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
huntingtin protein, as well as the highly pathogenic and aggregation-prone exon 1
huntingtin 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
huntingtin protein can be tracked, to better understand the molecular pathogenesis, and to assess the impact of
huntingtin protein-lowering approaches in preclinical studies and clinical trials.
Huntingtin protein bioassays for soluble and aggregated forms of
huntingtin protein are in widespread use on the homogeneous time-resolved fluorescence and Meso Scale Discovery platforms, but these do not distinguish between exon 1
huntingtin protein and full-length
huntingtin protein. In addition, they are frequently used to quantify
huntingtin protein 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
huntingtin protein bioassays to ensure that all soluble
huntingtin protein isoforms could be distinguished. We utilized the zQ175
Huntington's disease mouse model that has ∼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
polyglutamine-length-matched standard, the vast majority of soluble mutant
huntingtin protein assays cannot be used for quantitative purposes, as the highly expanded
polyglutamine tract decreased assay performance. The combination of our novel assays, with those already in existence, provides a tool-kit to track: total soluble mutant
huntingtin protein, soluble exon 1
huntingtin protein, soluble mutant
huntingtin protein (excluding the exon 1
huntingtin protein) and total soluble full-length
huntingtin protein (mutant and wild type). Several novel aggregation assays were also developed that track with
disease progression. These selected assays can be used to compare the levels of
huntingtin protein 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.