Abstract: Reducing the burden of mutant Huntingtin (mHTT) protein in brain cells is a strategy for treating Huntington's disease (HD). However, it is still unclear what pathological changes can be reproducibly reversed by mHTT lowering. We previously found that lipid changes that occur with HD progression could be prevented by attenuating HTT transcription of the mutant allele in a genetic mouse model (LacQ140) with inducible whole body lowering. Here, we tested whether intrastriatal injection of a therapeutic capable of repressing the mutant HTT allele with expanded CAG can provide similar protection against lipid changes in HD mice with a deletion of neo cassette (zQ175DN). Methods: Wild-type or zQ175DN mice were injected with AAV9 bearing a cDNA for a zinc finger protein (ZFP) which preferentially targets mutant HTT (ZFP-HTT) to repress transcription (Zeitler et al., 2019). Proteins were analyzed using western blot, capillary electrophoresis, and nitrocellulose filtration methods. Lipid analyses were conducted by liquid chromatography and mass spectrometry (LC-MS). Somatic expansion index was assessed using capillary gel electrophoresis of PCR products. Conclusions: Lowering mHTT levels by 43% for 4 months prevented numerous changes in lipids of caudate-putamen in zQ175DN mice. Our data support the idea that mHTT lowering can provide meaningful benefits and support brain health. Furthermore, our data demonstrate that analyzing lipid signatures is a valuable method for evaluating potential therapies in a preclinical model of HD. Key words: AAV9, striatum, Huntington's disease, transcription, metabolomics, gene therapy
Abstract HTT1a has been identified in human and mouse HD brain as the pathogenic exon 1 mRNA generated from aberrant splicing between exon 1 and 2 that contributes to aggregate formation and neuronal dysfunction (Sathasivam et al., 2013). Detection of the HTT exon 1 protein (HTTex1p) has been accomplished with surrogate antibodies in fluorescence-based reporter assays (MSD, HTRF), and immunoprecipitation assays, in HD postmortem cerebellum and knock-in mice but direct detection by SDS-PAGE and western blot assay has been lacking. Here proteins in subcellular fractions prepared from human and mouse HD brain were separated by SDS-PAGE and probed by western blot with neo-epitope monoclonal antibodies (P90-1B12 and 11G2) directed to the C-terminal 8 residues of HTTex1p. In human HD putamen and cortex, HTTex1p migrated at 56-60 kD and at higher molecular masses (HMM) consistent with the presence of CAG repeat expansion in HTT1a . HTTex1p in control brain was low or undetectable. Immunofluorescence labeling of human HD cortex using P90-11G2 revealed small aggregates that sparsely populated the neuropil in layers 3 and 5. In caudate putamen of 6 month old HD knock-in mice (Q50, Q80, Q111, Q140 and Q175) HTTex1p migration was inversely correlated with CAG repeat length and appeared as a SDS soluble high molecular mass (HMM) smear in HD Q111, Q140 and Q175 mice but not in Q50 and Q80 mice indicating a CAG repeat size threshold for detecting HTTex1p aggregation. Migration of HTTex1p and HMM smear changed with age in caudate putamen of Q111, Q175 and YAC128 mice. Treating HD Q111 mice with siRNA to MSH3, a modifier of CAG repeat expansion, significantly reduced levels of the HMM smear indicating that the effects of curbing CAG repeat expansion was quantifiable. These results show that P90 antibodies can be used in western blot assays and immunostaining to track and quantify HTTex1p levels, subcellular localization, and solubility.