NITRIC OXIDE DEPENDENT MITOCHONDRIAL DAMAGE PRECEDES APOPTOTIC NEURONAL DEATH IN A PRENATAL HYPOXIC MODEL

GIUSTI, SEBASTIÁN; FISZER DE PLAZAS, SARA

Charleston

Congreso; American Society for Neurochemistry; 2009

American Society for Neurochemistry

NO-mediated toxicity contributes to neuronal damage after hypoxia; however, the involved molecular mechanisms are still a matter of controversy. Since mitochondria play a key role in signaling neuronal death, we aimed to determine the role of nitrative stress in hypoxic-induced mitochondrial damage. Therefore, we analyzed the biochemical and ultrastructural impairment of these organelles in the optic lobe of chick embryos after in vivo hypoxia/reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. Transient but remarkable increase of mtNOS content and activity was observed at 0-2h post-hypoxia resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations of nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-NI (7-nitroindazole) 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. Importantly, 7-NI significantly reduced cytochrome c release and DNA fragmentation, two markers of hypoxic induced apoptotic cell death. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage leading to apoptosis by increasing mitochondrial NO in the developing brain. in vivo hypoxia/reoxygenation. Also, we studied the NO-dependence of damage and examined modulation of mitochondrial nitric oxide synthase (mtNOS) after the hypoxic event. Transient but remarkable increase of mtNOS content and activity was observed at 0-2h post-hypoxia resulting in accumulation of nitrated mitochondrial proteins measured by immunoblotting. However, no variations of nNOS content were observed in the homogenates, suggesting an increased translocation to mitochondria and not a general de novo synthesis. In parallel with mtNOS kinetics, mitochondria exhibited prolonged inhibition of maximal complex I activity and ultrastructural phenotypes associated with swelling, namely, fading of cristae, intracristal dilations and membrane disruption. Administration of the selective nNOS inhibitor 7-NI (7-nitroindazole) 20 min before hypoxia prevented complex I inhibition and most ultrastructural damage. Importantly, 7-NI significantly reduced cytochrome c release and DNA fragmentation, two markers of hypoxic induced apoptotic cell death. In conclusion, we show here for the first time that hypoxia induces NO-dependent complex I inhibition and ultrastructural damage leading to apoptosis by increasing mitochondrial NO in the developing brain.