Characterisation of novel cell models of Huntington's disease : insights into pathogenesis

Huntington’s disease (HD) is an adult-onset, autosomal dominant neurodegenerative disease characterised by progressive movement disorder, psychiatric and cognitive symptoms. The causative genetic mutation is an abnormally expanded CAG triplet repeat near the N-terminus of exon 1 of the huntingtin gene (HTT), and longer CAG repeat lengths are associated with earlier age of disease onset. The aim of this thesis was to create a HTT allelic series cell model of HD in which the effects of increasing CAG repeat length, against a stable genetic background, could be observed within human neurons. Initially, recombinant adeno-associated virus (rAAV) was used to knock-in different CAG expansions into an immortalised human neural stem cell line (ReNcellVM), but due to low rates of homologous recombination, this was not successful. Instead, work was undertaken to optimise and characterise a transgenic HTT exon 1 allelic series in the ReNcellVM line; there is much evidence to suggest that HTT exon 1 is the pathogenic species in HD and generates phenotypes over a faster timescale than full-length HTT models. In ReNcellVM neurons the expression of pathogenic HTT exon 1 led to the formation of mutant HTT aggregates in a proportion of cells, in a manner that was related to CAG-repeat length and levels of HTT exon 1 expression. No overt cell-death phenotypes were seen but subtle differences between control and mutant lines were observed. To complement the HTT exon 1 model, an induced pluripotent stem cell (iPSC) model was generated from an HD family who carried a range of CAG-repeat length mutations. These cells were differentiated into medium spiny neurons (MSNs) and both models were used to study the trafficking of huntingtin within cells; whilst differences were observed between the trafficking of control and mutant HTT exon 1, these were not apparent in the MSNs which express full-length HTT.