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Characterisation of a cortical neuronal cell model for SGCE-mutation positive Myoclonus Dystonia

Sperandeo, Alessandra 2021. Characterisation of a cortical neuronal cell model for SGCE-mutation positive Myoclonus Dystonia. PhD Thesis, Cardiff University.
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Dystonia is a hyperkinetic movement disorder caused by co-contraction of antagonistic muscles, resulting in abnormal positions and postures. It is one of the most common and functionally disabling movement disorders, with significant associated lifetime disability. Due to a limited understanding of the underlying pathophysiology of the disorder, there are few therapeutic options available and many patients are resistant to currently available treatments. Myoclonus Dystonia (MD) is caused by mutations to the autosomal dominantly inherited epsilon-sarcoglycan gene (SGCE) which encodes the transmembrane e-sarcoglycan protein. Although SGCE is a maternally imprinted gene, mutations are fully penetrant when paternally inherited, making a neuronal cell model of this disorder an ideal platform to further characterise dystonia pathophysiology. This thesis investigates the impact of loss of expression of the SGCE gene upon the development of excitatory cortical neurons in vitro. Using the CRISPR/Cas9 gene editing technique, a human embryonic stem cell (hESC) compound heterozygous SGCE knockout line (SGCEko) was derived. Differentiation of the SGCEko line towards a cortical glutamatergic neuronal lineage demonstrated no significant differences in neural progenitor cell number and cortical layer markers when compared to their isogenic wild-type control cells. However, differences between the two lines were observed across multiple functional analyses. These included calcium imaging and whole-cell patch clamp techniques, with higher overall levels of calcium activity, smaller amplitudes, longer rise and shorter fall times in the SGCEko hESC-derived cortical neurons compared to controls using the former technique. A similar pattern of increased excitability was observed in whole-cell patch clamp studies, with SGCEko neurons displaying more frequent and longer trains in response to current step injection, as well as action potentials with shorter half-widths,faster rise times and more rapid rates of repolarisation. Subsequent morphological examination of neurite arborisation also identified differences in neuronal architecture, with SGCEko neurons demonstrating a more extensive and complex dendritic arbor morphology. Overall, these results suggest that loss of epsilon-sarcoglycan expression in excitatory glutamatergic cortical neurons results in a hyperexcitable and more complex dendritic branching phenotype, which may in part contribute to the observed clinical phenotype. Future work will include replication of these findings in patient-derived induced pluripotent stem cell (iPSC) models, as well as neuronal models of other Mendelian inherited dystonic disorders.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 24 May 2021
Last Modified: 24 May 2022 01:30

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