Figure 10

Dampening of autophagy activation in Wdfy3-deficient neuronal progenitor cells is accompanied by decreased mitophagy. (A) Wdfy3 immunofluorescent labeling in striatal neuronal progenitor cells (NPCs) shows ubiquitous expression (scrambled). A significant down regulation of Wdfy3 was observed in siRNA-transfected cells. Quantification of Wdfy3 protein levels was based on fluorescent signal intensity normalized by cell area. Levels of Wdfy3 mRNA expression were assessed by qPCR normalized to Gapdh mRNA. (B) The effect of rapamycin (20 nM) addition was evaluated by quantifying LC3 immunostaining normalized by cell area, as well as the number of LC3 puncta per cell. Statistical analyses were performed by two-tailed Mann-Whitney test. (C) Total mitochondrial mass was visualized in scrambled and siRNA striatal cells by staining with antibodies to ATP5B (green) whereas polarized mitochondria were stained with MitoTracker (red). Co-localization of functional and total mitochondria is shown in yellow (overlay). Insets show details of the mitochondrial morphology and co-localization of ATP5B and MitoTracker in the two genotypes. Red channel intensity has been increased for siRNA-transfected cells, due to the low MitoTracker fluorescence signal emitted by non-polarized mitochondria. Scale bars = 10 µm. Panels (D–H). Mitochondrial content (D) was quantified as the cell area occupied by mitochondria (in %). Mitochondrial footprint (E) was expressed as functional mitochondria (area) normalized by total mitochondria area. (F) Average area per mitochondrion in scrambled- and si-RNA striatal cells. Mitochondrial morphology (G) was reported as circularity index, with mitochondria having a more tubular shape being closer to a value of 1. All measurements shown in panels D,G were performed using a macro for ImageJ (see Methods). (H,I) Assessment of the mitochondrial network integrity in scrambled- and siRNA striatal neurons was performed with the MiNA macro for ImageJ and reported as number of networks per cell (H) and mean branch length (I). Statistical analysis for panels D–I was performed with a 2-tailed Mann-Whitney test. (J) Surface plots representing levels and mitochondrial distribution in scrambled (left) and siRNA (right) striatal cells were obtained with the ImageJ feature “surface plot”. All the box plots shown in panels F–I were obtained analyzing confocal images of mitochondria stained with MitoTracker.