N-arachidonoyl glycine, an abundant endogenous lipid, potently drives directed cellular migration through GPR18, the putative abnormal cannabidiol receptor

Microglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB2 receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB1 or CB2 receptors, but functions as a selective agonist at this Gi/o-coupled GPCR. N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration.

NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.

Using Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEK-GPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonists N-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA. Conclusions: NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.