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Title: Effect of C9orf72 hexanucleotide repeat expansions on human induced pluripotent stem cell derived oligodendrocytes
Author: Cleary, Elaine Marie
ISNI:       0000 0004 7223 8042
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2017
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A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Genetic testing for this pathogenic mutation is challenging due to its GC rich, repetitive nature. I developed PCR based assays to detect the presence of the pathogenic variant, which were used in screening an archival cohort of Scottish ALS patients, and have also been implemented within a diagnostic setting. These PCR assays allow amplification of larger repeat expansions than have previously been reported, and can determine whether a C9orf72 expansion of greater than 100 repeats is present or not. It is not well understood how the repeat expansion leads to disease, but several potential mechanisms have been hypothesised, including reduced expression, RNA toxicity and protein toxicity via dipeptide repeat proteins produced through repeat associated non-AUG translation. Motor neurons are an understandably well studied target in amyotrophic lateral sclerosis, however the role of glia, particularly oligodendrocytes, in the pathogenesis of the disease has recently been highlighted from studies on rodent models and post mortem tissue. To investigate the effect of the C9orf72 repeat expansion on oligodendrocytes, we have applied a differentiation protocol to hiPSCs with the expansion and controls, including an isogenic control which has been generated in the lab. There was no difference in the production of neuronal and glial cell types between these cell lines. I went on to look for evidence of the main proposed pathological mechanisms of C9orf72 repeat expansions: loss of function or gain of function through either RNA or protein toxicity. hiPSC derived oligodendrocytes from both carrier and control showed low expression of C9orf72 mRNA, and there was no difference due to the presence of a repeat expansion. Carrier hiPSC derived oligodendrocytes displayed sense RNA foci, which did not appear to have an effect on cellular morphology. The detection of dipeptide repeat proteins proved challenging, and the results were inconclusive as to their presence in hiPSC derived oligodendrocytes. I went on to show there was no evidence of mislocalisation of TDP-43 in C9orf72 carrier oligodendrocytes. Finally, the study showed similar levels of cell death in basal conditions in carrier and control cells, and no clear difference in the response to endoplasmic reticulum stress. Further research will be required to elucidate the role of oligodendrocytes in C9orf72 related amyotrophic lateral sclerosis.
Supervisor: Chandran, Siddharthan ; Warner, Jon ; Porteous, Mary Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: C9orf72 gene ; amyotrophic lateral sclerosis ; frontotemporal dementia ; screening ; PCR assays ; oligodendrocytes