An Unbiased Approach to Identifying Changes in the Gene Expression Profile of Sensory Neurons in Painful Diabetic Neuropathy

Painful diabetic neuropathy (PDN) is an intractable and debilitating disease characterized by neuropathic pain and small-fiber degeneration. Given the prevalence of the disease, there is a dire need to identify new targets for the development of disease-modifying therapeutics for PDN. In patients with PDN, dorsal root ganglion (DRG) nociceptors become hyperexcitable and eventually degenerate, but the molecular mechanism underlying the phenomenon is unknown. We aim to identify the gene expression profile of the DRG neurons in PDN pathology to facilitate the discovery of novel druggable targets. Using a well-established mouse model of PDN, mice were either fed a regular diet (RD) or a high-fat diet (HFD) for 10 weeks. We used a single-cell RNA (scRNA-seq) sequencing approach to capture the changes in the DRG in an unbiased fashion. As expected, analysis of the scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes. Interestingly, we saw two closely related non-peptidergic clusters expressing a Mas-related G protein-coupled receptor (Mrgprd). While there were no differences in the expression of Mrgprd in one of the clusters (NP1 Type1), there seemed to be a significant increase in the expression of Mrgprd in a cluster we refer to as the NP1 Type2. To determine the functional relevance of the overexpression of Mrgprd, we used in vivo 2-photon calcium imaging with Nav1.8 Cre-GCaMP6 animals fed an RD or HFD and examined whether administration of β-alanine (a known agonist of Mrgprd) in the hind paw would directly activate Mrgprd positive DRG neurons. We observed an increase in the number, as well as an increase in the magnitude of response in the HFD indicating the hyperexcitability of neurons expressing Mrgprd. Taken together, our data highlights an important role of the Mrgprd receptor in the generation and maintenance of hyperexcitability in a mouse model of PDN. NS104295-01.