RESUMEN
Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, alpha-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis here, we used in vivo and in vitro models of chronic low-grade complex I inhibition with the pesticide rotenone. Chronic rotenone exposure in vivo caused oxidative modification of DJ-1, accumulation of alpha-synuclein, and proteasomal impairment. Interestingly, the effects become more regionally restricted such that systemic complex I inhibition eventually results in highly selective degeneration of the nigrostriatal pathway. DJ-1 modifications, alpha-synuclein accumulation, and proteasomal dysfunction were also seen in vitro and these effects could be prevented with alpha-tocopherol. Thus, chronic exposure to a pesticide and mitochondrial toxin brings into play three systems, DJ-1, alpha-synuclein, and the ubiquitin-proteasome system, and implies that mitochondrial dysfunction and oxidative stress link environmental and genetic forms of the disease.
Asunto(s)
Degeneración Nerviosa/inducido químicamente , Proteínas Oncogénicas/efectos de los fármacos , Trastornos Parkinsonianos/inducido químicamente , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Rotenona/toxicidad , Ubiquitina/efectos de los fármacos , alfa-Sinucleína/efectos de los fármacos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Complejo I de Transporte de Electrón/efectos de los fármacos , Complejo I de Transporte de Electrón/fisiología , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/fisiología , Humanos , Insecticidas/toxicidad , Masculino , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Oncogénicas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/fisiopatología , Peroxirredoxinas , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Desglicasa DJ-1 , Ratas , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Sustancia Negra/fisiopatología , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
Huntington's disease (HD) is associated with expansion of polyglutamine tract in a protein named huntingtin (htt) that is expressed in virtually all body tissues. Thus mutated htt (HD-htt) might affect all organs, although clinical manifestations of HD are associated with selective loss of corticostriatal neurons of the brain. In this work we studied how HD-htt affects mitochondria in human peripheral blood cells. We compared various functions of mitochondria isolated from cultured lymphoblastoid cells derived from three HD patients with juvenile onset of the disease (HD-LBM) and three age-matched control (C-LBM) individuals. Respiratory parameters in different metabolic states, with succinate and glutamate plus malate were the same for all control and HD cell lines. State 4 membrane potential in HD-LBM was slightly lower than in C-LBM. The calcium retention capacity (CRC) of mitochondria was estimated using simultaneously several methods to register permeability transition (PT). We found that LBM do not undergo swelling upon Ca2+-induced PT, and do not increase CRC in the presence of ADP + oligomycin. Although each cell line had different CRC values, qualitatively PT was different in C-LBM and HD-LBM. With C-LBM cyclosporin A (CsA) increased CRC significantly, while with HD-LBM CsA was ineffective. In C-LBM depolarization of mitochondria and a large pore opening (PT) always occurred simultaneously. In HD-LBM depolarization occurred at 20-50% lower Ca2+ loads than PT. We suggest that HD-htt promotes low H+ conductance of the mitochondria by interacting with proteins at the contacts sites without directly promoting PT or hampering mitochondrial oxidative phosphorylation.
Asunto(s)
Calcio/farmacología , Enfermedad de Huntington/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Adenosina Difosfato/farmacología , Adolescente , Animales , Cationes Bivalentes/farmacología , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Proteína Huntingtina , Leucocitos/efectos de los fármacos , Leucocitos/fisiología , Mitocondrias/efectos de los fármacos , Dilatación Mitocondrial , Oligomicinas/farmacología , Permeabilidad/efectos de los fármacos , RatasRESUMEN
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.