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Characterisation of Immunometabolic Responses in Astrocytes

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Characterisation of Immunometabolic Responses in Astrocytes Characterisation of immunometabolic responses in astrocytes Submitted by Josephine L. Robb to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Medical Studies in January 2020. This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that any material that has previously been submitted and approved for the award of a degree by this or any other University has been acknowledged. Signature: ……………………………………………………………………… 1 Abstract Astrocytes play a role in the central nervous system (CNS) inflammatory response. In many immune cell types cellular inflammation and metabolism are linked, a phenomenon termed ‘immunometabolism’. This has led to attempts to reduce chronic inflammation through manipulating cellular metabolism. Proteins of interest for this approach include transcription factor nuclear factor-kappa B (NF-κB) and mitochondrial protein ‘translocator protein (18 kDa)’ (TSPO). TSPO is of particular interest as a therapy for CNS disease as many TSPO ligands can access the CNS and have been demonstrated to have anti-inflammatory effects. However, immunometabolism has not been well described in astrocytes, and the function of TSPO is currently disputed. In this thesis, mouse primary astrocytes were used to characterise immunometabolic responses following treatment with the pro-inflammatory stimulus lipopolysaccharide (LPS). An initial increase in glycolytic metabolism was measured, prior to a shift towards oxidative phosphorylation and away from glucose metabolism, in part mediated by a decrease in glucose transporter GLUT1 expression. Pharmacological inhibition of NF-κB signalling demonstrated that this pathway is important in mediating the cellular metabolic response to inflammation in astrocytes, and may play a role in maintaining basal metabolic function in these cells. Pharmacological or genetic modulation of TSPO signalling in human astroglioma (U373) cells and/or mouse primary astrocytes demonstrated that while TSPO suppressed fatty acid oxidation and promoted glycolytic metabolism, this did not appear to acutely alter the inflammatory response of these cells after LPS treatment; however, the longer term effects remain to be explored. Together these data demonstrate that inflammation and metabolism are intrinsically linked in astrocytes and TSPO plays an important role in regulating metabolism in these cells. 2 Table of Contents Abstract ............................................................................................................... 2 List of Figures ................................................................................................... 12 List of Tables..................................................................................................... 18 Appendices ....................................................................................................... 19 Publications arising from thesis........................................................................ 19 Authors Declaration .......................................................................................... 20 Acknowledgements .......................................................................................... 21 List of Abbreviations ......................................................................................... 22 Chapter 1. Introduction ......................................................................................... 27 1.1 Astrocytes as regulators of CNS function .................................................. 27 1.1.1 Maintenance of the extracellular environment .................................... 28 1.1.2 Maintenance of neuronal function ....................................................... 29 1.2 Inflammation ............................................................................................... 31 1.2.1 Chronic inflammation in disease ......................................................... 32 1.2.2 Inflammation in the CNS ..................................................................... 34 1.2.3 Astrocytes in CNS inflammation.......................................................... 35 1.2.4 Regulation of inflammation .................................................................. 39 1.2.5 Nuclear Factor-kappa B regulation of inflammation ........................... 40 1.2.6 Astrocytic NF-κB signalling in the regulation of neuronal function ..... 44 1.2.7 The energetic cost of inflammation ..................................................... 45 1.3 Immunometabolism: Fuelling inflammation ............................................... 46 3 1.3.1 Glycolysis in immunometabolism ........................................................ 47 1.3.2 Mitochondria in energy metabolism .................................................... 60 1.3.3 TCA cycle in immunometabolism ........................................................ 61 1.3.4 Oxidative phosphorylation in immunometabolism .............................. 64 1.3.5 Mitochondrial dynamics in immunometabolism .................................. 68 1.3.6 Fatty acid oxidation in immunometabolism ......................................... 70 1.4 Translocator protein 18 kDa (TSPO) ......................................................... 75 1.4.1 Expression of TSPO ............................................................................ 76 1.4.2 Synthetic TSPO ligands ...................................................................... 81 1.4.3 Functions of TSPO .............................................................................. 85 1.4.4 TSPO ligand safety and clinical use ................................................... 91 1.5 Summary of introduction ............................................................................ 92 1.6 Aims of investigation .................................................................................. 93 Chapter 2. Methods .............................................................................................. 94 2.1 Materials ..................................................................................................... 94 2.1.1 Equipment ............................................................................................ 94 2.1.2 Reagents .............................................................................................. 95 2.1.3 Animals and ethics .............................................................................. 96 2.2 Methods ...................................................................................................... 97 2.2.1 Genotyping ........................................................................................... 97 2.2.2 Primary astrocyte isolation and cell culture ...................................... 100 2.2.3 Human astroglioma cell line (U373) culture ...................................... 103 4 2.2.4 Cell treatments ................................................................................... 104 2.2.5 Immunocytochemistry and culture purity .......................................... 104 2.2.6 Mitochondrial network characterisation ............................................ 105 2.2.7 Cell viability assay ............................................................................. 106 2.2.8 Protein isolation ................................................................................. 107 2.2.9 Protein estimation .............................................................................. 109 2.2.10 Immunoblotting ................................................................................ 109 2.2.11 Cell culture media cytokine concentrations .................................... 112 2.2.12 Metabolic analyses .......................................................................... 112 2.2.13 Glucose uptake rate measurement ................................................. 120 2.2.14 Intracellular ATP measurements ..................................................... 120 2.2.15 Mitochondrial membrane potential .................................................. 121 2.2.16 Immunoprecipitation ........................................................................ 122 2.3 Data presentation and statistics ............................................................... 124 Chapter 3. Metabolic changes due to inflammation are essential for cytokine release in mouse primary astrocytes ................................................................. 125 3.1 Introduction ............................................................................................... 125 3.2 Hypothesis ................................................................................................ 129 3.3 Results ...................................................................................................... 129 3.3.1 Lipopolysaccharide (LPS) stimulated NF-κB signalling in mouse primary astrocytes ....................................................................................... 129 3.3.2 LPS increased cytokine secretion from mouse primary astrocytes . 132 5 3.3.3 Lipopolysaccharide did not decrease viability of mouse primary astrocytes .................................................................................................... 135 3.3.4 LPS induced an acute increase in glycolytic rate in mouse primary astrocytes ...................................................................................................
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