Truncation of mutant huntingtin in knock-in mice via CRISPR-Cas9 uncovers exon1 huntingtin as a key pathogenic form
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA608647 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR11177807_1.fastq.gz | Fastqsanger.gz | |||
| SRR11177807_2.fastq.gz | Fastqsanger.gz | |||
| SRR11177808_1.fastq.gz | Fastqsanger.gz | |||
| SRR11177808_2.fastq.gz | Fastqsanger.gz | |||
| SRR11177809_1.fastq.gz | Fastqsanger.gz |
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Project description:It remains unknown how polyglutamine expansion in widely expressed proteins can cause selective neurodegeneration. In Huntington’s disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Considerable efforts have been devoted to investigating how HTT is cleaved and whether blocking its cleavage is therapeutically beneficial. However, using CRISPR-Cas9 to truncate full-length mutant Htt in HD140Q knock-in (KI) mice, we found that exon1 Htt is stably present in the brain, regardless of truncation sites in full-length Htt. This N-terminal Htt led to similar HD phenotypes and age-dependent Htt accumulation in striatum in different KI mice. Exon1 Htt is constantly generated but its selective accumulation in the striatum is caused by the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function accounts for the selective neuropathology in HD and highlight therapeutic importance in regulating this function.
2020-04-21 | GSE145879 | GEO
Project description:Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.
| S-EPMC7244548 | biostudies-literature
Project description:An autosomal recessive disease is caused by biallelic loss-of-function mutations. However, when more than two disease-causing variants are found in a patient’s gene, it has been challenging to determine which two of the variants are responsible for the disease phenotype. To decipher the pathogenic variants by precise haplotyping, we applied nanopore Cas9-targeted sequencing (nCATS) to three truncation COL7A1 variants detected in a patient with recessive dystrophic epidermolysis bullosa (EB). The distance between the most 5’ and 3’ variants was around 19 kb at the level of genomic DNA. nCATS successfully delineated that the most 5’ and 3’ variants were located in one allele while the variant in between was in the other allele. Intriguingly, the proband’s mother, who was phenotypically intact, was heterozygous for the allele that harbored the two truncation variants, which could otherwise be misinterpreted as those of typical recessive dystrophic EB. Our study illuminates nCATS as a useful tool to determine haplotypes of complicated genetic cases. Haplotyping of multiple variants in a gene can tell which variant should be therapeutically targeted when nucleotide-specific gene therapy is applied.
2022-05-02 | GSE196673 | GEO
Project description:Cas9-guided haplotyping of three truncation variants in autosomal recessive disease
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Project description:FGFR2 C-terminal truncation in cancer
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Project description:Huntingtin protein, mutated in Huntington disease, is implicated in nucleic acid- mediated processes, yet evidence for direct huntingtin-nucleic acid interaction is limited. Here we show wildtype and mutant huntingtin co-purify with nucleic acids, primarily RNA, and interact directly with G-rich RNAs in in vitro assays. Huntingtin RNA immunoprecipitation sequencing from patient-derived fibroblasts and neuronal progenitor cells expressing wildtype and mutant huntingtin revealed NEAT1 as a significantly enriched transcript. Altered NEAT1 levels were evident in Huntington’s disease cells and postmortem brain tissues, and huntingtin knockdown decreased NEAT1 levels. Huntingtin co-localized with NEAT1 in paraspeckles, and we identified a high-affinity RNA motif preferred by huntingtin. This study highlights NEAT1 as a novel huntingtin interactor, demonstrating huntingtin’s involvement in RNA-mediated functions and paraspeckle regulation.
2024-12-31 | GSE267435 | GEO
Project description:Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a CAG triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) stretch in the protein product which becomes aggregation-prone. The mutant Htt (mHtt) aggregates are associated with components of the Ubiquitin-Proteasome System (UPS), suggesting that mHtt is marked for proteasomal degradation and that for reasons still debated are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and its subsequent cellular response. We identified two lysine residues - 6 and 9 - in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size, but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Furthermore, proteomic analysis indicated that compared to cells expressing the protein that can be modified by ubiquitin, cells expressing the lysine-less protein did not activate protein synthetic pathways that enable the cell to cope with stress. Taken together, the findings suggest a novel role for ubiquitination - attenuation of the pathogenic effect of mHtt.
2020-06-30 | PXD019553 | Pride
Project description:Huntington's disease is a fatal neurodegenerative disorder characterized by the aggregation of polyglutamine-expanded huntingtin into oligomers and fibrils. How protein aggregation leads to cellular dysfunction is not well understood. To address this question, we combined in-cell single molecule fluorescence spectroscopy with quantitative proteomics to define how huntingtin engages in aberrant protein interactions during its progressive aggregation. We find that huntingtin interacts preferentially with key components of distinct cellular processes as aggregation proceeds from soluble oligomers to end-stage inclusions. The aberrant interactions of soluble oligomers are highly enriched on RNA-binding proteins and with proteins functioning in ribosome biogenesis, translation, transcription, and vesicle transport. A significant characteristic of these interactors is the presence of extended low-complexity sequence regions. Compared to the soluble aggregates, the interactome of insoluble inclusions is significantly less complex and is enriched in protein quality control components. Our results suggest a 'multiple hit' model for polyglutamine interactions in pathogenesis, with detrimental effects on cell function occurring in an aggregation stage-dependent manner.
2017-01-31 | PXD003446 | Pride
Project description:This SuperSeries is composed of the following subset Series: GSE38000: Polyglutamine expanded huntingtin dramatically alters the genome-wide binding of HSF1 (ChIP-Seq) GSE38001: Polyglutamine expanded huntingtin dramatically alters the genome-wide binding of HSF1 (mRNA) Refer to individual Series
2012-06-18 | E-GEOD-38002 | biostudies-arrayexpress
Project description:Huntington’s disease (HD) is a progressive incurable neurodegenerative disorder characterized by motor and neuropsychiatric symptoms. It is caused by expansion of a CAG triplet in the N-terminal domain of exon 1 in the huntingtin (HTT) gene that codes for an expanded polyglutamine (polyQ) stretch in the protein product which becomes aggregation-prone. The mutant Htt (mHtt) aggregates are associated with components of the Ubiquitin-Proteasome System (UPS), suggesting that mHtt is marked for proteasomal degradation and that for reasons still debated are not properly degraded. We used a novel HD rat model, proteomic analysis, and long-term live neuronal imaging to characterize the effects of ubiquitination on aggregation of mHtt and its subsequent cellular response. We identified two lysine residues - 6 and 9 - in the first exon of mHtt that are specifically ubiquitinated in striatal and cortical brain tissues of mHtt-transgenic animals. Expression of mHtt exon 1 lacking these ubiquitination sites in cortical neurons and cultured cells was found to slow aggregate appearance rates and reduce their size, but at the same time increase the number of much smaller and less visible ones. Importantly, expression of this form of mHtt was associated with elevated death rates. Furthermore, proteomic analysis indicated that compared to cells expressing the protein that can be modified by ubiquitin, cells expressing the lysine-less protein did not activate protein synthetic pathways that enable the cell to cope with stress. Taken together, the findings suggest a novel role for ubiquitination - attenuation of the pathogenic effect of mHtt.
2020-06-30 | PXD019552 | Pride