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The development of multiple sclerosis, a major neurodegenerative disease, is due to both genetic and environmental factors that might trigger aberrant epigenetic changes of the genome. In this study, we analysed global DNA methylation in the brain of mice upon induction of experimental autoimmune encephalomyelitis (EAE), and the effect of environmental enrichment (EE). We demonstrate that global DNA methylation decreased in the striatum, but not in the cortex, of EAE mice compared to healthy controls, in particular in neuronal nitric oxide synthase (nNOS)-positive interneurons of this brain area. Also, in the striatum but again not in the cortex, decreased DNA methylation of the nNOS downstream effector, dexamethasone-induced Ras protein 1 (Dexras 1), was observed in EAE mice, and was paralleled by an increase in its mRNA. Interestingly, EE was able to revert EAE effects on mRNA expression and DNA methylation levels of Dexras 1 and reduced gene expression of nNOS and 5-lipoxygenase (Alox5). Conversely, interleukin-1β (IL-1β) gene expression was found up-regulated in EAE mice compared to controls and was not affected by EE. Taken together, these data demonstrate an unprecedented epigenetic modulation of nNOS-signaling in the pathogenesis of multiple sclerosis, and show that EE can specifically revert EAE effects on Dexras 1 along this pathway.

The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1. Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases.

BACKGROUND AND PURPOSE The CB1 cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB1 receptor by analysing the functional consequences of site-specific mutation of Cys415, the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling. EXPERIMENTAL APPROACH The palmitoylation state of CB1 receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys415 was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [35S]GTPγS, cAMP and co-immunoprecipitation assays. KEY RESULTS Endogenous CB1 receptors in rat brain were palmitoylated. Mutation of Cys415 prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB1 receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins. CONCLUSIONS AND IMPLICATIONS CB1 receptors were post-translationally modified by palmitoylation. Mutation of Cys415 provides a receptor that is functionally impaired in terms of membrane targeting and signalling. LINKED ARTICLES This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7

Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB 1 R and CB 2 R) cannabinoid receptors. CB 1 R is negatively regulated by membrane cholesterol, while CB 2 R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB 1 R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB 2 R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB 1 R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.

G protein-coupled receptor 55 (GPR55) is activated by endogenous, plant-derived and synthetic cannabinoids. Recent studies reported a broad tissue distribution for GPR55 and found prominent roles for this receptor in inflammatory pain, gut and bone physiology, as well as cancer. However, little is known about the expression and function of GPR55 in immune cells. To address this question, we performed a detailed characterization of GPR55 in different human innate and adaptive immune populations using polychromatic flow cytometry and we found that monocytes and NK cells expressed remarkable levels of this receptor compared to several cells of adaptive immunity. GPR55 activation by the specific agonist O-1602 boosted IL-12 and TNF-α production, and decreased endocytic activity, in LPS-activated monocytes. In addition, it increased CD69 activation marker expression, granzyme B and CD107a-dependent cytotoxicity and IFN-γ and TNF-α production in NK cells activated by both IL-2 and IL-12. These over-stimulatory effects of GPR55 were antagonized by its selective antagonist cannabidiol. Altogether, our data thus unveil a proinflammatory role for GPR55 in innate immunity that may be important for the design of new immune therapeutic strategies.

Platelets modulate vascular system integrity, and their loss is critical in haematological pathologies and after chemotherapy. Therefore, identification of molecules enhancing platelet production would be useful to counteract thrombocytopenia. We have previously shown that 2-arachidonoylglycerol (2-AG) acts as a true agonist of platelets, as well as it commits erythroid precursors toward the megakaryocytic lineage. Against this background, we sought to further interrogate the role of 2-AG in megakaryocyte/platelet physiology by investigating terminal differentiation, and subsequent thrombopoiesis. To this end, we used MEG-01 cells, a human megakaryoblastic cell line able to produce in vitro platelet-like particles. 2-AG increased the number of cells showing ruffled surface and enhanced surface expression of specific megakaryocyte/platelet surface antigens, typical hallmarks of terminal megakaryocytic differentiation and platelet production. Changes in cytoskeleton modeling also occurred in differentiated megakaryocytes and blebbing platelets. 2-AG acted by binding to CB1 and CB2 receptors, because specific antagonists reverted its effect. Platelets were split off from megakaryocytes and were functional: they contained the platelet-specific surface markers CD61 and CD49, whose levels increased following stimulation with a natural agonist like collagen. Given the importance of 2-AG for driving megakaryopoiesis and thrombopoiesis, not surprisingly we found that its hydrolytic enzymes were tightly controlled by classical inducers of megakaryocyte differentiation. In conclusion 2-AG, by triggering megakaryocyte maturation and platelet release, may have clinical efficacy to counteract thrombocytopenia-related diseases.

There is clear evidence on the neuroprotective role of the endocannabinoid (eCB) signaling cascade in various models of epilepsy. In particular, increased levels of eCBs protect against kainic acid (KA)-induced seizures. However, the molecular mechanisms underlying this effect and its age-dependence are still unknown. To clarify this issue, we investigated which step of the biosynthetic and catabolic pathways of the eCBs may be responsible for the eCB-mediated neuroprotection in the hippocampus of P14 and P56-70 KA-treated rats. We found that both anandamide and N-palmitoylethanolamine, together with their biosynthetic enzyme significantly increased in the hippocampus of younger KA-treated rats, while decreasing in adults. In contrast, the levels of the other major eCB, 2-arachidonoylglycerol, similar to its biosynthetic enzyme, were higher in the hippocampus of P56-70 compared to P14 rats. In line with these data, extracellular field recordings in CA1 hippocampus showed that enhancement of endogenous AEA and 2-AG significantly counteracted KA-induced epileptiform bursting in P56-70 and P14 rats, respectively. On the contrary, while the CB1R antagonist SR141716 per se did not affect the population spike, it did worsen KA-induced bursts, confirming increased eCB tone upon KA treatment. Altogether these data indicate an age-specific alteration of the eCB system caused by KA and provide insights for the protective mechanism of the cannabinoid system against epileptiform discharges.

Objective Cannabinoid receptors are activated in murine macrophages upon exposure to oxidized low-density lipoproteins (oxLDL), and type-1 cannabinoid receptor (CB 1 R) is considered as a risk factor in atherosclerosis, because it promotes cholesterol accumulation and release of inflammatory mediators. Conversely, accumulated evidence suggests a protective role for type-2 cannabinoid receptor (CB 2 R). Here, we sought to ascertain whether different elements of the endocannabinoid system (ECS) were activated in human lipid-laden macrophages, and whether CB 2 R played any role in atherogenesis and inflammation of these cells. Methods and results Human macrophages were exposed to oxLDL in order to obtain lipid-laden foam cells. Liquid chromatography/mass spectrometry (LC/MS) was used to measure the production of the endocannabinoids in both macrophages and foam cells, and radiometric assays were performed to measure cannabinoid receptor binding and activity of endocannabinoid metabolizing enzymes. OxLDL accumulation was investigated by confocal imaging, and cytokine production and release were measured by means of flow cytometry and ELISA. The results showed that human macrophages possess a fully functional ECS, which was modulated by oxLDL. Selective CB 2 R activation reduced cellular oxLDL accumulation, which was associated with decreased expression of CD36 scavenger receptor, and decreased production of TNFα, IL-12 and IL-10. These anti-atherogenic and anti-inflammatory effects were reverted by the selective CB 2 R antagonist SR144528. Conclusions A fully active ECS is present in human macrophages and macrophage-derived foam cells. Selective activation of CB 2 R reduces CD36-dependent oxLDL accumulation and modulates production of inflammatory cytokines, thus representing a potential therapeutic strategy to combat atherosclerosis.

Endocannabinoid signaling modulates a variety of neuroinflammatory and neurodegenerative diseases, mainly through the activation of type-1 and type-2 (CB(1)R and CB(2)R) cannabinoid receptors. CB(1)R is negatively regulated by membrane cholesterol, while CB(2)R is unaffected. Here, we identified in the transmembrane helix 7 of human CBRs a consensus sequence already known in other proteins as cholesterol recognition/interaction amino acid sequence and consensus pattern. As this motif is different in the two CBR subtypes, we mutated lysine 402 of CB(1)R into glycine, to obtain a cholesterol recognition/interaction amino acid sequence and consensus similar to that of CB(2)R. Both mutated and wild-type receptors were transiently expressed in human neuronal SH-SY5Y cells, and their localization and functioning were investigated using biochemical assays and immunofluorescence labelling. We found a reduced propensity of the mutant CB(1)R to reside in cholesterol-rich microdomains and, by means of fluorescence recovery after photobleaching analysis, we documented its loss of sensitivity to increased membrane cholesterol content. These results seem to uncover the existence of a new structural determinant in cannabinoid receptors, that is likely implicated in directing their interaction with cholesterol-rich microdomains of cell membranes.

The G protein-coupled receptor GPR55 has been proposed as a new cannabinoid receptor associated with bone remodelling, nervous system excitability, vascular homeostasis as well as in several pathophysiological conditions including obesity and cancer. However, its physiological role and underlying mechanism remain unclear. In the present work, we demonstrate for the first time its presence in human macrophages and its increased expression in ox-LDL-induced foam cells. In addition, pharmacological activation of GPR55 by its selective agonist O-1602 increased CD36- and SRB-I-mediated lipid accumulation and blocked cholesterol efflux by downregulating ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, as well as enhanced cytokine- and pro-metalloprotease-9 (pro-MMP-9)-induced proinflammatory responses in foam cells. Treatment with cannabidiol, a selective antagonist of GPR55, counteracted these pro-atherogenic and proinflammatory O-1602-mediated effects. Our data suggest that GPR55 could play deleterious role in ox-LDL-induced foam cells and could be a novel pharmacological target to manage atherosclerosis and other related cardiovascular diseases.