BackgroundMicroglia 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.ResultsUsing 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.ConclusionsNAGly 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.

Cannabinoids have been shown to exert anti-inflammatory activities in various in vivo and in vitro experimental models as well as ameliorate various inflammatory degenerative diseases. However, the mechanisms of these effects are not completely understood. Using the BV-2 mouse microglial cell line and lipopolysaccharide (LPS) to induce an inflammatory response, we studied the signaling pathways engaged in the anti-inflammatory effects of cannabinoids as well as their influence on the expression of several genes known to be involved in inflammation. We found that the two major cannabinoids present in marijuana, ⌬ 9 -Tetrahydrocannabinol (THC) 3 is a major constituent of Cannabis and serves as an agonist of the cannabinoid receptors CB1 (located mainly in neural cells) and CB2 (located mainly on immune cells). The second major constituent of Cannabis extract is cannabidiol (CBD), which is virtually inactive at the CB1 and CB2 receptors (1). Thus, because of its negligible activity at the CB1 receptor, CBD lacks the psychoactive effects that accompany the use of THC. Moreover, CBD was demonstrated to antagonize some undesirable effects of THC, including intoxication, sedation, and tachycardia, while sharing neuroprotective, anti-oxidative, anti-emetic, and anti-carcinogenic properties (2-4). Both THC and CBD have been shown to exert anti-inflammatory properties and to modulate the function of immune cells, including suppression of humoral response, immune cell proliferation, maturation, and migration, and antigen presentation (5-9). Despite increasing amounts of such observations, the molecular mechanisms involved in these cannabinoid-mediated effects are not yet fully understood.Microglial cells are resident macrophages of the central nervous system and serve as early host defense against pathogens. Activation of microglial cells leads to the release of proinflammatory and neurotoxic factors and serves as part of the neuroinflammatory process (10). The BV-2 murine microglial cell line is known to retain morphological, phenotypic, and functional properties associated with freshly isolated microglia such as expression of nonspecific esterase activity, phagocytic ability, and the absence of peroxidase activity (11,12). Furthermore, these cells release lysozyme and, when stimulated, interleukin (IL)-1 and tumor necrosis factor ␣ (11, 12). Close similarities between BV-2 and primary microglia in mechanisms mediating microglial stimulations, e.g. by lipopolysaccharide (LPS), S100B, or ␤-amyloid, were reported (13). These properties make BV-2 cells an appropriate model for studying the activation of microglia in vitro. It has recently been shown that BV-2 cells express elements of the cannabinoid signaling systems, including the presence of endocannabinoids, i.e. anandamide and 2-arachidonoylglycerol, and cannabinoid or cannabinoid- 3 The abbreviations used are: THC, ⌬ 9 -tetrahydrocannabinol; CBD, cannabidiol; abn-CBD, abnormal cannabidiol; STAT, signal transducers and activators of transcription; IL, interleukin; IFN, ...

BACKGROUND AND PURPOSECannabis extracts and several cannabinoids have been shown to exert broad anti-inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACHWe used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation. KEY RESULTSTreatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB1 and CB2 receptors. CONCLUSIONS AND IMPLICATIONSCBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.

a b s t r a c tWe examined how lipopolysaccharide (LPS) and interferon gamma (IFN-c), known to differentially activate microglia, affect the expression of G protein-coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV-2 mouse microglial cell line, and that LPS down-regulates this message. Conversely, IFN-c slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV-2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV-2 stimulated with IFN-c, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli-induced changes in GPR55 expression are similar to those observed with CB 2 -R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.

Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD.