Enteric Glia Display Regional and Phenotype-specific MicroRNA Signatures

Abstract

Background: Enteric glia constitute a heterogeneous and highly plastic population of peripheral neuroglia distributed throughout the gastrointestinal tract. The molecular intricacies coupled to enteric glia plasticity are incompletely understood, however, increasing evidence has delineated that microRNA post-transcriptional regulation provides a layer of precise spatial and temporal control that is critical for 'tuning' cellular dynamics. To date, specific microRNA profiles have not been assigned to enteric glia, and the role of microRNAs in controlling enteric glia function during gastrointestinal homeostasis and disease is unknown. Methods: To identify the microRNA landscape of enteric glia at homeostasis, we performed comprehensive microRNA profiling in enteric glia from the small intestine and colon of naïve adult mice. Employing fluorescence-activated cell sorting with an enteric glial reporter mouse model, enteric glia and non-glial cells were isolated from naïve Sox10-CreERT2;nuclGFP adult mice and subjected to small RNA sequencing (RNAseq). To discern whether microRNAs are linked with enteric glial plasticity during gastrointestinal disease, small RNAseq was performed in enteric glia isolated from Heligmosomoides polygyrus-infected adult mice (Progatzky et al., 2021). MicroRNA candidates were validated in human and murine gut samples, and in an in vitro 'reactive' enteric glia model, using qPCR and fluorescence in situ hybridization (miRNAscope). Results: Principal component analysis (PCA) of the microRNA transcriptional landscape of enteric glia and non-glial cells, at homeostasis, revealed that enteric glia and non-glial cells from the small intestine and colon clustered disparately on a microRNA transcriptional level. At homeostasis, normalised microRNA expression levels from small RNAseq detected 39 and 18 significantly, differentially expressed microRNAs in enteric glia, in the small intestine and colon, respectively, with miR-21-5p and miR-9-5p being two of the most enriched microRNAs. PCA of enteric glia exposed to the Heligmosomoides polygyrus challenge showed that 'reactive' and steady-state enteric glia differed on a microRNA transcriptional level. A total of 17 microRNAs were upregulated and 33 microRNAs were downregulated in 'reactive' enteric glia relative to steady state, including miR-21-5p. Conclusion: We present the first microRNA expression atlas of enteric glia during homeostasis and gastrointestinal disease. Enteric glial cells of both the small intestine and colon exhibit unique transcriptional profiles during steady state and 'reactive' phenotypes, alluding to a pivotal role of specific microRNAs in instructing and facilitating enteric glia plasticity.