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Proteolysis resistant huntingtin isoform induced by antisense oligonucleotide maintains normal huntingtin function in mouse

ABSTRACT: Huntington’s disease (HD) is a late onset neurological disorder for which no neuroprotective therapies efficiently delay onset or progression. A key pathological mechanism in disease involves the proteolysis of polyglutamine (polyQ)-expanded mutant huntingtin (mHTT) generating polyQ-containing N-terminal fragments that are crucial contributors to HD pathogenesis since inhibition of the cleavage at the putative caspase-6 site reduces pathology in a HD mouse model. Interestingly, a naturally occurring alternative spliced form of HTT lacking exon12 (HTTΔ12) leads to a protein with an internal deletion of the caspase-6 N-terminal cleavage site. Here, we have taken a multidisciplinary approach to characterize the therapeutic potential of targeting exon12 of HTT. We show that HTTΔ12 is fully resistant to caspase-6 cleavage in both cell-free and tissue lysate assays, while maintaining overall biochemical and structural properties similar to wild-type (wt)-HTT. We generated mouse deleted for exon12 and found that exon12 is dispensable for HTT main physiological functions including embryonic development and intracellular trafficking such as BDNF transport. Finally, we pharmacologically induced HTTΔ12 using an antisense oligonucleotide (ASO). QRX-704 ASO efficiently distributes in the whole brain, is stable several months, and reduces pathogenic proteolysis. Thus, ASO-induced exon12 deletion of HT We performed gene expression profiling analysis using data obtained from RNA-seq of brain tissue (cortex and striatum) samples originating from mouse littermates from HttΔ12 (Δ12;KI) and HttKO (KO) lines at 3 month of age.

ORGANISM(S): Mus musculus

PROVIDER: GSE209893 | GEO | 2022/07/29

REPOSITORIES: GEO

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Project description:The polyglutamine expansion in huntingtin (Htt) protein is a cause of Huntington’s disease (HD). Htt is an essential gene as deletion of the mouse Htt gene homolog (Hdh) is embryonic lethal in mice. Therefore, in addition to elucidating the mechanisms responsible for polyQ-mediated pathology, it is also important to understand the normal function of Htt protein for both basic biology and for HD. To systematically search for a mouse Htt function, we took advantage of the Hdh +/- and Hdh-floxed mice and generated four mouse embryonic fibroblast (MEF) cells lines which contain a single copy of the Hdh gene (Hdh-HET) and four MEF lines in which the Hdh gene was deleted (Hdh-KO). The function of Htt in calcium (Ca2+) signaling was analyzed in Ca2+ imaging experiments with generated cell lines. We found that the cytoplasmic Ca2+ spikes resulting from the activation of inositol 1,4,5-trisphosphate receptor (InsP3R) and the ensuing mitochondrial Ca2+ signals were suppressed in the Hdh-KO cells when compared to Hdh-HET cells. Furthermore, in experiments with permeabilized cells we found that the InsP3-sensitivity of Ca2+ mobilization from endoplasmic reticulum was reduced in Hdh-KO cells. These results indicated that Htt plays an important role in modulating InsP3R-mediated Ca2+ signaling. To further evaluate function of Htt, we performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray. Our results revealed that 106 unique transcripts were downregulated by more than two-fold with p < 0.05 and 173 unique transcripts were upregulated at least two-fold with p < 0.05 in Hdh-KO cells when compared to Hdh-HET cells. The microarray results were confirmed by quantitative real-time PCR for a number of affected transcripts. Several signaling pathways affected by Hdh gene deletion were identified from annotation of the microarray results. The unbiased approach used in our study provides novel and unique information about the normal function of Htt in cells, which may contribute to our understanding and treatment of HD. Keywords: cell type comparison we generate four Hdh-HET MEF cell lines and four Hdh-KO MEF cell lines, and performed genome-wide transcription profiling of generated Hdh-HET and Hdh-KO cells by microarray.