 1.0, n = 3, aSyn/TOM20: p = 0.6086; aSyn/Hsp60: p = 0.2716).