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The Role of GPR65 and GALC in Multiple Sclerosis

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The role of GPR65 and GALC in Multiple Sclerosis Establishing the functional consequences of an MS associated variant on chromosome 14q31.3 Wenjia Liao Peterhouse Department of Clinical Neuroscience October 2018 This dissertation is submitted for the degree of Master of Science. Declarations This dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. The word count is less than 60000. 1 Abstract – The role of GPR65 and GALC in Multiple Sclerosis Wenjia Liao Genome-wide association studies have identified an extensive catalogue of unequivocally associated genetic variants, each of which provides a clue to the aetiology of multiple sclerosis (MS). Unfortunately, for the vast majority of these quite how the genetic change contributes to the development of the disease is unknown. However, preliminary work undertaken by one of my predecessors suggested that the functionally relevant variant underlying the association identified on chromosome 14q31.3 might exert its effect by influencing T cell activation in a pH dependent manner. From a genetics perspective the ImmunoChip study showed that the lead most strongly associated single nucleotide polymorphism (SNP) tagging this association was rs74796499, which lies in a region of genome containing two protein coding genes; the lysosomal enzyme galactosylceramidase (GALC) and acid sensing surface receptor G-protein coupled receptor 65 (GPR65). In my thesis I attempted to validate and replicate this finding, and also to explore the possibility of other pH dependent effects of this SNP. In order to undertake this work, I first developed a system capable of maintain cultured cells at a stable pH for prolonged periods. Using this system I was able to show that the activation of human lymphocytes to stimulation with anti-CD3/CD28 antibodies is maximal at neutral pH and reduced in both acidic and alkaline conditions. Unfortunately, I was unable to replicate the provisional finding that carrying the protective allele at rs74796499 resulted in greater inhibition of CD25 expression in acidic conditions. However, I did observe nominally significant evidence that the expression of the early activation marker CD69 was induced by culturing cells in unstimulated but acidic conditions, and that this expression was highest in individuals carrying the protective allele at rs74796499. In my first attempt to validate and replicate this new finding I not only found further evidence to support this effect on CD69 in unstimulated cells but also found nominally significant evidence that the expression of lactosylceramide (LacCer, CD17) was lower in ex-vivo cells from individuals carrying the protective allele at rs74796499. Unfortunately, in my final confirmatory experiments I was unable to replicate either of these effects. In summary despite carefully studying over 160 healthy subjects, I was unable to find any statistically significant evidence that rs74796499 genotype exerts any effects the expression of either CD25, CD69 or LacCer in ex-vivo cells or stimulated and unstimulated cells cultured under a range of pH conditions. 2 Table of Contents Declarations ............................................................................................................................... 1 Abstract ...................................................................................................................................... 2 Table of Contents ....................................................................................................................... 3 Abbreviation .............................................................................................................................. 8 Chapter 1: Introduction ............................................................................................................ 11 1.1 Multiple sclerosis ...................................................................................................... 11 Pathogenesis of multiple sclerosis ..................................................................... 11 Environmental factors ........................................................................................ 11 Genetic factors ................................................................................................... 12 Current disease modifying therapies for MS ..................................................... 13 1.2 Immunology of multiple sclerosis ............................................................................. 14 CD4+ helper T cells in MS ................................................................................ 14 CD8+ T cells in MS ........................................................................................... 14 Th17 cells and Treg in MS................................................................................. 15 NK and NKT cells in MS................................................................................... 15 Cytokines and chemokines in MS...................................................................... 16 1.3 The genetic analysis of multiple sclerosis ................................................................. 18 Genome-wide studies ......................................................................................... 18 The association of chromosome of 14q31.3 and MS ......................................... 19 1.4 GALC ........................................................................................................................ 23 The role of GALC in myelin metabolism .......................................................... 23 The role of GALC in the immune system .......................................................... 23 GALC and MS ................................................................................................... 24 1.5 GPR65 ....................................................................................................................... 25 The function of GPR65 ...................................................................................... 25 Function of GPR65 in acidic microenvironment ............................................... 26 3 GPR65 is a psychosine receptor ........................................................................ 26 GPR65 and autoimmune diseases ...................................................................... 27 1.6 Preliminary assessment of GALC and GPR65 ......................................................... 27 1.7 Summary ................................................................................................................... 28 Chapter 2: Materials and Methods ........................................................................................... 29 Human PBMC isolation and cell separation ............................................................. 29 Immunocytochemistry (ICC) .................................................................................... 29 Pierce bicinchoninic acid (BCA) protein assay......................................................... 30 Western blot .............................................................................................................. 30 Protein complex immunoprecipitation (Co-IP) and Mass Spectrometry analysis .... 31 siRNA transfection .................................................................................................... 32 DMEM pH measurement and adjustment ................................................................. 32 Cell culture ................................................................................................................ 33 Flow cytometry ......................................................................................................... 33 Flow cytometry gating strategy ................................................................................. 34 Lysosomal pH measurement ..................................................................................... 37 DNA/RNA extraction ................................................................................................ 37 DNase treatment and clean-up .................................................................................. 38 Bioanalyzer for RNA integrity number ..................................................................... 38 cDNA synthesis ......................................................................................................... 39 quantitative PCR ....................................................................................................... 40 Genotyping ................................................................................................................ 41 Buffers and mediums ................................................................................................ 42 Antibodies and dyes .................................................................................................. 44 Chapter 3: Alternate attempts to characterise GPR65 ............................................................. 45 3.1 Introduction ............................................................................................................... 45 3.2 Results ....................................................................................................................... 46 4 3.2.1 Immunocytochemistry ....................................................................................... 46 3.2.2 Western blot ......................................................................................................
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