Mutant huntingtin exon 1 protein detected in mouse brain with neoepitope antibody: effects of CAG repeat expansion, MutS Homolog 3 silencing and aggregation.

Abstract

HTT1a was identified in human and mouse Huntington's disease brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 of HTT that contributes to aggregate formation and neuronal dysfunction. Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with Meso Scale Discovery, Homogeneous Time Resolved Fluorescence and immunoprecipitation assays in Huntington's disease knock-in mice, but direct detection in homogenates by gel electrophoresis and western blot assay has been lacking. Subcellular fractions prepared from mouse and human Huntington's disease brain were separated by gel electrophoresis and probed by western blot with neoepitope monoclonal antibodies 1B12 and 11G2 directed to the C-terminal eight residues of HTT1a. In caudate putamen of an allelic series of 6-month-old Huntington's disease knock-in mice (Q50, Q80, Q111, Q140 and Q175), HTT1a migration was inversely correlated with CAG repeat length and appeared as a sodium dodecyl sulphate soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in wild-type mice or Q50 indicating a CAG repeat size threshold for detecting HTT1a. HTT1a immunoreactivity diminished if 1B12 and 11G2 antibodies were preincubated with an eight amino acid peptide containing the C-terminus of HTT1a but not with an unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Reducing levels of MutS Homolog 3 (MSH3) protein >84% in Q111 mice caudate putamen with small interfering RNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear suggesting that the effects of curbing CAG repeat expansion on HTT1a were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntington's disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinson's disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate huntingtin (HTT) proteins from either Huntington's disease mouse or human brain lysates using conditions that pulled down full-length HTT with anti-HTT antibody 2B7. Altogether, these data show that 11G2 and 1B12 antibodies can be used in western blot assays to track and quantify immunoreactive HTT1a levels, solubility and subcellular localization in Huntington's disease mouse brain.