Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.820867
Title: Characterisation of novel genetic variants of amyotrophic lateral sclerosis
Author: Moll, Tobias
ISNI:       0000 0004 9356 9588
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2020
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Abstract:
Background: Amyotrophic lateral sclerosis (ALS) is an invariably fatal and relatively common neurodegenerative disorder without effective therapy. Identified genetic variants cluster in biological pathways including RNA processing, axonal transport, and protein homeostasis. Discovery of new genetic variants within new biological pathways highlights new disease biology, and can lead to novel therapeutic targets. This project will focus on the development of cell and animal models to characterise novel ALS-associated mutations associated with GLT8D1, CAV1 and CAV2. Aims and objectives: i) To evaluate the relative toxicity of ALS-associated GLT8D1 mutations in neuronal and non-neuronal cell lines via MTT and lactate dehydrogenase assays. ii) To investigate the effect of mutations on the enzyme activity of GLT8D1 using a UDP-Glo glycosyltransferase assay. iii) To test whether mutant GLT8D1 causes fragmentation of the Golgi network using immunocytochemistry. iv) To model GLT8D1 mutations in zebrafish larvae via RNA microinjection. v) To model CAV1/CAV2 enhancer mutations in neuronal and non-neuronal cells via CRISPR/Cas9 genome editing. vi) To measure ganglioside expression in human cells expressing GLT8D1, CAV1 and CAV2 mutations using live cell imaging. Results: I studied in detail two ALS-associated GLT8D1 mutations: R92C and G78W. The relative toxicity of the mutations in model systems mirrors the clinical severity. Mutated GLT8D1 exhibits in vitro cytotoxicity and induces motor deficits in zebrafish larvae consistent with ALS. Identified GLT8D1 mutations are proximal to the substrate-binding site; both R92C and G78W mutations impair GLT8D1 enzyme activity. An R92C mutation reduces membrane ganglioside expression, which is indicative of dysregulated neurotrophic signalling. Ganglioside biosynthesis occurs in the Golgi; GLT8D1 localises to the Golgi in neuronal and non-neuronal cells, and preliminary data suggests an R92C mutation causes Golgi fragmentation. The second stage of this project follows the identification of ALS-associated variation within an enhancer linked to expression of CAV1/CAV2. CAV1 and CAV2 encode major components of caveolae, which organise membrane lipid rafts (MLR) important for neurotrophic signalling. Gangliosides are a key component of MLR. Discovered enhancer mutations reduce CAV1/CAV2 expression and disrupt ganglioside expression within MLR in patient-derived cells; and CRISPR/Cas9 perturbation proximate to a patient-mutation is sufficient to reduce CAV1/CAV2 expression in neurons. Conclusions: These results place dysregulated ganglioside metabolism upstream in the pathogenesis of ALS. I propose that GLT8D1 and CAV1/CAV2 share a common pathway of pathogenesis in ALS via disruption of ganglioside recruitment to MLR and impaired neurotrophic signalling.
Supervisor: Cooper-Knock, Johnathan ; Shaw, Pamela ; Kirby, Janine Sponsor: Lee Newton Studentship ; Sheffield NIHR Biomedical Research Centre (BRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.820867  DOI: Not available
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