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Regulation of the Microglial NADPH Oxidase by Neurotransmitters: Implications for Microglial – Regulation of the microglial NADPH oxidase by neurotransmitters: implications for microglial – neuronal interactions Emma Louise Mead UCL Institute of Neurology A thesis submitted for the degree of Doctor of Philosophy (Ph.D) 1 I, Emma Louise Mead, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Acknowledgements First, I would like to thank my supervisor Dr Jennifer Pocock for giving me the opportunity to work in her lab as well as for her guidance and support throughout my research. I would also like to thank my secondary supervisor Professor Simon Heales for his advice. I would like to acknowledge the Medical Research Council and the Brain Research Trust, who funded my Ph.D. Many thanks go to Dr Simon Eaton (UCL, Institute of Child Health) for his expertise and guidance with the HPLC analysis and also to Dr Angelina Mosely (UCL, Institute of Neurology) for her expertise and support with the Flow Cytometry analysis. I would also like to thank Dr Claudie Hooper (Kings College London, Institute of Psychiatry) for her help and guidance with microglial preparations, and for the gift ofthe BV2 microglial cell line, and also for her friendship and advice. Many thanks also go to Mr Thomas Piers, and Dr Ioanna Sevastou for their friendship and encouragement, and for making the last three years thoroughly enjoyable. I would finaly like to thank Kieran, for his continued support and understanding, particularly in the final year of my studies, and many thanks go to my parents for their support, patience, understanding and encouragement throughout my Ph.D and with all decisions that I have made. 3 Abstract 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 4 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. 5 Table of Contents Title …………………………………………………………………………………………...1 Declaration ……………………………………………………………………………………2 Acknowledgements …………………………………………………………………………...3 Abstract………………………………………………………………………………………..4 List of Figures and Tables ………………………………………………………………….10 List of Abbreviations ………………………………………………………………………...16 1. Introduction…………………………………………………………………………..21 1.1 Cells of the Central Nervous System ............................................................................ 22 1.1.1 Neurons ................................................................................................................ 222 1.1.2 Glia ...................................................................................................................... 255 1.1.2.1 Astrocytes .............................................................................................................. 26 1.1.2.2 Oligodendrocytes ................................................................................................... 28 1.1.2.3 Ependymal cells ..................................................................................................... 30 1.1.2.4 Brain macrophages ................................................................................................ 30 1.2 Microglia in heath and disease .................................................................................. 31 1.3 Micrioglial activation in neurodegeneration ................................................................. 35 1.4 The immune system ...................................................................................................... 37 1.4.1 The brain as an immune privileged organ ............................................................. 37 1.5 Reactive oxygen species in the central nervous system ................................................ 41 1.5.1 Pathology and physiology of ROS and the immune response............................... 42 1.5.2 Production of superoxide by the NADPH oxidase ................................................ 44 1.5.3 NADPH oxidase isoforms ..................................................................................... 48 1.5.4 NADPH oxidase in the CNS ................................................................................. 50 1.5.4.1 Microglial expression of the NADPH oxidase ...................................................... 51 1.5.4.1.1 Microglial NADPH oxidase in physiological conditions ............................... 52 1.5.4.1.2 Microglial NADPH oxidase in pathology ...................................................... 54 1.6 Neurotransmitter and neurotransmitter receptor dysregulation in neurodegeneration . 60 1.6.1 Glutamate............................................................................................................... 61 1.6.2 GABA .................................................................................................................... 63 1.6.3 ATP ........................................................................................................................ 64 6 1.6.4 Neurotransmitter receptor modulation of microglial activation in neurodegeneration and involvement of ROS ....................................................................... 67 1.7 Aims and objectives ...................................................................................................... 80 2. Materials and Methods.........................................................................................................82 2.1 Materials ....................................................................................................................... 83 2.2 Methods......................................................................................................................... 85 2.2.1 Cell Culture ............................................................................................................... 85 2.2.1.1 BV2 cell culture ..................................................................................................... 85 2.2.1.2 Primary microglial culture ..................................................................................... 86 2.2.1.3 Cerebellar Granule Cell (CGC) culture ............................................................... 889 2.2.2 Cell treatments........................................................................................................... 90 2.2.2.1 Treatment of primary and BV2 microglia ............................................................. 90 2.2.2.2 Treatment of CGCs ................................................................................................ 92 2.2.2.3 Conditioned media assays ..................................................................................... 92 2.2.3 Cell imaging .............................................................................................................. 93 2.2.3.1 Immunocytochemistry ........................................................................................... 94 2.2.3.2 Assessment of cell death ........................................................................................ 95 2.2.4 Measurement of superoxide production .................................................................... 96 2.2.4.1 NBT detection of intracellular superoxide ............................................................ 98 2.2.4.1.1 NBT assay optimisation ................................................................................... 100 2.2.4.1.2 Optimised
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