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Microglial Glutathione and Glutamate: Regulation Mechanisms

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Microglial Glutathione and Glutamate: Regulation Mechanisms Microglial glutathione and glutamate: Regulation mechanisms Victoria Anne Honey Fry UCL Institute of Neurology A thesis submitted for the degree of Doctor of Philosophy (Ph.D.) 1 I, Victoria Fry, 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 Abstract Microglia, the immune cells of the central nervous system (CNS), are important in the protection of the CNS, but may be implicated in the pathogenesis of neuroinflammatory disease. Upon activation, microglia produce reactive oxygen and nitrogen species; intracellular antioxidants are therefore likely to be important in their self-defence. Here, it was confirmed that cultured microglia contain high levels of glutathione, the predominant intracellular antioxidant in mammalian cells. The activation of microglia with lipopolysaccharide (LPS) or LPS + interferon- was shown to affect their glutathione levels. GSH levels in primary microglia and those of the BV-2 cell line increased upon activation, whilst levels in N9 microglial cells decreased. - Microglial glutathione synthesis is dependent upon cystine uptake via the xc transporter, which exchanges cystine and glutamate. Glutamate is an excitatory neurotransmitter whose extracellular concentration is tightly regulated by excitatory amino acid transporters, as high levels cause toxicity to neurones and other CNS cell types through overstimulation of - glutamate receptors or by causing reversal of xc transporters. Following exposure to LPS, increased extracellular glutamate and increased levels of messenger ribonucleic acid - (mRNA) for xCT, the specific subunit of xc , were observed in BV-2 and primary microglial cells, suggesting upregulated GSH synthesis. An activation-induced decrease in N9 GSH levels suggests that this cell line is more susceptible to oxidative damage, and may be less able to upregulate GSH synthesis. Albumin, to which microglia may be exposed following blood-brain barrier damage, increased iNOS expression, glutamate release, xCT mRNA levels and intracellular levels of GSH and ATP in BV-2 and primary microglia. Primary and BV-2 microglial conditioned medium contained low levels of GSH, suggesting that microglia may release GSH. Modulation of microglial metabotropic glutamate receptors (mGluRs) may alter microglial activation and neurotoxicity. Here, stimulation of the neuroprotective mGluR5 and group III mGluRs caused a decline in GSH levels in BV-2 and N9 microglia, respectively. In contrast mGluR1 stimulation may increase BV-2 GSH levels. The work presented in this thesis therefore extends current knowledge regarding microglial GSH and its regulation, and contributes to the understanding of microglial neurotoxicity and neuroprotection. 3 Table of contents List of tables.............................................................................................................. 10 List of figures............................................................................................................ 11 Acknowledgements .................................................................................................... 18 Abbreviations ............................................................................................................. 19 Chapter 1 - Introduction 1.1. The Central Nervous System .............................................................................. 26 1.1.1. Neurones ...................................................................................................... 26 1.1.2. Astrocytes ..................................................................................................... 27 1.1.3. Oligodendrocytes ......................................................................................... 28 1.1.4. Microglia ...................................................................................................... 28 1.1.5. The Blood-Brain Barrier .............................................................................. 29 1.2. Microglia ............................................................................................................. 31 1.2.1. Origin ........................................................................................................... 31 1.2.2. Morphology of microglia ............................................................................. 32 1.2.3. Microglial activation .................................................................................... 34 1.2.4. Microglia and the immune system ............................................................... 36 1.2.5. Expression and release profiles of activated microglia ................................ 37 1.2.6. Neurone-microglia interactions: neuroprotection and neurotoxicity ........... 41 1.2.7. Microglia in ageing, neuroinflammatory and neurodegenerative disease ... 42 1.3. Neuroinflammatory disease ................................................................................ 44 1.3.1. Multiple sclerosis ......................................................................................... 44 1.3.1.1. MS pathology and pathogenesis ........................................................... 45 1.3.1.2. Microglia in MS .................................................................................... 46 1.3.1.3. Albumin as an in vitro model of BBB damage in MS .......................... 50 1.3.2. Alzheimer‟s disease ..................................................................................... 51 1.3.2.1. Aβ and the amyloid cascade hypothesis ............................................... 51 1.3.2.2. Oxidative stress in AD .......................................................................... 53 1.3.2.3. Microglia in AD .................................................................................... 54 1.3.2.4. Treatment of AD ................................................................................... 59 1.4. Glutamate ............................................................................................................ 60 1.4.1. Glutamate receptors ..................................................................................... 61 4 1.4.1.1. Ionotropic glutamate receptors .............................................................. 61 1.4.1.2. Metabotropic glutamate receptors ......................................................... 64 1.4.2. Glutamate transporters ................................................................................. 76 1.4.2.1. Excitatory amino acid transporters ....................................................... 76 - 1.4.2.2. xc glutamate/cystine transporter system ............................................... 78 1.4.2.3. Microglial glutamate transporters ......................................................... 79 1.4.2.4. Glutamate transporters in AD and MS .................................................. 80 1.5. Glutathione .......................................................................................................... 83 1.5.1. Functions of glutathione ............................................................................... 83 1.5.2. Glutathione synthesis and consumption ....................................................... 84 1.5.3. Glutathione in the CNS ................................................................................ 85 1.5.4. Glutathione and related enzymes in ageing, AD and MS ............................ 88 1.5.5. Astrocytes release GSH................................................................................ 90 1.5.6. Microglial glutathione .................................................................................. 90 1.6. Aims .................................................................................................................... 94 Chapter 2 - Materials and methods 2.1 Materials ............................................................................................................... 97 2.1.1 Animals ......................................................................................................... 97 2.1.2 Reagents ........................................................................................................ 97 2.1.3 Consumables ................................................................................................. 98 2.2 Methods ................................................................................................................ 99 2.2.1 Cell culture .................................................................................................... 99 2.2.1.1 Culture of primary rat microglia ............................................................ 99 2.2.1.2. Microglial cell lines............................................................................. 103 2.2.2 Determination of intracellular reduced glutathione levels with reverse-phase HPLC ................................................................................................................... 105 2.2.2.1 Principle ............................................................................................... 105 2.2.2.2 Preparation of cells ............................................................................... 106 2.2.2.3 Measurement of reduced glutathione ................................................... 109 2.2.2.4 Measurement of oxidised and total glutathione ................................... 111 2.2.3 Determination of intracellular glutathione levels by
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