Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms

Abstract

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.

Small, Seeding-Competent Huntingtin Fibrils Are Prominent Aggregate Species in Brains of zQ175 Huntington’s Disease Knock-in Mice

“…Our investigations suggest that seeding-competent mHTT aggregate species indeed are detected with MW8 in ELISAs. However, it is important to note that current antibody-based mHTT aggregate detection assays ( Reindl et al, 2019 ; Landles et al, 2021 ), in strong contrast to the FRASE method applied here, might also detect non-fibrillar mHTT assemblies in tissue samples. Such assemblies were previously described in different HD model systems ( Sathasivam et al, 2010 ) and are thought to play a key role in pathogenesis ( Hoffner and Djian, 2014 ).…”

“…R6/2 and zQ175 mice were genotyped and CAG repeat sizing was performed as previously described ( Landles et al, 2021 ). The mean CAG repeat size ± SD was 190.23 ± 4.53 for 6-month-old zQ175, 194.45 ± 3.15 for 12-month-old zQ175, and 181.0 ± 4.0 for 12-week-old R6/2 mice.…”

Alternative processing of humanHTTmRNA with implications for Huntington’s disease therapeutics

“…We have recently established a series of HTRF assays to track changes in soluble and aggregated HTT isoforms in Huntington’s disease mouse tissues. 21 Therefore, we applied these bioassays to investigate how soluble and aggregated HTT levels might change with disease progression in brain regions from YAC128 mice. HTRF relies on the detection of a fluorescent signal that is emitted when two antibodies recognizing specific HTT epitopes are in close proximity.…”

“…Homogeneous time-resolved FRET (HTRF) was performed as previously described. 21 A detailed account of the optimization and use of assays for YAC128 tissues and MEFs is provided in the Supplementary material .…”

Use of high-content imaging to quantify transduction of AAV-PHP viruses in the brain following systemic delivery

“…Mouse husbandry was as previously described. 20 Mice were housed in individually ventilated cages with up to five mice per cage, dependent on sex, with Aspen Chips 4 Premium bedding (Datesand) and environmental enrichment, which included chew sticks and play tunnel (Datesand). On arrival from the supplier, mice were handled daily and acclimatised to the facility for one week prior to the initiation of experiments.…”