2261: Pain in Osteoarthritis - Identification of GPR34 and GPR150 as Novel Targets in the Dorsal Root Ganglia

Matthew Wood¹, Terese Geraghty², David Thomas³, Alia Obeidat¹, Jun Li², Richard Miller⁴, Rachel Miller¹ and Anne-Marie Malfait⁵, ¹Rush University Medical Center, Chicago, IL, ²Rush University, Chicago, IL, ³University of Minnesota Internal Medicine Residency, St. Paul, MN, ⁴Northwestern University, Evaston, IL, ⁵Rush University, Oak Park, IL

Background/Purpose: Osteoarthritis (OA) is a leading cause of chronic pain worldwide. Currently available pain-alleviating therapies are often inadequate and associated with serious adverse effects, resulting in a major unmet need for effective analgesics in OA. To discover new analgesic targets, we performed single cell RNA-sequencing (scRNA-seq) of lumbar dorsal root ganglia (DRG) in a mouse model of OA, induced by destabilization of the medial meniscus (DMM). We focused on druggable targets in both neuronal and non-neuronal DRG cells. For non-neuronal cells, we focused on macrophages, because we have previously shown that DRG macrophages are involved in chronic pain in this model.

Methods: scRNA-seq was performed on L3-L5 DRGs (containing cell bodies from knee afferents) from naïve mice, or from mice 8 weeks after DMM. Partial meniscectomy (PMX) or sham surgery was performed in 10-week old male and female mice (n=3/group), and ipsilateral L4 DRG were collected 12 weeks later. Macrophage numbers were assessed by immunofluorescent staining for F4/80. Gpr34 was assessed by RNA in situ hybridization (ISH) using RNAscope. For mouse DRG, Adgre1and Gpr34 probes were used. Human DRG were removed postmortem from a male (age 82) and female (age 86) donor and flash frozen. RNA ISH was performed using GPR34 and CD14 probes. Spatial gene expression was performed using the 10X Genomics Visium platform. Protein expression of GPR34 and GPR150 was confirmed in purified protein extracted from 4 human DRGs (2 male, 2 female) using RayBiotech L4000 glass slide protein array.

Results: scRNAseq revealed a number of druggable candidates in the DRG of mice 8 weeks post DMM. Of these targets, we identified the rhodopsin class GPCR, Gpr34, which is expressed by macrophages and microglia, as well as Gpr150 expressed by nociceptors. Mice of both sexes developed joint damage by 12 weeks post PMX, as described. We observed an increase in the number of F4/80+ cells in DRGs after PMX, compared to sham controls. We confirmed the expression of Gpr34 specifically by Adgre1+ (F4/80) cells by RNAscope in both sham and PMX. When assessing the expression level of Gpr34 by Adgre1+ cells, we noted a significant increase in expression in DRG macrophages after PMX compared to sham controls. In human DRGs, both male and female, we confirmed that macrophages (CD14+ cells) express GPR34 and both GPR34 and GPR150 gene expression was confirmed and visualized in human DRGs using spatial gene expression technology. Finally, GPR34 and GPR150 protein were highly expressed in human DRGs.

Conclusion: We identified two GPCRs as targets in the DRG: Gpr34 as a macrophage target and Gpr150 as a neuronal target. We have shown that the number of DRG macrophages is increased in an experimental model of OA (PMX), and that Gpr34 is specifically expressed by DRG macrophages. Since these are druggable targets, our findings open new avenues for exploring the effect of targeting either non-neuronal cells (macrophages expressing GPR34) or neuronal cells (GRP150) in models of OA pain. Furthermore, we found both receptors to be highly expressed in human DRGs, providing further support that these receptors should be explored as putative targets.

Disclosures: M. Wood, None; T. Geraghty, None; D. Thomas, None; A. Obeidat, None; J. Li, None; R. Miller, None; R. Miller, None; A. Malfait, None.