Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 11, 44

N-arachidonoyl Glycine, an Abundant Endogenous Lipid, Potently Drives Directed Cellular Migration Through GPR18, the Putative Abnormal Cannabidiol Receptor

Affiliations

N-arachidonoyl Glycine, an Abundant Endogenous Lipid, Potently Drives Directed Cellular Migration Through GPR18, the Putative Abnormal Cannabidiol Receptor

Douglas McHugh et al. BMC Neurosci.

Abstract

Background: 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.

Results: 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.

Figures

Figure 1
Figure 1
NAGly-induced directed BV-2 microglial migration. (A) BV-2 microglial migration in response to basal conditions; vh (0.1% DMSO); 1 μM fMLP; 1 μM LPA; 0.1 nM - 300 μM NAGly. * = P < 0.05, ** = P < 0.01 compared to 1 μM fMLP; one-way ANOVA; n = 8. Insert is a filter photograph of one random field of view at ×40 magnification indicating the migration produced by 100 nM NAGly. The 10 μm diameter pores can be discerned as the clear unstained circles. (B) BV-2 microglial migration in response to basal conditions; vh (0.1% DMSO); 1 μM fMLP ± concentration gradient; 0.1 nM - 10 μM NAGly ± concentration gradient. ** = P < 0.01 compared to the corresponding concentration gradient; Student's unpaired t-test; n = 3. Insert is a filter photograph of one random field of view at ×40 magnification indicating the migration produced by 100 nM NAGly in the absence of a concentration gradient.
Figure 2
Figure 2
NAGly-induced BV-2 microglial migration is concentration- and structure-dependent. (A) BV-2 microglial migration in response to 0.1 nM - 10 μM concentrations of NAGly; AEA; 2-AG; PEA; PALGly; Abn-CBD; O-1602; LPI; n = 3. (B) Filter photographs of one random field of view at ×40 magnification indicating the migration produced by 10 μM concentrations of NAGly, O-1602, 2-AG, Abn-CBD, AEA and LPI.
Figure 3
Figure 3
NAGly-induced BV-2 cell proliferation and MAPK enzyme activation. (A) BV-2 microglial proliferation in response to 0.01 nM - 100 μM concentrations of NAGly; AEA; 2-AG; n = 3. (B) p44/42 MAPK activation in BV-2 microglia in response to vh (0.1% DMSO) for 3 hours; 10 nM - 10 μM NAGly for 3 hours; 10 μM Ionomycin for 5 min. ** = P < 0.01 compared to vh; one-way ANOVA; n = 3. (C) p38 MAPK activation in BV-2 microglia in response to vh (0.1% DMSO) for 3 hours; 10 nM - 10 μM NAGly for 3 hours; 10 μM Ionomycin for 5 min. ** = P < 0.01 compared to vh; one-way ANOVA; n = 3. (D) JNK MAPK activation in BV-2 microglia in response to vh (0.1% DMSO) for 3 hours; 10 nM - 10 μM NAGly for 3 hours; 10 μM Ionomycin for 5 min. ** = P < 0.01 compared to vh; one-way ANOVA; n = 3.
Figure 4
Figure 4
NAGly-induced BV-2 microglial migration is Gi/o-receptor mediated and can be antagonized. (A) BV-2 microglial migration in response to basal conditions; vh (0.1% DMSO); 1 μM fMLP; 1 μM fMLP + 100 nM SR144528; 1 μM NAGly; 1 μM NAGly + 1 μM SR141716A; 1 μM NAGly + 100 nM SR141716A; 1 μM NAGly + 100 nM SR144528;. ** = P < 0.01 compared to 1 μM NAGly; tt = P < 0.01 compared to 1 μM fMLP; Student's unpaired t-test; n = 3. (B) BV-2 microglial migration in response to 0.1 nM - 10 μM NAGly ± 1 μM O-1918, ± 1 μM ARA-S, or ± 24 h pre-treatment with 1 μg/ml PTX; n = 3.
Figure 5
Figure 5
BV-2 microglia express GPR18 mRNA and GPR18 receptors. (A) Gel electrophoresis of BV-2 microglia and HEK293-GPR18 RT-qPCR products. RT-qPCR products were collected from the RT-qPCR run, loading buffer was added to the samples, and samples were run on a 2% agarose gel. No template control (NTC) and a control without reverse transcription (NRT) were used as controls. (B) Representative qPCR amplification curves showing the different amounts of mRNAs for GPR18 in primary microglia and BV-2 cells; n = 3.
Figure 6
Figure 6
BV-2 microglia and HEK293-GPR18 transfected, but not HEK293 wildtype, cells express GPR18. Immunofluorescent confocal microscopy was conducted using an antibody against the GPR18 C-terminus (1:150; green), phalloidin to label actin (1:40; red), and DAPI (1.5 μg/ml) to label the nucleus (blue). a, HEK293 wildtype with DAPI and phalloidin. b, HEK293 wildtype with GPR18 antibody and phalloidin. c, HEK293 wildtype with GPR18 antibody, DAPI, and phalloidin. d, HEK293-GPR18 transfected with DAPI and phalloidin. e, HEK293-GPR18 transfected with GPR18 antibody and phalloidin. f, HEK293-GPR18 transfected with GPR18 antibody, DAPI, and phalloidin. g, BV-2 microglia with DAPI and phalloidin. h, BV-2 microglia with GPR18 antibody and phalloidin. i, BV-2 microglia with GPR18 antibody, DAPI, and phalloidin.
Figure 7
Figure 7
NAGly-induced migration of HEK293-GPR18 and HEK293-GPR55 cells. (A) HEK293 wildtype and HEK293-GPR18 transfected cell migration in response to 0.1 nM - 10 μM NAGly; n = 3. (B) Filter photographs of one random field of view at ×40 magnification indicating the migration produced by 10 μM concentrations of NAGly in HEK293-GPR18 and HEK293 wildtype cells. The 10 μm diameter pores can be discerned as the clear unstained circles. (C) HEK293-GPR18 cell migration in response to Vh (0.1% DMSO); 1 μM NAGly; 1 μM NAGly ± 1 μM O-1918; 1 μM NAGly ± 1 μM ARA-S. *** = P < 0.001 compared to 1 μM NAGly; one-way ANOVA; n = 3. HEK293-GPR18 cell migration in response to 1 μM Abn-CBD; 1 μM Abn-CBD ± 1 μM O-1918; 1 μM Abn-CBD ± 1 μM ARA-S; ttt = P < 0.001 compared to 1 μM Abn-CBD; n = 3. HEK293-GPR18 cell migration in response to 1 μM O-1602; 1 μM O-1602 ± 1 μM O-1918; 1 μM O-1602 ± 1 μM ARA-S. §§§ = P < 0.001 compared to 1 μM O-1602; one-way ANOVA; n = 3. (D) BV-2 microglia and HEK293-GPR18 cell migration in response to Vh (0.1% DMSO); 1 μM NAGly; 1 μM NAGly = 1 μM CBD; *** = P < 0.001 compared to 1 μM NAGly; one-way ANOVA; n = 3. (E) p44/42 MAPK activation in HEK293-GPR18 cells in response to vh (0.1% DMSO) for 3 hours; 10 nM - 10 μM NAGly for 3 hours; 10 μM Ionomycin for 5 min. ** = P < 0.01 compared to vh; one-way ANOVA; n = 3. (F) HEK293-GPR55 cell migration in response to 0.1 nM - 10 μM concentrations of LPI, Abn-CBD, NAGly, O-1602 and Vh (0.1% DMSO); n = 3.
Figure 8
Figure 8
GPR18 antibody recognizes hGPR18 receptors stably expressed in HEK293-GPR18 cells. Immunofluorescent confocal microscopy was conducted using HEK293 cells stably transfected with HA11-tagged hGPR18 and HA11 (1:500) and GPR18 (1:500) antibodies. a, HA11 antibody (detected with Texas Red secondary; red) staining shows HA11-hGPR18 transfected cells. b, hGPR18 antibody (detected with FITC secondary; green) staining of the same cells identified with the HA11 antibody. c, HA11 antibody (detected with Texas Red secondary; red) staining shows HA11-hGPR18 transfected cells. d, hGPR18 antibody staining (detected with FITC secondary; green) was blocked when the GPR18 antibody was co-incubated with immunizing protein (30 μg/ml) for 1 hour before administration. DAPI (1.5 μg/ml; blue) was used to label the nucleus of all HEK293-GPR18 cells.

Similar articles

See all similar articles

Cited by 103 PubMed Central articles

See all "Cited by" articles

References

    1. Fetler L, Amigorena S. NEUROSCIENCE: Brain Under Surveillance: The Microglia Patrol. Science. 2005;309:392–393. doi: 10.1126/science.1114852. - DOI - PubMed
    1. Raivich G. Like cops on the beat: the active role of resting microglia. Trends Neurosci. 2005;28:571–573. doi: 10.1016/j.tins.2005.09.001. - DOI - PubMed
    1. Block ML, Zecca L, Hong J-S. Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci. 2007;8:57–69. doi: 10.1038/nrn2038. - DOI - PubMed
    1. Trapp BD. Evidence for synaptic stripping by cortical microglia. Glia. pp. 360–368. - DOI - PubMed
    1. Garden GA, Moller T. Microglia biology in health and disease. J Neuroimmune Pharmacol. 2006;1:127–137. doi: 10.1007/s11481-006-9015-5. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources

Feedback