Keratinocyte Gq-linked GRCRs Activation Regulates Nerve Degeneration via Semaphorin Signaling

Background & Aims

Painful diabetic neuropathy (PDN) is one of the most common and intractable complications of diabetes. PDN is characterized by?small-fiber degeneration and neuropathic pain. Uncovering the mechanisms underlying?neurodegeneration in PDN remains a major challenge to finding effective and disease-modifying therapy. Keratinocytes are closely juxtaposed to cutaneous nerve terminals, potentially enabling communication between keratinocytes and cutaneous afferents. ?The aim of this study is to explore mechanisms by which keratinocytes communicate with cutaneous afferents and how this communication impacts dorsal root ganglion (DRG) neuron axonal degeneration underlying neuropathic pain in PDN

Methods

We genetically expressed Gq-linked G-Protein Coupled Receptors (Gq GPCRs) DREADD (hM3Dq) receptors into basal keratinocytes (KCs) expressing K14 in mice as a tool for mimicking the activation of Gq-linked GPCRs in basal keratinocytes. We used an unbiased approach to investigate the communication between KCs and cutaneous innervation, including single-cell RNA sequencing (scRNA-seq) of the epidermis and DRGs to capture DRG and keratinocytes single gene expression profiles. We integrate epidermis and DRG scRNA-seq data using CellChat to generate interactome maps and highlight ligand-receptor interactions between keratinocytes and DRG neuron subtypes

Results

We have shown that activation of Gq DREADDs in basal K14 Kcs reduced innervation of the epidermis, indicating that activation of K-14 Gq-linked GPCRs can regulate nerve fiber degeneration in the epidermis.?? Moreover, we found that activated KCs in hM3Dq-K14 mice induced an expansion of all four layers of the epidermis, including the granule cell layer (GRN), thereby enhancing the thickening of the epidermis. ScRNA-seq of the epidermis identified several clusters of KCs and immune cells, including several differentially expressed genes (DEGs). We found that the expression of several differentiation marker genes (Flg, Lor, and Lce1) was associated with GRN KCs. We further identified altered expression of factors that affect innervations, such as semaphorins and their receptors. To integrate epidermis and DRG scRNA-seq data, we used Cell-Chat, and identified changes in semaphorin-plexin signaling pathways known to be involved in axonal degeneration and regeneration.

Conclusions

Our findings indicate that?activating?basal keratinocytes Gq-linked GPCRs?can result in?profound changes in DRG neuronal transcriptomic profile, including relevant axonal degeneration and regeneration pathways, such as the semaphorin-plexin signaling pathways. Furthermore, our results suggest possible therapeutic effects of activating or blocking specific keratinocyte Gq-linked GPCRs for promoting axon regeneration and small fiber neuropathy in PDN. Topical drug application for treating PDN is a very appealing possibility. Doing so should bypass drug side effects associated with systemic drug administration.

References

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