Dysregulation of mitochondrial homeostasis in PARK2 iPSC-derived neurons. (A) Electron micrographs of control A (B7), control B (WD39) and PARK2 (PA9 and PB2) iPSC-derived neurons. Boxed areas are shown in the enlarged images to the right. Control mitochondria showed a characteristically long, cylindrical profile with well-organized cristae, and the electron density of the matrix was relatively low (white arrowheads). By contrast, increased electron density of the matrix was evident in PARK2 mitochondria (black arrowheads), and the cristae often appeared swollen. As shown in PB2, some of the neurons contained both morphologically intact (white arrowheads) and abnormal (black arrowheads) mitochondria. Furthermore, abnormal tubulovesicular structures (asterisks) were observed adjacent to the Golgi cisternae (G). (B) The relative perikaryal volume of the abnormal mitochondria was significantly increased, and that of the normal mitochondria was decreased, in PARK2 neurons compared with control neurons. (C) Double labeling for the IMM marker, ComplexIII coreI (CIII-Core I; magenta) and βIII-tubulin (green) of control A (B7), control B (WD39) and PARK2 (PA9 and PB2) iPSC-derived neurons. The volume of the IMM area was reduced in control neurons treated with CCCP, but not in PARK2 neurons treated with CCCP. Administration of Baf A1 rescued the CCCP-induced phenotype in control neurons. (D) The CCCP/DMSO ratio in control A (B7 and YA9) and B (WD39) neurons was reduced after CCCP treatment. This reduction was not observed in PARK2 (PA1, 9 and 22, and PB2 and 20) iPSC-derived neurons (black bars indicate CCCP/DMSO ratio; white bars indicate Baf A1+CCCP/Baf A1 ratio). ** indicates P < 0.01 compared with the control; ¶¶ indicates P < 0.01 when comparing the black and white bars (Mann–Whitney U-test). At least three experiments were performed for each group, with 5–36 cells quantified per experiment. Scale bars: a, 1 μm; c, 10 μm. Error bars represent the SEM. N.D., not detected.