Fig. 7

Knock-down of tau rescues mitochondrial abnormalities and presynaptic loss in glucolipotoxicity-stressed RGCs. RGCs were transfected with a Cy5-labeled siRNA (si-Tau or si-sc) and treated with HG + PA. (a) Representative immunocytochemistry for Ac-tubulin (green, Ac-tubulin; red, Cy5; scale bar, 40 μm). (b) Western blotting for Ac-tubulin and Tau 5. Intensities were normalized against the level of GAPDH and expressed as fold changes of protein abundance with si-Tau relative to si-sc control. (c) Mitochondrial transport in siRNA-transfected RGCs was performed by infecting cultured RGCs with Ad-GFP-Mito and treated with HG + PA. Representative fluorescence images (upper panels; scale bar, 10 μm) for GFP-labeled mitochondria (GFP, green) within axons in siRNA-transfected cells (Cy5, red) and kymograph images of axonal mitochondrial movement (middle and bottom panels) are shown. Traces of moving mitochondria are indicated with white arrow. The average transport speed of movable mitochondria was calculated. (d) Activity of mitochondrial complex I and complex IV. (e) Western blotting for complex I, complex IV and synaptophysin from whole cell lysates (Total) or synaptosome fractions (Syn). Intensities were normalized against the level of GAPDH and expressed as fold changes of protein abundance with si-Tau relative to si-sc control. (f) Representative fluorescence images for synaptophysin (green) in siRNA-transfected cells (Cy5, red). Areas boxed in for synaptophysin immunostaining are shown at higher magnification. Scale bar, 40 μm. Data are means ± SEM of three independent experiments. ^*P < 0.05 and ^**P < 0.01 vs control si-sc