title: An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-synuclein metabolism and oxidative damage.
abstract: 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 ubiquitin- and alpha-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, approximately 5% of cells began to undergo apoptosis. Short-term rotenone treatment (1 week) elevated soluble alpha-synuclein protein levels without changing message levels, suggesting that alpha-synuclein degradation was retarded. Chronic rotenone exposure (4 weeks) increased levels of SDS-insoluble alpha-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 alpha-synuclein and ubiquitin, progressive oxidative damage, and caspase-dependent death, mechanisms that may be central to PD pathogenesis.
authors.affiliation: Center for Neurodegenerative Disease and Department of Neurology, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Todd B
authors.initials: TB
authors.lastname: Sherer
authors.forename: Ranjita
authors.initials: R
authors.lastname: Betarbet
authors.forename: Amy K
authors.initials: AK
authors.lastname: Stout
authors.forename: Serena
authors.initials: S
authors.lastname: Lund
authors.forename: Melisa
authors.initials: M
authors.lastname: Baptista
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Panov
authors.forename: Mark R
authors.initials: MR
authors.lastname: Cookson
authors.forename: J Timothy
authors.initials: JT
authors.lastname: Greenamyre
mesh_terms: D000818:Animals; D000978:Antiparkinson Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D019069:Cell Respiration / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004357:Drug Synergism; D042967:Electron Transport Complex I; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D010300:Parkinson Disease / Q000378:metabolism* / Q000473:pathology; D010302:Parkinson Disease, Secondary / Q000139:chemically induced; D012402:Rotenone / Q000494:pharmacology*; D051843:Synucleins; D013995:Time; D014407:Tumor Cells, Cultured; D025801:Ubiquitin / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology; D051844:alpha-Synuclein
chemical_list: D000978:Antiparkinson Agents; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D016877:Oxidants; C497604:SNCA protein, human; D051843:Synucleins; D025801:Ubiquitin; D014475:Uncoupling Agents; D051844:alpha-Synuclein; D012402:Rotenone; D006861:Hydrogen Peroxide; D009247:NADH, NADPH Oxidoreductases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D042967:Electron Transport Complex I; D005978:Glutathione