Figure 6

Rapamycin treatment or overexpression of PINK1 or Parkin rescues cellular phenotypes associated with loss of mortalin function (a) Cells were treated with control siRNA or mortalin siRNA and analyzed for apoptotic nuclei and activation of caspase 3 via immunofluorescence studies. The induction of autophagy by treatment with Rapamycin resulted in a significantly lower amount of caspase 3 activation as well as apoptotic nuclei in the mortalin knockdown condition compared with the control condition. (b) Cells were treated with control siRNA or mortalin siRNA and analyzed for apoptotic cell death via Annexin V staining measured by FACS analysis. Knockdown of mortalin resulted in an increased Annexin V signal. The inhibition of lysosomal function by treatment with BafA1 enhanced the Annexin V signal in both control and mortalin knockdown condition. (c) Fluorescence microscopy in fixed cells (n=197 cells) was used to analyze the number of autophagosome by overexpressing GFP-LC3 (green) in mortalin and control siRNA-treated SH-SY5Y. Cells were transfected with wt mortalin, PINK1 or Parkin in the ratio of 1 : 4 together with GFP-LC3 (controls for positive transfection of the mortalin plasmid). Mortalin siRNA-treated cells have a higher number of GFP-LC3 puncta compared with control siRNA-treated cells. The overexpression of wt mortalin resulted in a decreased number of GFP-LC3 puncta, whereas the overexpression of PINK1 or Parkin was followed by an enhanced number of GFP-LC3 puncta compared with the control condition. (d) Fluorescence microscopy in fixed cells (n=214 cells) was used to analyze mitochondrial morphology in mortalin and control siRNA-treated SH-SY5Y with or without overexpressed wt mortalin, PINK1 or Parkin. Mortalin siRNA-treated cells have a smaller mitochondrial form factor, aspect ratio and mitochondrial size (area per mitochondrion). The reintroduction of either wt mortalin, PINK1 or Parkin rescued this mitochondrial fragmentation phenotype