Iron Activation of Cellular Oxidases: Modulation of Neuronal Viability (In Vitro)

Iron contributes to oxidative stress in a number of conditions. The reactive oxygen species (ROS) generated by iron can activate the NADPH oxidase (NOX) enzymes, which are expressed predominantly in microglia in the brain and contribute to activation of pro-inflammatory pathways and the production of even more ROS. This feed forward paradigm may explain chronic pro-inflammatory polarization of microglia following traumatic brain or spinal cord injury. Currently no research has explored the relationship between iron and NOX. Therefore we hypothesize that exogenous iron stimulates NOX to generate ROS, perpetuating a pro-inflammatory phenotype in microglia that diminishes neuronal viability in vitro.  To test this hypothesis, we propose three specific aims.  

The first aim will characterize the response of microglia to iron in vitro. Preliminary experiments have revealed that iron increased ROS production and the pro-inflammatory polarization shift in lipopolysaccharide (LPS)-stimulated microglia in a dose-dependent fashion. We now propose to fully investigate the pro-inflammatory response of microglia exposed to iron.  

The second aim will investigate the effect of NOX inhibition on microglia following iron exposure. Preliminary data suggests that NOX inhibition reduces the microglial ROS production induced by iron and LPS.  We propose utilizing pharmacological NOX inhibitors and genetic manipulation of NOX expression to evaluate the relationship between NOX, oxidative stress and iron.  

Finally, aim 3 will determine neuronal viability after exposure to iron-stimulated microglia. Pro-inflammatory microglia are known to be toxic to neurons; we suspect this phenomenon may be exacerbated by iron.  We therefore propose to expose neurons to microglia following exposure to iron with and without NOX inhibitors, followed by detailed evaluation of neuronal viability.  Ultimately, this research will determine the impact of excess iron, potentially from blood transfusions, on inflammation and neuronal viability.  

This proposal will be the first study to evaluate the role of iron in NOX mediated oxidative stress and the first to evaluate a potential therapeutic approach of inhibition of this enzyme.