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的细胞集 生理角色；但是，RNA或DNA签名不能始终与唯一的表面标记相关， 阻碍这些细胞子集的重新分离以进行深入分析。此外，传统的单细胞 方法需要测序大量的细胞来表征稀有子集。我们会在这里 开发和应用搜索seq，一种高通量细胞仪法，可检测使用RNA或DNA标记 单分子敏感性允许靶细胞快速分离以深入转录组，基因组或 表观基因组分析。我们将使用该方法研究控制星形胶质细胞的调节机制 亚群的特征在于剪接的XBP1转录本，该转录物促进了疾病病理学 多发性硬化症（MS）。这种亚群也体现在实验前临床小鼠模型中 自身免疫性脑脊髓炎（EAE）。将搜索seq与有条件的敲除小鼠结合在一起 Vivo CRISPR/CAS9驱动的扰动研究以及小鼠EAE和人类MS样品的RNA-seq分析 我们将表征这些细胞的作用及其与核受体NR3C2及其的相互作用 CorePressor NCOR2在限制XBP1驱动的病原体星形胶质细胞反应中。总而言之，搜索seq是 新颖，敏感和高吞吐量方法，用于捕获难以研究的稀有脑细胞子集 现有技术，可能具有与MS发病机理有关的治疗靶向机制。