Identification of Regulatory Mechanisms Operating in Rare Pathogenic Astrocyte Subsets in Multiple Sclerosis with a Novel Genomic Technology

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.