Characterisation of the transcriptomic and proteomic profile of astrocytes in multiple sclerosis

Multiple sclerosis (MS) is a chronic, neuroinflammatory demyelinating disease of the central nervous system (CNS). Typical white matter lesions (WML) in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex subtle pathology, including blood brain barrier (BBB) dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, are known to support neuronal function, maintain homeostasis within the CNS and regulate neurotransmission. Yet conversely, can promote an inflammatory response, inhibit myelin repair and support the production of autoreactive T cells in MS. This thesis aimed to investigate the transcriptomic and proteomic profile of astrocytes in MS NAWM to determine whether specific astroglial changes exist, which may contribute/prevent disease progression in MS. Initial data presented in this thesis demonstrated a change in astrocyte phenotype within different pathological regions of the CNS in MS, as observed by the distinct immunoprofile of a variety of known astrocyte markers. Being able to isolate cell types from human tissue is fundamental in beginning to define a particular cell’s role in disease pathogenesis. An immuno-laser capture microdissection (LCM) method was developed to enable the isolation of glial cells from human post mortem (PM) CNS tissue. In the current study glial fibrillary acidic protein (GFAP) positive astrocytes were isolated from MS NAWM and control WM via immuno-LCM and microarray analysis completed to compare their transcriptome. Significantly differentially expressed genes were associated with the immune response, cell signaling, cytoskeletal changes and regulation of homeostasis which relate to the distinct roles of astrocytes. Interestingly, from the top 20 significant differentially upregulated genes, six of them were related to the regulation of iron homeostasis and oxidative stress, including metallothionein I-II (MT-I+II), ferritin light chain (FTL) and transferrin (TF). Subsequent transcriptomic and proteomic investigations were carried out on candidate genes using polymerase chain reaction, immunohistochemistry, western blotting and mass spectrometry to investigate the neuroprotective role of astrocytes in regulating iron homeostasis and oxidative stress in MS NAWM. Evidence presented in this thesis demonstrates the importance of astrocytes in the pathogenesis of MS. The results indicate that further investigations into the protective roles of astrocytes in regulating iron and oxidative stress in MS NAWM are warranted