An In Vitro Model of Parkinson’s Disease: Linking Mitochondrial Impairment to Altered -Synuclein Metabolism and Oxidative Damage

Chronic systemic complex I inhibition caused by rotenone exposure induces features of Parkinson’s disease (PD) in rats, including selective nigrostriatal dopaminergic degeneration and formation of ubiquitinand -synuclein-positive inclusions (Betarbet et al., 2000). To determine underlying mechanisms of rotenone-induced cell death, we developed a chronic in vitro model based on treating human neuroblastoma cells with 5 nM rotenone for 1–4 weeks. For up to 4 weeks, cells grown in the presence of rotenone had normal morphology and growth kinetics, but at this time point, 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble -synuclein protein levels without changing message levels, suggesting that -synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDSinsoluble -synuclein and ubiquitin. After a latency of 2 weeks, rotenone-treated cells showed evidence of oxidative stress, including loss of glutathione and increased oxidative DNA and protein damage. Chronic rotenone treatment (4 weeks) caused a slight elevation in basal apoptosis and markedly sensitized cells to further oxidative challenge. In response to H2O2, there was cytochrome c release from mitochondria, caspase-3 activation, and apoptosis, all of which occurred earlier and to a much greater extent in rotenone-treated cells; caspase inhibition provided substantial protection. These studies indicate that chronic low-grade complex I inhibition caused by rotenone exposure induces accumulation and aggregation of -synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.