The apoptosis of cerebellar granule neurons (CGN) induced by low potassium in the extracellular medium is a model of neuronal apoptosis where an overshot of reactive oxygen species (ROS) triggers the neuronal death. In this work, using dihydroethidium and L-012 as specific dyes for superoxide anion detection we show that this ROS overshot can be accounted by an increased release of superoxide anion to the extracellular medium. The amplitude and time course of the increase of superoxide anion observed early during apoptosis correlated with the increase of the content of soluble cytochrome b(5), a substrate of the NADH-dependent oxidase activity of the cytochrome b(5) reductase associated with lipid rafts in CGN. Western blotting and immunofluorescence microscopy approaches, including fluorescence energy transfer, pointed out an enhanced clustering of cytochrome b(5) reductase within caveolins-rich lipid rafts microdomains. Protein/protein docking analysis suggests that cytochrome b(5) reductase can form complexes with caveolins 1α, 1β and 2, playing electrostatic interactions a major role in this association. In conclusion, our results indicate that overstimulation of cytochrome b(5) reductase associated with lipid rafts can account for the overshot of plasma membrane-focalized superoxide anion production that triggers the entry of CGN in the irreversible phase of apoptosis. This article is part of a Special Issue entitled: Proteomics: The clinical link.
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