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Gene expression profiling and functional studies of astrocytes in SOD1-related amyotrophic lateral sclerosis

Baker, David (2015) Gene expression profiling and functional studies of astrocytes in SOD1-related amyotrophic lateral sclerosis. PhD thesis, University of Sheffield.

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Abstract

Amyotrophic Lateral Sclerosis (ALS) is the most common adult onset motor neuron (MN) disorder, characterised by muscle wasting due to MN death. Astrocytes play an important role in disease progression in the SOD1G93A transgenic mouse model and patients of ALS. Although astrocytes display a selective toxicity to MN, the toxic factor(s) have not been identified. We hypothesise that differential gene expression in SOD1-ALS astrocytes will reveal targets for therapeutic intervention. Microarray analysis was performed upon Laser Capture Microdissected astrocytes isolated from spinal cord of symptomatic (90 day) and late-stage (120 day) SOD1G93A mice and non-transgenic (NTg) littermates, and from post-mortem human SOD1-ALS and control spinal cord. Functional studies were performed using enzymatic activity assays and immunohistochemistry upon spinal cord and in vitro validation studies were performed using murine neonatal cultures of astrocytes, microglia and embryonic MNs and “i-astrocytes” directly converted from human ALS fibroblasts. In murine astrocytes annotation clustering analysis showed increased expression of lysosomal transcripts which were validated by qPCR . Using functional experiments, we have found significantly higher activity of the lysosomal enzyme β hexosamindase in the spinal cord of SOD1G93A mice. Immune response and phagocytic pathways are also enriched within both datasets, and phagocytosis assays using fluorescently labelled NSC34 cell debris show that SOD1G93A astrocytes engulf significantly higher amounts of neuronal debris compared to NTg controls, highlighting an increased reactivity of astrocytes at symptom onset. Human SOD1-astrocytes show down-regulation of transcripts involved in tight junction formation such as ZO-2, Claudin-5 and Occludin, and we hypothesise that ALS-astrocytes contribute to the breakdown in blood-brain-barrier (BBB) integrity seen in ALS. qPCR confirmed differential expression of BBB-influencing genes such as claudin-5, junctional adhesion molecule 2 and transforming growth factor beta-2. Model BBBs made with i-astrocytes from human patients co-cultured with endothelia show significantly lower transendothelial electrical resistance and dextran-permeability values pointing to an astrocyte role in increased BBB permeability during disease. These studies show the breadth of behaviours displayed by astrocytes during ALS disease progression and will provide an important guide for future therapeutic intervention.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Medicine (Sheffield)
Identification Number/EthosID: uk.bl.ethos.648019
Depositing User: Mr David Baker
Date Deposited: 08 Jun 2015 08:13
Last Modified: 12 Oct 2018 09:22
URI: http://etheses.whiterose.ac.uk/id/eprint/9173

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