Oxidative stress-induced parkin auto-ubiquitination and aggregation in PD cell-based and animal models. (A) Auto-ubiquitination of parkin examined in parkin-overexpressing SH-SY5Y cells exposed to H2O2. H2O2 induced dose-dependent and biphasic effects on parkin activity with an initial increase in activity at 2 hours followed by a decrease in activity by 6 hours. (B) and (C) H2O2 involvement in MPP+-induced parkin aggregation in parkin-overexpressing SH-SY5Y cells. Cells plated onto cover slips in 24-well culture plates were immunostained with anti-myc mAb for parkin followed by second antibody conjugated to Cy3 fluorescence dye. Cells were then counterstained for nuclear DNA with Hoechst dye 33342. Inset: Higher magnification of cells showing parkin immunoreactive aggregates after MPP+ exposure. Data are expressed as mean ± SEM; n = 3, *p < 0.01 for MPP+ vs. Control; #p < 0.01 for MPP+ vs. MPP+ + Catalase by post-hoc ANOVA. Catalase protected parkin from aggregation in parkin-overexpressing SH-SY5Y cells exposed to MPP+, indicating that H2O2-mediated parkin sulfonation may play a role in formation of parkin aggregates. (D) Increased parkin aggregation (arrows) in striatum of rats exposed to rotenone. Inset: High-power view showing parkin immunoreactive aggregates. (E) Increased parkin aggregation in monkeys exposed to MPTP. hNSCs transplanted into MPTP-lesioned monkeys appeared to diminish parkin aggregation (arrow) in the host nigrostriatal system including substantia nigra, similar to that of nonlesioned monkeys with or without hNSCs. Scale bar: D, 100 μm; E, 50 μm.