Detection of HTTex1p by western blot and immunostaining of HD human and mouse brain using neo-epitope antibody P90 highlights impact of CAG repeat expansion on its size, solubility, and response to MSH3 silencing

Abstract

HTT1a was identified in human and mouse Huntingtons 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.1 Detection of the huntingtin exon 1 protein (HTT1a) has been accomplished with fluorescence-based reporter assays (Meso Scale Discovery, Homogeneous Time Resolved Fluorescence) and immunoprecipitation assays in Huntingtons 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 Huntingtons disease brain were separated by gel electrophoresis and probed by western blot with neo-epitope 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 Huntingtons disease knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTT1a migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass smear in Q111, Q140 and Q175 mice but weakly in Q80 and not in WT 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 unrelated peptide sequence. Migration of HTT1a and its high molecular mass smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the high molecular mass smear indicating that the effects of curbing CAG repeat expansion were quantifiable. A prominent 56-60 kDa doublet detected by 1B12 and 11G2 antibodies in lysates from human Huntingtons disease brain was not blocked by preincubation with C-terminal HTT1a blocking peptide and also appeared in brains of Parkinsons disease patients. 1B12 and 11G2 antibodies did not immunoprecipitate HTT proteins from either Huntingtons 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 Huntingtons disease mouse brain. Abbreviated SummarySapp et al., report that pathogenic exon 1 protein HTT1a is detected in brain of mouse models of Huntingtons disease by direct western blot assay using monoclonal antibodies 11G2 and 1B12. Lowering MSH3 mRNA in the caudate putamen to prevent CAG repeat expansion reduced levels of HTT1a.