Identification of astrocyte regulators by nucleic acid cytometry

• Published in: Nature (London) Vol. 614; no. 7947; pp. 326 - 333
• Main Authors: Clark, Iain C., Wheeler, Michael A., Lee, Hong-Gyun, Li, Zhaorong, Sanmarco, Liliana M., Thaploo, Shravan, Polonio, Carolina M., Shin, Seung Won, Scalisi, Giulia, Henry, Amy R., Rone, Joseph M., Giovannoni, Federico, Charabati, Marc, Akl, Camilo Faust, Aleman, Dulce M., Zandee, Stephanie E. J., Prat, Alexandre, Douek, Daniel C., Boritz, Eli A., Quintana, Francisco J., Abate, Adam R.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 09.02.2023; Nature Publishing Group
• Subjects: 38/22; 38/62; 38/90; 38/91; 631/1647/514; 631/250/371; Acids; Animals; Astrocytes; Astrocytes - metabolism; Astrocytes - pathology; Autoimmune diseases; Biomarkers; Cells; Central nervous system; Cytometry; Droplets; Encephalomyelitis, Autoimmune, Experimental; Experimental allergic encephalomyelitis; Gene Editing; Gene expression; Gene Expression Regulation; Gene sequencing; Genomes; Glial cells; Humanities and Social Sciences; Humans; Hydrogels; In vivo methods and tests; Inflammatory diseases; Interrogation; Mice; Mice, Knockout; Microfluidics; Microfluidics - methods; multidisciplinary; Multiple sclerosis; Multiple Sclerosis - pathology; Nervous system; Nucleic acids; Nucleic Acids - analysis; Pathogenesis; Pathology; Perturbation; Science; Science (multidisciplinary); Single-Cell Gene Expression Analysis - methods; Splicing; Surface markers; Target detection; Therapeutic targets; Thermal cycling; Transcription activation; Transcription factors; Transcriptomics
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/s41586-022-05613-0

Multiple sclerosis is a chronic inflammatory disease of the central nervous system 1 . Astrocytes are heterogeneous glial cells that are resident in the central nervous system and participate in the pathogenesis of multiple sclerosis and its model experimental autoimmune encephalomyelitis 2 , 3 . However, few unique surface markers are available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; these limitations are further amplified by the rarity of pathogenic astrocytes. Here, to address these challenges, we developed focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), a high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyses of cells of interest at single-cell resolution. We applied FIND-seq to study the regulation of astrocytes characterized by the splicing-driven activation of the transcription factor XBP1, which promotes disease pathology in multiple sclerosis and experimental autoimmune encephalomyelitis 4 . Using FIND-seq in combination with conditional-knockout mice, in vivo CRISPR–Cas9-driven genetic perturbation studies and bulk and single-cell RNA sequencing analyses of samples from mouse experimental autoimmune encephalomyelitis and humans with multiple sclerosis, we identified a new role for the nuclear receptor NR3C2 and its corepressor NCOR2 in limiting XBP1-driven pathogenic astrocyte responses. In summary, we used FIND-seq to identify a therapeutically targetable mechanism that limits XBP1-driven pathogenic astrocyte responses. FIND-seq enables the investigation of previously inaccessible cells, including rare cell subsets defined by unique gene expression signatures or other nucleic acid markers. The pathogenic function of XBP1-expressing astrocytes in experimental autoimmune encephalomyelitis and multiple sclerosis have been studied using FIND-seq, a new method combining microfluidics cytometry, PCR-based detection of nucleic acids and cell sorting for in-depth single-cell transcriptomics analyses of rare cells.