Investigating the mechanism of the incomplete splicing of huntingtin in models of Huntington’s disease

Huntington’s disease (HD) is a neurodegenerative disorder caused by an expanded CAG repeat in the first exon of huntingtin (HTT). The disease starts in adulthood or in rarer cases, childhood, or adolescence, and is characterised by motor, cognitive and psychiatric disturbances. Despite the monogenic nature of HD, the molecular basis of many disease mechanisms remains to be uncovered. One such mechanism is incomplete splicing, whereby HTT RNA does not splice from exon 1 to exon 2 and instead terminates and polyadenylates in intron 1. This has been observed in HD mouse models, including zQ175 knock-in mice, and cells and post-mortem tissue from HD patients. It results in the production of the Htt1a transcript, which encodes the highly toxic exon 1 HTT protein. Previous studies predicted serine/arginine- rich splicing factor 6 (SRSF6) to be a modifier of this process. In the experiments described in this thesis, a novel constitutive Srsf6 knockout mouse model was used to reduce SRSF6 in mice. Homozygosity for Srsf6 knockout was found to be embryonic lethal, which limited these experiments to the use of the heterozygous mice, for which a 50 % reduction in Srsf6 mRNA and SRSF6 protein was observed. However, heterozygosity for Srsf6 knockout was not sufficient to modify levels of incomplete splicing in zQ175 mouse brains, which was measured using a QuantiGene multiplex gene expression assay. These experiments led to the development of zQ175 mouse embryonic fibroblasts (MEFs) to allow the reduction of Srsf6 further using siRNAs. Incomplete splicing of Htt was readily detected in the zQ175 MEFs using the QuantiGene multiplex assay however SRSF6 ablation did not alter Htt1a levels. Heterogeneous ribonuclearprotein C (hnRNP C) was also tested in the MEFs, as this protein had previously been found to prevent intronic polyadenylation of specific genes; but reduction of hnRNP C did not modify incomplete splicing levels. Although it has not been possible to shed light on the underlying mechanisms of incomplete splicing, a methodological workflow, using zQ175 MEFs has been developed, whereby a possible role for RNA- binding proteins that may underlie this process can be screened. Moreover, these MEFs can be, and have been, used to examine the efficacy of Htt- lowering therapeutics e.g. U1 adaptors