Regulation of the microglial NADPH oxidase by neurotransmitters : implications for microglial-neuronal interactions

Neurotransmitter dysregulation and reactive oxygen species (ROS) are a hallmark of neurodegenerative disease. Microglia, the immune cells of the CNS, express three NADPH oxidase isoforms (Nox1, Nox2 and Nox4), which produce superoxide that is used as an intracellular signalling molecule, mediating the production of neurotoxic and neurotrophic factors. Microglia also express a range of neurotransmitter receptors, enabling them to respond to physiological and pathological levels of neurotransmitters. As both microglial superoxide production and neurotransmitter dysregulation are common to many neurodegenerative conditions, the interaction between microglial neurotransmitter receptor modulation and NADPH oxidase activation was investigated. Superoxide production was assessed in primary and BV2 microglia using flow cytometry, HPLC, fluorescence microscopy, and a colorometric assay. Glutamate (1 μM), GABA (100 μM) or BzATP (250 μM) induced NADPH oxidase derived superoxide production. Furthermore, antagonism of the group I mGluRs induced Nox1 and Nox2 dependent superoxide production through PKC / PI3-K pathways and p44/42ERK activation. Activation of mGluR3 induced Nox2 and Nox4 activation in a p44/42ERK and p38MAPK dependent manner, whilst activation of the group III mGluRs induced Nox2 and Nox4 activation dependent on p38MAPK signalling. Microglial NMDA receptor activation promoted superoxide production that was dependent on p38MAPK activation. Modulation of the microglial glutamate receptors mediated protection of cerebellar granule neurons, as deomstrated using microglial conditioned media assays. Activation of the microglial GABAA receptor induced Nox1 activation through PKC and p38MAPK signalling which mediated TNFα release and neurotoxicity, whilst P2Y2/4 receptor activation mediated Nox1 activation through PI3-K and p38MAPK with neurotoxic consequences. The findings in this thesis show that microglial Nox2 and Nox4 activation has neuroprotective consequences, whereas microglial Nox1 activation mediates neurotoxicity. Modulation of the microglial neurotransmitter receptors therefore has ramifications for the survival of neurons in degenerative diseases, which could have important consequences for the production of future therapies for neurodegenerative conditions.