Identification of astrocyte regulators by nucleic acid cytometry

Area of Science:

• Neuroscience
• Immunology
• Genomics

Background:

• Multiple sclerosis (MS) is a chronic central nervous system (CNS) inflammatory disease.
• Astrocytes, CNS glial cells, contribute to MS pathogenesis, but their subsets are poorly understood due to a lack of unique surface markers.
• Identifying and analyzing rare pathogenic astrocyte subsets is crucial for understanding MS and developing therapies.

Purpose of the Study:

• To develop a novel method for high-throughput transcriptomic analysis of rare and specific cell populations.
• To investigate the role of X-box binding protein 1 (XBP1) in astrocyte responses in MS and its animal model.
• To identify novel regulators of pathogenic astrocyte responses in MS.

Main Methods:

• Focused Interrogation of Cells by Nucleic Acid Detection and Sequencing (FIND-seq): a microfluidic method combining cell encapsulation, PCR, and droplet sorting for single-cell transcriptomics.
• Application of FIND-seq to study XBP1-regulated astrocytes in mouse experimental autoimmune encephalomyelitis (EAE) and human MS samples.
• Integration with conditional-knockout mice, CRISPR-Cas9 genetic perturbations, and bulk/single-cell RNA sequencing.

Main Results:

• FIND-seq successfully enabled in-depth transcriptomic analysis of rare cells at single-cell resolution.
• Identified a previously unknown role for the nuclear receptor NR3C2 and its corepressor NCOR2.
• Demonstrated that NR3C2 and NCOR2 limit XBP1-driven pathogenic astrocyte responses in MS.

Conclusions:

• FIND-seq is a powerful tool for investigating previously inaccessible rare cell subsets.
• The NR3C2-NCOR2 pathway represents a novel, therapeutically targetable mechanism to limit pathogenic astrocyte activity in MS.
• This study advances the understanding of astrocyte heterogeneity and its role in CNS inflammatory diseases.