Excess ROS production causes oxidative damage and is associated with neurodegeneration. Therefore, there is considerable interest in determining the extent and dynamics of cellular ROS production under normal and pathophysiological conditions. Mitochondria are the major producer of ROS and their dysfunction leads to excessive ROS output. So far redox-sensitive dyes have been used, but their oxidation is irreversible, they are cytotoxic and autooxidize easily. In cultured rat hippocampal neurons transfected with innovative redox sensitive GFP (roGFP1) we succeeded to monitor dynamic changes in ROS levels. Cytosolic roGFP was reversibly oxidized by H2O2, •O2- produced by xanthine/xanthine oxidase and •OH-radicals generated by FeSO4 plus H2O2. CN- immediately increased ROS production, while N2-mediated anoxia and DPI had the opposite effect. Since rotenone did not cause a clear ROS production, the effects of DPI seem to reflect inhibition of cytosolic oxidases rather than inhibition of complex I. 3-NPA also increased ROS generation; antimycin A and FCCP elicited delayed and inconsistent effects. Enabling the continuous probing of ROS levels in central neurons, roGFP will prove valuable in unveiling the signalling role of cytosolic ROS and their mechanistic contribution to neurodegenerative diseases.

Supported by the DFG (SFB 406, CMPB)