A015 Mutant Huntingtin protein levels decrease with CAG repeat expansion: implications for therapeutics and bioassays

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Background The Huntington’s disease CAG repeat expansion is unstable and expands in brain and peripheral tissues throughout life. The rate of somatic expansion drives the age of onset and rate of disease progression. Mutant HTT pre-mRNA can be alternatively processed to generate the HTT1a transcript, encoding the highly pathogenic HTTexon1 protein; a mechanism through which somatic expansion may exert pathogenicity since the longer the CAG repeat, the more HTT1a and HTTexon1 protein is produced. Aims The measurement of HTT protein isoforms in disease models and clinical samples is crucial for the interpretation of mechanistic studies and therapeutic interventions. The allelic series of knock-in mouse models: HdhQ20, HdhQ50, HdhQ80, HdhQ111, CAG140 and zQ175 can be used to model the molecular and cellular consequences of CAG repeat expansion within a single neuron. Here, the relationship between CAG repeat length and levels of mutant HTT protein has been investigated. Methodologies: Western blot, Homogenous time-resolved FRET (HTRF), Mesoscale Discovery (MSD). Results By western blotting of cortical lysates, we have found that as the mutant CAG repeat expands, the level of mutant HTT protein decreases dramatically. To identify optimal bioassays for detecting the full-length HTT and HTTexon1 isoforms, we interrogated a comprehensive analysis of 32-pairwise HTT antibody combinations using the HTRF and MSD platforms; almost none of which recapitulated the western blot data. Conclusions HTT bioassays detecting either full-length mutant HTT or HTTexon1, that avoids detecting more than one HTT isoform, are the most appropriate for development. The increase in HTTexon1 levels, whilst full-length mutant HTT decreases with CAG expansion, has significant implications for therapeutic interventions. If mutant HTT levels in cells containing (CAG)200 repeats are only 10% of wild-type, HTT-lowering strategies that only target full-length HTT will predominantly lower wild-type HTT, as mutant HTT levels are already depleted. These data supports therapeutic strategies that lowers HTT1a, depleting levels of the HTTexon1 protein. Funding This work was supported by the CHDI Foundation.

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