Abstract 1735 Dissection of astrocyte transcriptional circuits underlying behavior by single-cell nucleic acid cytometry

Infrequent cells in human tissue may have catalyst behavior regarding tissue pathology and physiology. Astrocytes are heterogeneous glial cells that reside in the central nervous system and participate in the pathogenesis of multiple neurological disorders. Major depression has been correlated with the prevalence of astrocyte cells, and are of primary interest for isolation and further investigation. There are, however, few unique surface markers available for the isolation of astrocyte subsets, preventing their analysis and the identification of candidate therapeutic targets; this is amplified by the rarity of pathogenic astrocytes marked by expression of XBP1, a transcription factor that undergoes RNA splicing to generate a fully functional isoform. Despite increasingly advanced databases of transcriptional markers that are composed with single cell approaches, the nucleic acid signature of astrocytes does not map to unique surface markers. It becomes fundamental, then, to develop a more individualized technique to interrogate the properties of astrocytes. Methods: To address these challenges, the development of focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq) was executed. This interrogation strategically implemented high-throughput microfluidic cytometry method that combines encapsulation of cells in droplets, to then apply a PCR-based detection of target nucleic acids and droplet sorting to enable in-depth transcriptomic analyzes of cells of interest at single-cell resolution (*Clark, *Wheeler et al., Nature 2023). The technique was adapted specifically to astrocytes, making it feasible to conduct a more detailed analysis of the genetic information present in such cells. Results: following the application of FIND-seq to study the regulation of astrocytes characterized by RNA splicing-driven activation of the transcription factor XBP1 and uncovered an antagonistic cross-talk with the mineralocorticoid receptor NR3C2, responsive to the stress-activated hormone corticosterone. Supporting this idea, cytokines induced in response to chronic stress increased the expression of activated Xbp1 whereas corticosterone limited XBP1 activation. Similarly, activation of XBP1 via the recently identified herbicide linuron, (Wheeler et al., Cell 2019) worsened behavioral phenotypes as it boosts astrocyte pathogenic activities regarding Central Nervous System inflammation used as an experimental model of depression. Conclusion: Altogether, these data support a model of reciprocal transcriptional antagonism mediated by NR3C2-XBP1 signaling with implications for mood disorders. The refined interrogation technique successfully identified a candidate receptor that may be investigated in future studies of other neurological diseases.