Identification of GPR63 and GPR153 as Novel Modulators of Opioid Antinociception in Pathological Pain and Their Associated Roles in Microglia

Abstract

Pathological pain is a significant public health issue driven by many complex biological mechanisms which are not fully understood, and current pharmacotherapies for long-term pain management are highly variable in their efficacy while also presenting numerous detrimental drawbacks. Many clinical drugs are designed to target GPCRs, but a sizeable proportion of GPCRs remain understudied despite presenting opportunities for new druggable targets. Our lab has chosen four orphan GPCRs – GPR63, GPR141, GPR150, and GPR153 – for investigation in the context of pain and opioid-induced antinociception across multiple mouse models of nociceptive and pathological pain. All four of these targets are known to be expressed in the mouse and human spinal cord according to transcriptomic data from other groups, but their endogenous ligands and functions are poorly understood. We performed CRISPR knockdown of all four receptors in the mouse spinal cord by delivering predesigned CRISPR constructs via the IT route and evaluated their effects in the hot water tail flick assay and CIPN and paw incision pain models with von Frey. We observed no effects of any knockdown on baseline nociceptive responses or the antinociceptive effects of a 3.2 mg/kg screening dose of morphine (SC) in the tail flick assay. GPR63 and GPR153 knockdowns completely ablated the analgesic effects of morphine in the CIPN model, however. Furthermore, GPR153 knockdown yielded a similar result in the paw incision model and showed impairments in recovery from mechanical allodynia post-surgery while GPR63 knockdown had no effect in either regard. Considering that the knockdown of GPR63 and GPR153 had no influence in acute nociceptive pain but did influence behavioral responses to morphine in pathological models, we hypothesized that these receptors may play roles in modulating glial cells in the spinal cord which are considered to play significant roles in the development and/or maintenance of persistent pathological pain. Colocalization studies of the expression of GPR63 and GPR153 mRNAs with markers for microglia (Aif1) and astrocytes (Gfap) demonstrated that both receptors are expressed in approximately 50% of each cell type in the spinal dorsal horns of naïve mice with the exception that GPR153 appears to be expressed in 80-90% of microglia. To interrogate if this expression in glial cells was biologically relevant to the behavioral outcomes we observed previously, we performed selective CRISPR knockdowns targeting GPR63 and GPR153 in microglia or astrocytes alone. We found that when both targets were knocked down in microglia only, the blockade of opioid-induced antinociception and impairments to recovery were recapitulated in CIPN and paw incision models whereas astrocyte-specific knockdown had no effect. From this finding we hypothesized that GPR63 and GPR153 knockdown are enhancing microgliosis in the spinal cord. To test this, we administered minocycline to the spinal cord after GPR63 and GPR153 knockdown to inhibit microglia and were able to rescue opioid analgesia in the CIPN model. Morphological analysis of microglia in GPR63 and GPR153 CRISPR-treated spinal cords also depicted microglia with more amoeboid soma and less complex arborization compared to negative control treatments, both phenotypic indicators of microgliosis. Together, the above findings provide a scientific premise for further investigation of GPR63 and GPR153 as novel targets for analgesic drug discovery through the modulation of microglia in pathological pain states.