Mitochondrial DNA damage and impaired mitochondrial function contribute toapoptosis of insulin-stimulated ethanol-exposed neuronal cells

Background: Ethanol inhibition of insulin signaling may contribute to impai red central nervous system development in fetal alcohol syndrome. An import ant consequence of ethanol inhibition of insulin signaling is increased apo ptosis due to reduced levels of insulin-stimulated phosphoinositol-3-kinase activity. Methods: We used viability assays, end-labeling, Western blot analysis, and MitoTracker (Molecular Probes, Eugene, OR) fluorescence labeling to determ ine whether ethanol-induced central nervous system neuronal cell death was mediated in part by increased mitochondrial (Mt) DNA damage and impaired Mt function. Results: In ethanol-exposed, insulin-stimulated PNET2, central nervous syst em-derived human neuronal cells, reduced viability was associated with incr eased Mt DNA damage, reduced Mt mass (manifested by reduced Mt protein expr ession and MitoTracker Green fluorescent labeling), and impaired Mt functio n (manifested by reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-dipheny ltetrazolium bromide activity, cytochrome oxidase-Complex IV, Subunit II ex pression, and MitoTracker Red fluorescence). The adverse effects of ethanol on Mt function were reduced by pretreating the cells with broad-spectrum c aspase inhibitors and nearly abolished by nerve growth factor stimulation, with or without concomitant treatment with global caspase inhibitors. Conclusions: These results suggest that ethanol-induced death of insulin-st imulated immature neuronal cells is mediated in part by impaired Mt functio n associated with Mt DNA damage and reduced Mt mass, and therefore it is li kely to contribute to neuronal loss associated with fetal alcohol syndrome. The findings also suggest that the adverse effects of ethanol on insulin-s timulated survival and metabolic function could be overcome by stimulating with growth factors that support Mt function through insulin-independent pa thways.