Huntington’s disease is caused by the expansion of a CAG repeat within exon 1 of the HTT gene, which is unstable, leading to further expansion, the extent of which is brain region and peripheral tissue specific. The identification of DNA repair genes as genetic modifiers of Huntington’s disease, that were known to abrogate somatic instability in Huntington’s disease mouse models, demonstrated that somatic CAG expansion is central to disease pathogenesis, and that the CAG repeat threshold for pathogenesis in specific brain cells might not be known. We have previously shown that the HTT gene is incompletely spliced generating a small transcript that encodes the highly pathogenic exon 1 HTT protein. The longer the CAG repeat, the more of this toxic fragment is generated, providing a pathogenic consequence for somatic expansion. Here, we have used the R6/2 mouse model to investigate the molecular and behavioural consequences of expressing exon 1 HTT with 90 CAGs, a mutation that causes juvenile Huntington’s disease, compared to R6/2 mice carrying ∼200 CAGs, a repeat expansion of a size rarely found in Huntington’s disease patient’s blood, but which has been detected in post mortem brains as a consequence of somatic CAG repeat expansion. We show that nuclear aggregation occurred earlier in R6/2(CAG)90 mice and that this correlated with the onset of transcriptional dysregulation in these lines. Whereas in R6/2(CAG)200 mice, cytoplasmic aggregates accumulated rapidly and closely tracked with the progression of behavioural phenotypes and with end-stage disease. We find that aggregate species formed in the R6/2(CAG)90 brains have different properties to those in the R6/2(CAG)200 mice. Within the nucleus, they retain a diffuse punctate appearance throughout the course of the disease, can be partially solubilised by detergents and have a greater seeding potential in young mice. In contrast, aggregates from R6/2(CAG)200 brains polymerise into larger structures that appear as inclusion bodies. These data emphasise that a subcellular analysis, using multiple complementary approaches, must be undertaken in order to draw any conclusions about the relationship between HTT aggregation and the onset and progression of disease phenotypes.

Huntington’s disease is caused by a CAG/polyglutamine repeat expansion. Mutated CAG repeats undergo somatic instability, resulting in tracts of several hundred CAGs in the brain; and genetic modifiers of Huntington’s disease have indicated that somatic instability is a major driver of age of onset and disease progression. As the CAG repeat expands, the likelihood that exon 1 does not splice to exon 2 increases, resulting in two transcripts that encode full-length HTT, as well as the highly pathogenic and aggregation-prone exon 1 HTT protein. Strategies that target the huntingtin gene or transcripts are a major focus of therapeutic development. It is essential that the levels of all isoforms of HTT can be tracked, to better understand molecular pathogenesis, and to assess the impact of HTT lowering approaches in preclinical studies and clinical trials. HTT bioassays for soluble and aggregated forms of HTT are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1 HTT and full-length HTT. In addition, they are frequently used to quantify HTT levels in the context of highly expanded polyglutamine tracts, for which appropriate protein standards do not currently exist. Here, we set out to develop novel HTT bioassays to ensure that all soluble HTT isoforms could be distinguished. We utilised the zQ175 Huntington’s disease mouse model that has approximately 190 CAGs, a CAG repeat size for which protein standards are not available. Initially, 30 combinations of six antibodies were tested on three technology platforms: homogeneous time-resolved fluorescence, amplified luminescent proximity homogeneous assay and Meso Scale Discovery, and a triage strategy was employed to select the best assays. We found that, without a polyQ-length matched standard, the vast majority of soluble mutant HTT assays cannot be used for quantitative purposes, as the highly expanded polyQ tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant HTT, soluble exon 1 HTT, soluble mutant HTT (excluding the exon 1 HTT protein) and total soluble full-length HTT (mutant and wild-type). Several novel aggregation assays were also developed that track with disease progression. These selected assays can be used to compare levels of HTT isoforms in a wide variety of mouse models of Huntington’s disease and to determine how these change in response to genetic or therapeutic manipulations.