Satellite glial cells (SGCs) play an important role in regulating the function of trigeminal ganglion (TG) neurons. Multiple mediators are involved in the bidirectional communication between SGCs and neurons in different physiological and pathological states. However, molecular insights into the transcript characteristics of SGCs are limited. Moreover, little is known about the heterogeneity of SGCs in TG, and a more in-depth understanding of the interactions between SGCs and neuron subtypes is needed. Here we show the single-cell RNA sequencing (scRNA-seq) profile of SGCs in TG under physiological conditions. Our results demonstrate TG includes nine types of cell clusters, such as neurons, SGCs, myeloid Schwann cells (mSCs), non-myeloid Schwann cells (nmSCs), immune cells, etc., and the corresponding markers are also presented. We reveal the signature gene expression of SGCs, mSCs and nmSCs in the TG, and analyze the ligand-receptor pairs between neuron subtypes and SGCs in the TG. In the heterogeneity analysis of SGCs, four SGCs subtypes are identified, including subtypes enriched for genes associated with extracellular matrix organization, immediate early genes, interferon beta, and cell adhesion molecules, respectively. Our data suggest the molecular characteristics, heterogeneity of SGCs, and bidirectional interactions between SGCs and neurons, providing a valuable resource for studying SGCs in the TG.

Orofacial inflammation leads to transcriptional alterations in trigeminal ganglion (TG) neurons. However, diverse alterations and regulatory mechanisms following orofacial inflammatory pain in different types of TG neurons remain unclear. Here, orofacial inflammation was induced by injection of complete Freund’s adjuvant (CFA) in mice. After 7 days, we performed single-cell RNA-sequencing on TG cells of mice from control and treatment groups. We identified primary sensory neurons, Schwann cells, satellite glial cells, oligodendrocyte-like cells, immune cells, fibroblasts, and endothelial cells in TG tissue. After principal component analysis and hierarchical clustering, we identified six TG neuronal subpopulations: peptidergic nociceptors (PEP1 and PEP2), non-peptidergic nociceptors (NP1 and NP2), C-fiber low-threshold mechanoreceptors (cLTMR) and myelinated neurons (Nefh-positive neurons, NF) based on annotated marker gene expression. We also performed differential gene expression analysis among TG neuronal subtypes, identifying several differential genes involved in the inflammatory response, neuronal excitability, neuroprotection, and metabolic processes. Notably, we identified several potential novel targets associated with pain modulation, including Arl6ip1, Gsk3b, Scn7a, and Zbtb20 in PEP1, Rgs7bp in PEP2, and Bhlha9 in cLTMR. The established protein–protein interaction network identified some hub genes, implying their critical involvement in regulating orofacial inflammatory pain. Our study revealed the heterogeneity of TG neurons and their diverse neuronal transcriptomic responses to orofacial inflammation, providing a basis for the development of therapeutic strategies for orofacial inflammatory pain.

Trigeminal ganglion (TG) is the first station of sensory pathways in the orofacial region. The TG neurons communicate with satellite glial cells (SGCs), macrophages and other cells forming a functional unit that is responsible for processing of orofacial sensory information. Purinergic signaling, one of the most widespread autocrine and paracrine pathways, plays a crucial role in intercellular communication. The multidirectional action of purinergic signaling in different cell types contributes to the neuromodulation and orofacial sensation. To fully understand the purinergic signaling in these processes, it is essential to determine the shared and unique expression patterns of genes associated with purinergic signaling in different cell types. Here, we performed single-cell RNA sequencing of 22,969 cells isolated from normal mouse TGs. We identified 18 distinct cell populations, including 6 neuron subpopulations, 3 glial subpopulations, 7 immune cell subpopulations, fibroblasts, and endothelial cells. We also revealed the transcriptional features of genes associated with purinergic signaling, including purinergic receptors, extracellular adenosine triphosphate (eATP) release channels, eATP metabolism-associated enzymes, and eATP transporters in each cell type. Our results have important implications for understanding and predicting the cell type-specific roles of the purinergic signaling in orofacial signal processing in the trigeminal primary sensory system.

Protein ubiquitination is a precisely controlled enzymatic cascade reaction belonging to the post-translational modification of proteins. In this process, E3 ligases catalyze the binding of ubiquitin (Ub) to protein substrates and define specificity. The neuronally expressed developmentally down-regulated 4 (NEDD4) subfamily, belonging to the homology to E6APC terminus (HECT) class of E3 ligases, has recently emerged as an essential determinant of multiple cellular processes in different tissues, including bone and tooth. Here, we place special emphasis on the regulatory role of the NEDD4 subfamily in the molecular and cell biology of osteogenesis. We elucidate in detail the specific roles, downstream substrates, and upstream regulatory mechanisms of the NEDD4 subfamily. Further, we provide an overview of the involvement of E3 ligases and deubiquitinases in the development, repair, and regeneration of another mineralized tissue—tooth.