Identification of regulatory mechanisms operating in rare pathogenic astrocyte subsets in multiple sclerosis with a novel genomic technology

利用新型基因组技术鉴定多发性硬化症中罕见致病性星形胶质细胞亚群的调节机制

• 批准号: 10737509
• 负责人: Adam R. Abate
• 金额: $ 57.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737509
• 关键词: Address; Alternative Splicing; Astrocytes; Atlases; Biological Markers; Biology; CRISPR/Cas technology; Cell Separation; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Cytometry; DNA; DNA Markers; Disease; Experimental Autoimmune Encephalomyelitis; Gene Expression Profile; Genetic; Genomics; Grant; Human; Investigation; Knockout Mice; Link; Methods; Multiple Sclerosis; Mus; Mutation; NCOR2 gene; NR3C2 gene; Nerve Degeneration; Nuclear Receptors; Nucleic Acids; Pathogenesis; Pathogenicity; Pathology; Physiological; Population; Pre-Clinical Model; Prions; RNA; RNA Splicing; RNA marker; Receptor Signaling; Reporting; Role; Sampling; Signal Transduction; Surface; Technology; Therapeutically Targetable; Transcript; Transcriptional Activation; Virus Integration; XBP1 gene; brain cell; cell type; conditional knockout; detection method; disorder control; epigenomics; genetic corepressor; in vivo; interest; knock-down; mouse model; nervous system disorder; novel; posttranscriptional; pre-clinical; response; single cell technology; single molecule; single-cell RNA sequencing; transcription factor; transcriptome sequencing; transcriptomics

ABSTRACT Single-cell genomic, epigenomic, and transcriptomic technologies can identify unique cell subsets with important physiologic roles; however, RNA or DNA signatures cannot always be linked to unique surface markers, hampering the re-isolation of these cell subsets for in-depth analyses. Moreover, conventional single-cell methods require sequencing prohibitively large numbers of cells to characterize rare subsets. Here we will develop and apply SEARCH-seq, a high-throughput cytometry method that detects RNA or DNA markers with single molecule sensitivity that allows the rapid isolation of target cells for in-depth transcriptomic, genomic, or epigenomic analyses. We will use the method to study the regulatory mechanisms controlling an astrocyte subpopulation characterized by an alternatively spliced XBP1 transcript, which promotes disease pathology in multiple sclerosis (MS). This subpopulation also manifests in the pre-clinical mouse model of experimental autoimmune encephalomyelitis (EAE). Using SEARCH-seq in combination with conditional knock-out mice, in vivo CRISPR/Cas9-driven perturbation studies, and RNA-seq analyses of mouse EAE and human MS samples, we will characterize the role of these cells and their interaction with the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, SEARCH-seq is a novel, sensitive, and high throughput method to capture rare brain cell subsets that are difficult to study with existing technology and may have therapeutically targetable mechanisms relevant to MS pathogenesis.

抽象的 单细胞基因组、表观基因组和转录组技术可以识别具有重要意义的独特细胞亚群 生理作用；然而，RNA 或 DNA 特征并不总是与独特的表面标记相关联， 阻碍了这些细胞亚群的重新分离以进行深入分析。此外，传统的单细胞 方法需要对大量细胞进行测序来表征罕见的亚群。在这里我们将 开发并应用 SEARCH-seq，这是一种高通量细胞计数方法，可检测 RNA 或 DNA 标记 单分子敏感性，可快速分离靶细胞以进行深入的转录组、基因组或 表观基因组分析。我们将使用该方法来研究控制星形胶质细胞的调节机制 以选择性剪接 XBP1 转录本为特征的亚群，该转录本可促进疾病病理学 多发性硬化症（MS）。该亚群也出现在实验性临床前小鼠模型中 自身免疫性脑脊髓炎（EAE）。使用 SEARCH-seq 与条件敲除小鼠相结合， 体内 CRISPR/Cas9 驱动的扰动研究，以及小鼠 EAE 和人类 MS 样本的 RNA 序列分析， 我们将描述这些细胞的作用及其与核受体 NR3C2 及其相互作用 辅阻遏物 NCOR2 限制 XBP1 驱动的致病性星形胶质细胞反应。综上所述，SEARCH-seq 是一个 新颖、灵敏、高通量的方法来捕获难以研究的稀有脑细胞子集 现有技术，并且可能具有与多发性硬化症发病机制相关的治疗靶向机制。