Rotenone-induced Cell Death Research Articles

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death.

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.

Prostaglandin A1 inhibits rotenone-induced apoptosis in SH-SY5Y cells.

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor-kappaB (NF-kappaB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-kappaB and then the activation of caspase-3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone-induced nuclear translocation of NF-kappaB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-kappaB nuclear translocation and caspase-3 activation. PGA1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-kappaB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition.

AnIn VitroModel of Parkinson's Disease: Linking Mitochondrial Impairment to Altered α-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 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.

An in vitro model of Parkinson's disease: linking mitochondrial impairment to altered alpha-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 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.

Activation of adenosine triphosphate-sensitive potassium channels confers protection against rotenone-induced cell death: therapeutic implications for Parkinson's disease.

It is anticipated that further understanding of the protective mechanism induced by ischemic preconditioning will improve prognosis for patients of ischemic injury. It is not known whether preconditioning exerts beneficial actions in neurodegenerative diseases, in which ischemic injury plays a causative role. Here we show that transient activation of ATP-sensitive potassium channels, a trigger in ischemic preconditioning signaling, confers protection in PC12 cells and SH-SY5Y cells against neurotoxic effect of rotenone and MPTP, mitochondrial complex I inhibitors that have been implicated in the pathogenesis of Parkinson's disease. The degree of protection is in proportion to the bouts of exposure to an ATP-sensitive potassium channel opener, a feature reminiscent of ischemic tolerance in vivo. Protection is sensitive to a protein synthesis inhibitor, indicating the involvement of de novo protein synthesis in the protective processes. Pretreatment of PC12 cells with preconditioning stimuli FeSO(4) or xanthine/xanthine oxidase also confers protection against rotenone-induced cell death. Our results demonstrate for the first time the protective role of ATP-sensitive potassium channels in a dopaminergic neuronal cell line against rotenone-induced neurotoxicity and conceptually support the view that ischemic preconditioning-derived therapeutic strategies may have potential and feasibility in therapy for Parkinson's disease.