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Guoge Han, Robert Casson, John P M Wood, Glyn Chidlow; The Mitochondrial Complex I Inhibitor, Rotenone, Induces Endoplasmic Reticulum Stress and Activation of GSK-3β in Cultured Rat Retinal Cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6318.
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Mitochondrial dysfunction is implicated in the pathogenesis of central nervous system neurodegenerative disease, including retinal degenerations.In this study, we examined the mechanisms of mitochondria complex I inhibitor rotenone induced cell death in rat retinal cell cultures, which comprise both neurons and glia.
7 day old retinal cultures (comprising neurons and glia) and 28 day old retinal cultures (comprising glia) were treated with the mitochondrial complex I inhibitor, rotenone, at a range of concentrations, for up to 24 hours. In some cases, the cells were treated with ER stress inducer thapsigargin, GSK 3β inhibitor (LiCl) or Calpain-µ inhibitor calpain III-µ. Cell viability was assessed using complementary techniques: immunocytochemistry, immunoblotting, cytotoxicity assay, and TUNEL. Cellular ATP levels in different cell cultures, reactive oxygen species (ROS) were also assessed.
Neurons were relatively more vulnerable to rotenone (1μM) as compared to glial cells; these cells were more likely to die rapidly via non-apoptotic means. Increased expression of the characteristic ER stress components, p-PERK, Bip, ATF4, CHOP was evident in the rotenone-treated cultures after 6 hours, mainly in vimentin-positive Müller glial cells. Positive TUNEL reactivity was also localized to Müller cells at this time. Furthermore, glycogen synthase kinase-3β (GSK3β) and calpain-µ were found to be activated in glia and neurons; inhibition of the former enzyme with LiCl was able to protect all cells, however, inhibition of the latter with calpain inhibitor III only protected neurons.
These data together demonstrate that a variety of mechanisms including ER stress, activation of GSK-3β and calpain stimulation all contributed to rotenone cytotoxicity in rat retinal cell cultures. Such mehanisms were dependent upon the concentration of rotenone, the time of its application and the type of cell which was being affected.
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