Project description:Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by abnormal expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin gene (HTT). The resultant mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin protein (HTT) levels alleviates HD-associated motor and neuropathological abnormalities, confirming the importance of huntingtin-lowering as a therapeutic approach. Several therapies in development repress HTT transcription or translation, including antisense oligonucleotides, virally-delivered microRNAs, and zinc finger protein transcription factors. However, they all require invasive procedures to reach the central nervous system (CNS) and do not distribute evenly to target areas in the brain. Systemically distributed therapeutics are needed to address the CNS and peripheral dysfunctions associated with HD. Here we report the discovery of small molecule splicing modifiers that lower HTT expression by selective modulation of pre-mRNA splicing. These compounds promote the inclusion of a pseudoexon containing a premature termination codon triggering HTT mRNA degradation and a reduction of HTT protein levels in vitro and in vivo. These orally bioavailable small molecules represent a non-invasive treatment option for HD and our findings support their continued development for the treatment of HD.
Project description:Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by abnormal expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats in the huntingtin gene (HTT). The resultant mutant protein is ubiquitously expressed and drives pathogenesis of HD through a toxic gain-of-function mechanism. Animal models of HD have demonstrated that reducing huntingtin protein (HTT) levels alleviates HD-associated motor and neuropathological abnormalities, confirming the importance of huntingtin-lowering as a therapeutic approach. Several therapies in development repress HTT transcription or translation, including antisense oligonucleotides, virally-delivered microRNAs, and zinc finger protein transcription factors. However, they all require invasive procedures to reach the central nervous system (CNS) and do not distribute evenly to target areas in the brain. Systemically distributed therapeutics are needed to address the CNS and peripheral dysfunctions associated with HD. Here we report the discovery of small molecule splicing modifiers that lower HTT expression by selective modulation of pre-mRNA splicing. These compounds promote the inclusion of a pseudoexon containing a premature termination codon triggering HTT mRNA degradation and a reduction of HTT protein levels in vitro and in vivo. These orally bioavailable small molecules represent a non-invasive treatment option for HD and our findings support their continued development for the treatment of HD.
Project description:Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, causative of Huntington’s disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, however none of these studies were designed to systematically characterize PTMs on the endogenous full-length Htt protein.We found that full-length endogenous Htt, immunoprecipitated from HD knock-in mouse and human post mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free mass spectrometry, we identified around 40 PTMs, of which half are novel. These findings will be instrumental in the further assembling the Htt PTM framework, and validate PTMs of Htt as promising therapeutic targets for HD.
Project description:Huntington's disease (HD) is characterized by the aggregation of polyglutamine-expanded huntingtin (HTT), proceeding from soluble oligomers to end-stage inclusions. The molecular mechanisms of how protein aggregation leads to neuronal dysfunction are not well understood. We employed mass spectrometry-based quantitative proteomics to dissect spatiotemporal mechanisms of neurodegeneration using the R6/2 mouse model of HD. We show that extensive remodeling of the soluble brain proteome correlates with changes in insoluble aggregate formation during disease progression. In-depth characterization of HTT inclusion bodies uncovered an unprecedented complexity of several hundred proteins. Sequestration to inclusions was dependent on protein expression levels and the presence of aggregation-prone amino acid sequence features, such as low-complexity regions or coiled-coil domains. Overexpression of several sequestered proteins ameliorated HTT toxicity and modified the aggregation behavior in an in vitro model of HD. Our study provides a comprehensive and spatiotemporally-resolved proteome resource of HD progression, indicating that widespread loss of protein function contributes to aggregate-mediated toxicity.
Project description:Huntington's Disease (HD) is a fatal neurodegenerative disorder caused by an extended polyglutamine repeat in the N-terminus of the huntingtin (Htt) protein. Reactive microglia and elevated cytokine levels are observed in the brains of HD patients, but the extent to which neuroinflammation results from extrinsic or cell-autonomous mechanisms is unknown. Furthermore, the impact of microglia activation on the pathogenesis of HD remains to be established. Using genome-wide approaches, we show that expression of mutant Htt in microglia promotes cell-autonomous pro-inflammatory transcriptional activation within microglia by increasing the expression and transcriptional activities of the myeloid lineage-determining factors PU.1 and C/EBPs. Elevated levels of PU.1 and its target genes are observed in the brains of mouse models and HD individuals. Moreover, mutant Htt expressing microglia exhibit an increased capacity to induce neuronal death ex vivo and in vivo in the presence of sterile inflammation. These findings suggest that expression of mutant Htt in microglia may contribute to neuronal pathology in Huntingtin disease. RNA-Seq and ChIP-Seq for PU.1, C/EBP, and H3K4me2 in BV2 cells and RNA-Seq in primary microglia and macrophages
Project description:Inflammation is a key component of pathological angiogenesis. Here we induce cornea neovascularisation using sutures placed into the cornea, and sutures are removed to induce a regression phase. We used whole transcriptome microarray to monitor gene expression profies of several genes