Deciphering Crosstalk between glia, immune cells, and neurons in dorsal root ganglia using Translating Ribosome Affinity Purification followed by RNA Sequencing (TRAP-seq)

Our recent transcriptome study using bulk RNA-sequencing identified crucial genes in peripheral sensory ganglia linked to visceral hypersensitivity in an animal model of chronic pelvic pain (CPP). Differential pathway changes in male and female animals were also identified, strongly suggesting that different cell populations in the sensory ganglia are responsible for the development of visceral pain symptoms in different sex. Moreover, a recent report using single nuclei sequencing and cell to cell communication (NeuronChat) reveals satellite glial cells communicate with all cell types including sensory neurons in dorsal root ganglia (DRGs). Therefore, in this study, we set out to characterize satellite glial translatome in lumbosacral dorsal root ganglia associated with CPP symptoms. Male and female glial-specific inducible mice (Sox10-CreERT2) and ribosome-tagged mice (L10-eGFP) were bred to generate Sox10-TRAP animals used in this study. Visceral hypersensitivity was induced by intravesical instillations of the mouse recombinant vascular endothelial growth factor A (VEGFA). Sox10-TRAP mice were sacrificed after visceral hypersensitivity was confirmed by Von Frey assay and their lumbosacral dorsal root ganglia (L1, L2, L6, S1 and S2) were dissected and homogenized. Around 15,000 Sox10-positive glia (satellite glial cells and a small number of Schwann cells) and ~400,000 Sox10-negative cells (sensory neurons, resident macrophages, pericytes, fibroblasts, and endothelial cells) are expected per mouse. After immunoprecipitation of the actively transcribing ribosomes using magnetic breads conjugated with eGFP antibody, TRAP-seq was performed at 50 million reads per sample. Bioinformatic analysis was done using Partek Flow Genomic Analysis Software and QIAGEN Ingenuity Pathway Analysis. Our samples showed successful enrichment of Sox10+ glia specific genes, particularly those of satellite glia. Our data shows that satellite glia are actively involved in cell-to-cell communication during the symptoms of CPP in a sex-specific manner. The TRAP-seq study strongly suggest satellite glial participation in VEGFA-induced bladder afferent hypersensitivity. NIDDKR01DK129260 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.