Figure 8.

Parkin prevents mitochondrial damage and dysfunction induced by ER stress. (a and b) Increased parkin expression suppresses ER stress-induced mitochondrial fragmentation. SH-SY5Y cells were transfected with parkin or mCherry (as a control). One day after transfection, the cells were treated with thapsigargin (TG, 1 μM for 5 or 16 h) or tunicamycin (TM, 2 μg/ml for 5 or 16 h). Mitochondria were visualized by life cell microscopy after incubating cells with the fluorescent dye DiOC6(3). (a) The mitochondrial morphology was classified as tubular or fragmented (small rod-like or spherical mitochondria). Shown is the percentage of cells with fragmented mitochondria. Quantifications were based on triplicates of three independent experiments. For each experiment ⩾300 cells per coverslip of triplicate samples were assessed. Expression levels of parkin were analyzed by immunoblotting using the anti-parkin pAb 2132 (lower panel). Loading was controlled by re-probing the blots for β-actin. (b) Examples of mitochondrial morphologies of the experiment described in (a). Treatment of cells with TG or TM cause a disruption of the tubular mitochondrial network, which can be suppressed by increased parkin expression. (c) Parkin deficiency increases ATP depletion in response to ER stress. SH-SY5Y cells were transfected with parkin or control siRNA duplexes. On day 2 after transfection, the cells were shifted to low-glucose medium containing 3 mM glucose instead of 25 mM. On day 3, the cells were treated with 2 μg/ml tunicamycin (TM) for 5 h and the steady-state cellular ATP levels were measured by a bioluminescence assay. Cultured cells derived from tumors derive almost all of their energy from aerobic glycolysis rather than mitochondrial oxidative phosphorylation; in addition, stimulation of glycolysis in the presence of glucose inhibits mitochondrial respiration. Therefore, low glucose concentrations in the medium forces the cells to relay on oxidative phosphorylation to generate sufficient ATP. ^***P<0.001, ^**P<0.01