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

McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM, Bradshaw HB - BMC Neurosci (2010)

Bottom Line: 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 is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405, USA.

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.

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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.
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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.

Mentions: Microglia in the adult CNS derive chiefly from a self-renewing population or rarely are replenished from adult bone marrow [31]. As they invade an injured region of the CNS, microglia can enter the cell cycle and proliferate via mitosis [5], e.g. elevated numbers of microglia are found in brains of patients with multiple sclerosis [33], Alzheimer's disease [34] and HIV [35]. The reduction of tetrazolium salts is widely accepted as a reliable way to examine cell proliferation. In the MTT reduction technique, the yellow tetrazolium 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan dye can be solubilised and quantified by spectrophotometric means. Using this means to quantify cell proliferation in response to NAGly, AEA and 2-AG, we found that NAGly increased the population of BV-2 microglia at picomolar to low nanomolar concentrations after 24 hours (Figure 3A). The rank order of potency was NAGly > 2-AG > AEA at stimulating BV-2 cell proliferation: a ~50% increase was achieved by 10 nM NAGly, which was significantly greater than the ~24% and ~21% seen with 10 nM AEA and 2-AG, respectively (P < 0.01; one-way ANOVA; n = 3) (Figure 3A). Decreased cell viability was observed for all three compounds at concentrations greater than 1 μM. Carrier et al had previously shown that 2-AG, but not AEA, exerted a M-CSF (macrophage-colony stimulating factor) dependent proliferative effect on rat RTMGL1 microglia via CB2 receptors [36]. They observed ~30% increase with 300 nM 2-AG 24 hours after treatment, and this was accompanied by an increase in active p44/42 MAPK (a.k.a. ERK1/2). MAPKs respond to extracellular stimuli/mitogens and regulate activities such as cell proliferation, differentiation, motility, and death. As migration is an activated-MAPK-dependent phenomenon and 'Abn-CBD' receptors have been shown to induce p44/42 MAPK phosphorylation [13,19], we investigated the effect of NAGly on p44/42, p38 and JNK MAPK enzymes using In-Cell Western assays (Figures 3B, C &3D). NAGly induced a marked concentration-dependent phosphorylation of p44/42 and JNK MAPK (Figure 3B &3D), reflecting activation of these kinases, whereas, p38 MAPK was only significantly activated by 10 nM NAGly (Figure 3C). Our findings extend those of Carrier et al, showing that NAGly, 2-AG and AEA independently induce BV-2 microglial mitosis, with NAGly being the most potent of the three. Given the association between cell migration and proliferation, and that both are MAPK-dependent, 'Abn-CBD' receptor-activated phosphorylation of p44/42 and JNK MAPK in response to NAGly likely underlies the migratory and proliferative phenomena in BV-2 microglia.


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

McHugh D, Hu SS, Rimmerman N, Juknat A, Vogel Z, Walker JM, Bradshaw HB - BMC Neurosci (2010)

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.
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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.
Mentions: Microglia in the adult CNS derive chiefly from a self-renewing population or rarely are replenished from adult bone marrow [31]. As they invade an injured region of the CNS, microglia can enter the cell cycle and proliferate via mitosis [5], e.g. elevated numbers of microglia are found in brains of patients with multiple sclerosis [33], Alzheimer's disease [34] and HIV [35]. The reduction of tetrazolium salts is widely accepted as a reliable way to examine cell proliferation. In the MTT reduction technique, the yellow tetrazolium 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is reduced by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazan dye can be solubilised and quantified by spectrophotometric means. Using this means to quantify cell proliferation in response to NAGly, AEA and 2-AG, we found that NAGly increased the population of BV-2 microglia at picomolar to low nanomolar concentrations after 24 hours (Figure 3A). The rank order of potency was NAGly > 2-AG > AEA at stimulating BV-2 cell proliferation: a ~50% increase was achieved by 10 nM NAGly, which was significantly greater than the ~24% and ~21% seen with 10 nM AEA and 2-AG, respectively (P < 0.01; one-way ANOVA; n = 3) (Figure 3A). Decreased cell viability was observed for all three compounds at concentrations greater than 1 μM. Carrier et al had previously shown that 2-AG, but not AEA, exerted a M-CSF (macrophage-colony stimulating factor) dependent proliferative effect on rat RTMGL1 microglia via CB2 receptors [36]. They observed ~30% increase with 300 nM 2-AG 24 hours after treatment, and this was accompanied by an increase in active p44/42 MAPK (a.k.a. ERK1/2). MAPKs respond to extracellular stimuli/mitogens and regulate activities such as cell proliferation, differentiation, motility, and death. As migration is an activated-MAPK-dependent phenomenon and 'Abn-CBD' receptors have been shown to induce p44/42 MAPK phosphorylation [13,19], we investigated the effect of NAGly on p44/42, p38 and JNK MAPK enzymes using In-Cell Western assays (Figures 3B, C &3D). NAGly induced a marked concentration-dependent phosphorylation of p44/42 and JNK MAPK (Figure 3B &3D), reflecting activation of these kinases, whereas, p38 MAPK was only significantly activated by 10 nM NAGly (Figure 3C). Our findings extend those of Carrier et al, showing that NAGly, 2-AG and AEA independently induce BV-2 microglial mitosis, with NAGly being the most potent of the three. Given the association between cell migration and proliferation, and that both are MAPK-dependent, 'Abn-CBD' receptor-activated phosphorylation of p44/42 and JNK MAPK in response to NAGly likely underlies the migratory and proliferative phenomena in BV-2 microglia.

Bottom Line: 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 is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN 47405, USA.

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.

Show MeSH
Related in: MedlinePlus