Activation of Keratinocyte Gq-linked G-Protein Coupled Receptors Remodels Dorsal Root Ganglion Neurons Single-Cell Transcriptomic Profile

Abstract

Keratinocytes are closely juxtaposed to cutaneous nerve terminals, enabling communication between keratinocytes and cutaneous nerves. We investigated potential mechanisms that mediate this communication. We genetically expressed stimulatory DREADDs (hM3Dq) into K14 basal keratinocytes (K14) in mice as a tool for mimicking the activation of Gq-linked G protein-coupled receptors (GPCRs) in K14 expressing cells. We have shown that activation of these DREADDs reduced innervation of the epidermis indicating that activation of K-14 Gq-linked GPCRs can regulate nerve fiber degeneration in the epidermis. We used bulk and single-cell RNA (scRNA-seq) sequencing from the skin and Dorsal Root Ganglion (DRG) taken from mice expressing hM3Dq into K14 keratinocytes comparing control mice receiving saline and mice receiving CNO to activate K14 keratinocytes expressing hM3Dq. Bulk RNA sequencing of the epidermis revealed changes in a set of genes associated with neurite outgrowth, including class III semaphorins, potentially impacting cutaneous nerve degeneration. Using RNAScope we found dramatic changes in the expression of semaphorins and their receptors in distinct DRG neurons subtypes. Using single-cell RNA-seq from lumbar DRG we profiled the transcriptome of 10,000 individual DRG cells and found that hM3Dq mediated stimulation of keratinocytes induced changes in DRG single-cell gene expression profile resulting in the identification of a novel DRG neuronal subtype among the nonpeptidergic nociceptors (NP1.2). Our findings indicate that activation of basal keratinocytes Gq-linked GPCRs can result in profound changes in DRG neuronal transcriptomic profile resulting in a phenotypical switch (plasticity) of DRG neurons. Furthermore, our results suggest possible therapeutic effects of activating or blocking specific keratinocyte Gq-linked GPCRs for promoting axon regeneration in small fiber neuropathy. Grant support from 1R01AR77691-01.