PROJECT SUMMARY
Huntington’s Disease (HD) is caused by expanded CAG repeats in exon 1 of the Huntingtin (HTT) gene, resulting
in transcription of mutant HTT mRNA and translation of mutant protein, both of which form nuclear aggregates
in neurons. The mechanisms linking expanded CAG repeats to molecular features and outcomes of HD –
including what contributes to HTT aggregation – is unclear. With the clinical failure of therapies that lower mutant
HTT protein, it is crucial to better understand HD mechanisms to identify additional therapeutic targets for HD.
The presence of an alternatively spliced HTT isoform, called HTT1a, is positively related to HTT CAG repeat
length (predictor of HD onset), and associates with mutant HTT mRNA and protein aggregates in HD mouse
models. How HTT1a and mutant HTT interact to affect aggregates, and whether this interaction can be disrupted,
is unknown. It is also unclear what mechanisms favor HTT1a production. In HD patients, HTT1a is only detected
in brain regions where further lengthening of the inherited HTT CAG tract had occurred over time, a process
called somatic expansion. Somatic expansion, which occurs when repeats misalign after transcription, directly
contributes to HD progression in mice. Whether HTT1a production is a cause or consequence of somatic
expansion, and the effect of modulating this relationship on HD outcomes, has not been directly tested.
This project seeks to use small interfering RNA (siRNA) to dissect the relationship between HTT1a expression,
HTT aggregation, and somatic expansion in HD mouse models. siRNA scaffolds – called di-siRNA and tri-siRNA
– have been optimized to enable widespread, potent gene silencing in the central nervous system (CNS) of mice.
Aim 1 will determine the contribution of HTT isoforms to nuclear mRNA and protein aggregates. Delivery of di-
siRNA targeting mutant HTT or HTT1a to the CNS of HD mice had no impact on aggregates, suggesting
simultaneous knockdown of both transcripts may be needed to disrupt aggregation. In Aim 1, di-siRNA targeting
mutant HTT, HTT1a, or both will be injected into HD mouse CNS. HTT isoform mRNA and protein expression,
subcellular localization, and aggregation will be measured 1 month later. The Aim will use di-siRNA that localize
to cytoplasm or nucleus to dissect the role of HTT isoforms in each subcellular compartment in aggregation. Aim
2 will dissect the relationship between somatic expansion and HTT isoform expression in HD. Somatic expansion
and HTT1a are associated in HD patient brains, and co-silencing Msh3 (modifier of somatic expansion) and
mutant HTT by siRNA further decreases somatic expansion compared to Msh3 silencing alone. Aim 2 will
examine an interplay between somatic expansion and HTT isoforms by injecting tri-siRNA targeting Msh3, HTT
isoforms, or both into HD mice, and measuring HTT isoform expression and subcellular localization, and somatic
expansion 2 months later. Top siRNA that block expansion and HTT isoform expression will be reinjected into
HD mice, and motor function and pathology will be measured. This work will reveal HTT1a mechanisms, inform
HD therapy design, and provide the fellow with training in therapeutic development, HD biology, and microscopy.
