Identification of astrocyte regulators by nucleic acid cytometry

Iain C. Clark, Michael A. Wheeler, Hong-Gyun Lee, Zhaorong Li, Liliana M. Sanmarco, Shravan Thaploo, Carolina M. Polonio, Seung Won Shin, Giulia Scalisi, Amy R. Henry, Joseph M. Rone, Federico Giovannoni, Marc Charabati, Camilo Faust Akl, Dulce M. Aleman, Stephanie E. J. Zandee, Alexandre Prat, Daniel C. Douek, Eli A. Boritz, Francisco J. Quintana () and Adam R. Abate ()
Additional contact information
Iain C. Clark: Harvard Medical School
Michael A. Wheeler: Harvard Medical School
Hong-Gyun Lee: Harvard Medical School
Zhaorong Li: Harvard Medical School
Liliana M. Sanmarco: Harvard Medical School
Shravan Thaploo: Harvard Medical School
Carolina M. Polonio: Harvard Medical School
Seung Won Shin: University of California Berkeley
Giulia Scalisi: Harvard Medical School
Amy R. Henry: National Institutes of Health
Joseph M. Rone: Harvard Medical School
Federico Giovannoni: Harvard Medical School
Marc Charabati: Harvard Medical School
Camilo Faust Akl: Harvard Medical School
Dulce M. Aleman: Harvard Medical School
Stephanie E. J. Zandee: Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM)
Alexandre Prat: Centre de Recherche du Centre Hospitalier de l’Université de Montréal (CRCHUM)
Daniel C. Douek: National Institutes of Health
Eli A. Boritz: National Institutes of Health
Francisco J. Quintana: Harvard Medical School
Adam R. Abate: University of California San Francisco

Nature, 2023, vol. 614, issue 7947, 326-333

Abstract: Abstract Multiple sclerosis is a chronic inflammatory disease of the central nervous system1. 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 encephalomyelitis2,3. 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 encephalomyelitis4. 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.

Date: 2023
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DOI: 10.1038/s41586-022-05613-0

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