Interrogating the role of splicing factors in Huntington’s Disease

Huntington’s disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (HTT) protein. Given the wide range of cellular interactions involving HTT, pathogenesis is attributed to both disruption of numerous cellular and metabolic pathways, as well as toxic gain-of-function effects, most notably a propensity of mutant HTT to misfold and aggregate. mRNA splicing defects have been observed in several neurological diseases, though the role of splicing in HD pathogenesis is unclear. A recent genetic modifier screen in baker’s yeast identified several splicing genes which suppress mutant HTT toxicity when overexpressed. I initially interrogated these candidate genes in mammalian cell lines to elucidate the mechanism(s) underlying this protection. Using HD cell models, I have found that overexpression of these splicing genes reduces the level of caspase3/7 activation, a mark for apoptosis. Furthermore, while I observed a mutant HTT-induced impairment of splicing, overexpression of the suppressors failed to ameliorate this defect. However, using automated aggregation analyses, these suppressors were found to modulate HTT aggregation dynamics- a possible mechanism of suppression. I have further interrogated these promising splicing gene hits in the nematode C. elegans, though the results were inconclusive. While characterising disease-relevant phenotypes in HD model mice, I uncovered a robust burrowing defect in these mice, which manifested earlier than impairments in the more widely-used rota-rod locomotor assay. As such, this test could provide a means of detecting early behavioural dysfunction in HD mouse models. Finally I sought to optimise intranasal delivery of lentivirus to the brain, as an alternative to a stereotactic approach. Our results were incredibly variable yet promising and with further optimisation, this technique could be a viable alternative to more invasive methods. Ultimately my work has provided insight into the modulatory potential of splicing factors in HD.