The evolution and comparative neurobiology of endocannabinoid signalling

CB₁- and CB₂-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB₁-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB₁/CB₂-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB₁/CB₂-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB₁/CB₂-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB₁/CB₂-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.     

CB2 Receptor Deficiency Increases Amyloid Pathology and Alters Tau Processing in a Transgenic Mouse Model of Alzheimer’s Disease

The endocannabinoid CB₂ receptor system has been implicated in the neuropathology of Alzheimer’s disease (AD). In order to investigate the impact of the CB₂ receptor system on AD pathology, a colony of mice with a deleted CB₂ receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB₂ receptor–sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2^−/− (Cnr2^tm1Dgen/J) mice to produce a colony of J20 CNR2^+/+ and J20 CNR2^−/− mice. Seventeen J20 CNR2^+/+ mice (12 females, 5 males) and 16 J20 CNR2^−/− mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aβ production and plaque deposition quantified with 6E10 staining, the effect of CB₂ receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aβ42 and plaque deposition were significantly increased in J20 CNR2^−/− mice relative to CNR2^+/+ mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2^−/− mice. Total tau was significantly suppressed in J20 CNR2^−/− mice relative to J20 CNR2^+/+ mice. The results confirm the constitutive role of the CB₂ receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB₂ to reduce Aβ; however, the results suggest that interventions may have a divergent effect on tau pathology.     

CB2 Receptor Deficiency Increases Amyloid Pathology and Alters Tau Processing in a Transgenic Mouse Model of Alzheimer’s Disease

The endocannabinoid CB₂ receptor system has been implicated in the neuropathology of Alzheimer’s disease (AD). In order to investigate the impact of the CB₂ receptor system on AD pathology, a colony of mice with a deleted CB₂ receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB₂ receptor–sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2^−/− (Cnr2^tm1Dgen/J) mice to produce a colony of J20 CNR2^+/+ and J20 CNR2^−/− mice. Seventeen J20 CNR2^+/+ mice (12 females, 5 males) and 16 J20 CNR2^−/− mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aβ production and plaque deposition quantified with 6E10 staining, the effect of CB₂ receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aβ42 and plaque deposition were significantly increased in J20 CNR2^−/− mice relative to CNR2^+/+ mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2^−/− mice. Total tau was significantly suppressed in J20 CNR2^−/− mice relative to J20 CNR2^+/+ mice. The results confirm the constitutive role of the CB₂ receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB₂ to reduce Aβ; however, the results suggest that interventions may have a divergent effect on tau pathology.     

Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

Modulation of type 1 cannabinoid receptor (CB₁) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB₁ are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)) on arrestin2-, Gα_i/o-, Gβγ-, Gα_s-, and Gα_q-mediated intracellular signaling in the mouse STHdh^Q7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB₁. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα_i/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB₁ levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα_q-dependent pathways. CP55,940 and CBD both signaled through Gα_s. CP55,940, but not CBD, activated downstream Gα_s pathways via CB₁ targets. THC and CP55,940 promoted CB₁ internalization and decreased CB₁ protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB₁ leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.     

Characterisation of the cannabinoid receptor system in synovial tissue and fluid in patients with osteoarthritis and rheumatoid arthritis

Introduction

Cannabis-based medicines have a number of therapeutic indications, including anti-inflammatory and analgesic effects. The endocannabinoid receptor system, including the cannabinoid receptor 1 (CB₁) and receptor 2 (CB₂) and the endocannabinoids, are implicated in a wide range of physiological and pathophysiological processes. Pre-clinical and clinical studies have demonstrated that cannabis-based drugs have therapeutic potential in inflammatory diseases, including rheumatoid arthritis (RA) and multiple sclerosis. The aim of this study was to determine whether the key elements of the endocannabinoid signalling system, which produces immunosuppression and analgesia, are expressed in the synovia of patients with osteoarthritis (OA) or RA.

Methods

Thirty-two OA and 13 RA patients undergoing total knee arthroplasty were included in this study. Clinical staging was conducted from x-rays scored according to Kellgren-Lawrence and Larsen scales, and synovitis of synovial biopsies was graded. Endocannabinoid levels were quantified in synovial fluid by liquid chromatography-mass spectrometry. The expression of CB₁ and CB₂ protein and RNA in synovial biopsies was investigated. Functional activity of these receptors was determined with mitogen-activated protein kinase assays. To assess the impact of OA and RA on this receptor system, levels of endocannabinoids in the synovial fluid of patients and non-inflamed healthy volunteers were compared. The activity of fatty acid amide hydrolase (FAAH), the predominant catabolic endocannabinoid enzyme, was measured in synovium.

Results

CB₁ and CB₂ protein and RNA were present in the synovia of OA and RA patients. Cannabinoid receptor stimulation of fibroblast-like cells from OA and RA patients produced a time-dependent phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 which was significantly blocked by the CB₁ antagonist SR141716A. The endocannabinoids anandamide (AEA) and 2-arachidonyl glycerol (2-AG) were identified in the synovial fluid of OA and RA patients. However, neither AEA nor 2-AG was detected in synovial fluid from normal volunteers. FAAH was active in the synovia of OA and RA patients and was sensitive to inhibition by URB597 (3'-(aminocarbonyl) [1,1'-biphenyl]-3-yl)-cyclohexylcarbamate).

Conclusion

Our data predict that the cannabinoid receptor system present in the synovium may be an important therapeutic target for the treatment of pain and inflammation associated with OA and RA.     

Cannabinoid Receptor CB2 Modulates Axon Guidance

Navigation of retinal projections towards their targets is regulated by guidance molecules and growth cone transduction mechanisms. Here, we present in vitro and in vivo evidences that the cannabinoid receptor 2 (CB₂R) is expressed along the retino-thalamic pathway and exerts a modulatory action on axon guidance. These effects are specific to CB₂R since no changes were observed in mice where the gene coding for this receptor was altered (cnr2 ^−/−). The CB₂R induced morphological changes observed at the growth cone are PKA dependent and require the presence of the netrin-1 receptor, Deleted in Colorectal Cancer. Interfering with endogenous CB₂R signalling using pharmacological agents increased retinal axon length and induced aberrant projections. Additionally, cnr2 ^−/− mice showed abnormal eye-specific segregation of retinal projections in the dorsal lateral geniculate nucleus (dLGN) indicating CB₂R’s implication in retinothalamic development. Overall, this study demonstrates that the contribution of endocannabinoids to brain development is not solely mediated by CB₁R, but also involves CB₂R.     

Transcriptional Regulation of Cannabinoid Receptor-1 Expression in the Liver by Retinoic Acid Acting via Retinoic Acid Receptor-γ

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB₁ cannabinoid receptors (CB₁R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB₁R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB₁R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227–235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB₁R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB₁R mRNA and protein, the most efficacious being the RARγ agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB₁R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARγ to the CB₁R gene 5′ upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB₁R expression was attenuated by small interfering RNA knockdown of RARγ in hepatocytes. We conclude that RARγ regulates CB₁R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.     

Hematopoietic Stem/Progenitor Cell Proliferation and Differentiation Is Differentially Regulated by High-Density and Low-Density Lipoproteins in Mice

Rationale

Hematopoietic stem/progenitor cells (HSPC) are responsible for maintaining the blood system as a result of their self-renewal and multilineage differentiation capacity. Recently, studies have suggested that HDL cholesterol may inhibit and impaired cholesterol efflux may increase HSPC proliferation and differentiation.

Objectives

We hypothesized that LDL may enhance HSPC proliferation and differentiation while HDL might have the opposing effect which might influence the size of the pool of inflammatory cells.

Methods and Results

HSPC number and function were studied in hypercholesterolemic LDL receptor knockout (LDLr^−/−) mice on high fat diet. Hypercholesterolemia was associated with increased frequency of HSPC, monocytes and granulocytes in the peripheral blood (PB). In addition, an increased proportion of BM HSPC was in G₂M of the cell cycle, and the percentage of HSPC and granulocyte-macrophage progenitors (GMP) increased in BM of LDLr^−/− mice. When BM Lin-Sca-1+cKit+ (i.e. “LSK”) cells were cultured in the presence of LDL in vitro we also found enhanced differentiation towards monocytes and granulocytes. Furthermore, LDL promoted lineage negative (Lin−) cells motility. The modulation by LDL on HSPC differentiation into granulocytes and motility was inhibited by inhibiting ERK phosphorylation. By contrast, when mice were infused with human apoA-I (the major apolipoprotein of HDL) or reconstituted HDL (rHDL), the frequency and proliferation of HSPC was reduced in BM in vivo. HDL also reversed the LDL-induced monocyte and granulocyte differentiation in vitro.

Conclusion

Our data suggest that LDL and HDL have opposing effects on HSPC proliferation and differentiation. It will be of interest to determine if breakdown of HSPC homeostasis by hypercholesterolemia contributes to inflammation and atherosclerosis progression.     

Modulation of the Endocannabinoids N-Arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) on Executive Functions in Humans

Animal studies point to an implication of the endocannabinoid system on executive functions. In humans, several studies have suggested an association between acute or chronic use of exogenous cannabinoids (Δ9-tetrahydrocannabinol) and executive impairments. However, to date, no published reports establish the relationship between endocannabinoids, as biomarkers of the cannabinoid neurotransmission system, and executive functioning in humans. The aim of the present study was to explore the association between circulating levels of plasma endocannabinoids N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) and executive functions (decision making, response inhibition and cognitive flexibility) in healthy subjects. One hundred and fifty seven subjects were included and assessed with the Wisconsin Card Sorting Test; Stroop Color and Word Test; and Iowa Gambling Task. All participants were female, aged between 18 and 60 years and spoke Spanish as their first language. Results showed a negative correlation between 2-AG and cognitive flexibility performance (r = −.37; p<.05). A positive correlation was found between AEA concentrations and both cognitive flexibility (r = .59; p<.05) and decision making performance (r = .23; P<.05). There was no significant correlation between either 2-AG (r = −.17) or AEA (r = −.08) concentrations and inhibition response. These results show, in humans, a relevant modulation of the endocannabinoid system on prefrontal-dependent cognitive functioning. The present study might have significant implications for the underlying executive alterations described in some psychiatric disorders currently associated with endocannabinoids deregulation (namely drug abuse/dependence, depression, obesity and eating disorders). Understanding the neurobiology of their dysexecutive profile might certainly contribute to the development of new treatments and pharmacological approaches.     

Anandamide Suppresses Proliferation and Cytokine Release from Primary Human T-Lymphocytes Mainly via CB2 Receptors

Background

Anandamide (AEA) is an endogenous lipid mediator that exerts several effects in the brain as well as in peripheral tissues. These effects are mediated mainly by two types of cannabinoid receptors, named CB₁R and CB₂R, making AEA a prominent member of the “endocannabinoid” family. Also immune cells express CB₁ and CB₂ receptors, and possess the whole machinery responsible for endocannabinoid metabolism. Not surprisingly, evidence has been accumulated showing manifold roles of endocannabinoids in the modulation of the immune system. However, details of such a modulation have not yet been disclosed in primary human T-cells.

Methodology/Significance

In this investigation we used flow cytometry and ELISA tests, in order to show that AEA suppresses proliferation and release of cytokines like IL-2, TNF-α and INF-γ from activated human peripheral T-lymphocytes. However, AEA did not exert any cytotoxic effect on T-cells. The immunosuppression induced by AEA was mainly dependent on CB₂R, since it could be mimicked by the CB₂R selective agonist JWH-015, and could be blocked by the specific CB₂R antagonist SR144528. Instead the selective CB₁R agonist ACEA, or the selective CB₁R antagonist SR141716, were ineffective. Furthermore, we demonstrated an unprecedented immunosuppressive effect of AEA on IL-17 production, a typical cytokine that is released from the unique CD4+ T-cell subset T-helper 17.

Conclusions/Significance

Overall, our study investigates for the first time the effects of the endocannabinoid AEA on primary human T-lymphocytes, demonstrating that it is a powerful modulator of immune cell functions. In particular, not only we clarify that CB₂R mediates the immunosuppressive activity of AEA, but we are the first to describe such an immunosuppressive effect on the newly identified Th-17 cells. These findings might be of crucial importance for the rational design of new endocannabinoid-based immunotherapeutic approaches.     

Differences in the Endocannabinoid System of Sperm from Fertile and Infertile Men

Male infertility is a major cause of problems for many couples in conceiving a child. Recently, lifestyle pastimes such as alcohol, tobacco and marijuana have been shown to have further negative effects on male reproduction. The endocannabinoid system (ECS), mainly through the action of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) at cannabinoid (CB₁, CB₂) and vanilloid (TRPV1) receptors, plays a crucial role in controlling functionality of sperm, with a clear impact on male reproductive potential. Here, sperm from fertile and infertile men were used to investigate content (through LC-ESI-MS), mRNA (through quantitative RT-PCR), protein (through Western Blotting and ELISA) expression, and functionality (through activity and binding assays) of the main metabolic enzymes of AEA and 2-AG (NAPE-PLD and FAAH, for AEA; DAGL and MAGL for 2-AG), as well as of their binding receptors CB₁, CB₂ and TRPV1. Our findings show a marked reduction of AEA and 2-AG content in infertile seminal plasma, paralleled by increased degradation: biosynthesis ratios of both substances in sperm from infertile versus fertile men. In addition, TRPV1 binding was detected in fertile sperm but was undetectable in infertile sperm, whereas that of CB₁ and CB₂ receptors was not statistically different in the two groups. In conclusion, this study identified unprecedented alterations of the ECS in infertile sperm, that might impact on capacitation and acrosome reaction, and hence fertilization outcomes. These alterations might also point to new biomarkers to determine male reproductive defects, and identify distinct ECS elements as novel targets for therapeutic exploitation of ECS-oriented drugs to treat male fertility problems.     

Genetic Background Can Result in a Marked or Minimal Effect of Gene Knockout (GPR55 and CB2 Receptor) in Experimental Autoimmune Encephalomyelitis Models of Multiple Sclerosis

Endocannabinoids and some phytocannabinoids bind to CB₁ and CB₂ cannabinoid receptors, transient receptor potential vanilloid one (TRPV1) receptor and the orphan G protein receptor fifty-five (GPR55). Studies using C57BL/10 and C57BL/6 (Cnr2 ^tm1Zim) CB₂ cannabinoid receptor knockout mice have demonstrated an immune-augmenting effect in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis. However, other EAE studies in Biozzi ABH mice often failed to show any treatment effect of either CB₂ receptor agonism or antagonism on inhibition of T cell autoimmunity. The influence of genetic background on the induction of EAE in endocannabinoid system-related gene knockout mice was examined. It was found that C57BL/6.GPR55 knockout mice developed less severe disease, notably in female mice, following active induction with myelin oligodendrocyte glycoprotein 35-55 peptide. In contrast C57BL/6.CB₂ (Cnr2 ^Dgen) receptor knockout mice developed augmented severity of disease consistent with the genetically and pharmacologically-distinct, Cnr2 ^tm1Zim mice. However, when the knockout gene was bred into the ABH mouse background and EAE induced with spinal cord autoantigens the immune-enhancing effect of CB₂ receptor deletion was lost. Likewise CB₁ receptor and transient receptor potential vanilloid one knockout mice on the ABH background demonstrated no alteration in immune-susceptibility, in terms of disease incidence and severity of EAE, in contrast to that reported in some C57BL/6 mouse studies. Furthermore the immune-modulating influence of GPR55 was marginal on the ABH mouse background. Whilst sedative doses of tetrahydrocannabinol could induce immunosuppression, this was associated with a CB₁ receptor rather than a CB₂ receptor-mediated effect. These data support the fact that non-psychoactive doses of medicinal cannabis have a marginal influence on the immune response in MS. Importantly, it adds a note of caution for the translational value of some transgenic/gene knockout and other studies on low-EAE susceptibility backgrounds with inconsistent disease course and susceptibility.     

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin−c-Kit+ and Lin−Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin−Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.     

The endocannabinoid system in neurodegeneration

Endocannabinoids are bioactive lipids, that comprise amides, esters and ethers of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the best studied endocannabinoids, and act as agonists of cannabinoid receptors. Thus, AEA and 2-AG mimic several pharmacological effects of the exogenous cannabinoid delta9-tetrahydrocannabinol, the psychoactive principle of hashish and marijuana. It is known that the activity of endocannabinoids at their receptors is limited by cellular uptake through specific membrane transporters, followed by intracellular degradation by a fatty acid amide hydrolase (for AEA and partly 2-AG) or by a monoacylglycerol lipase (for 2-AG). Together with AEA, 2-AG and congeners, the proteins that bind, transport and metabolize these lipids form the "endocannabinoid system". This new system will be briefly presented in this review, in order to put in a better perspective the role of the endocannabinoid pathway in neurodegenerative disorders, like Parkinson's disease, Huntington's disease, and multiple sclerosis. In addition, the potential exploitation of antagonists of endocannabinoid receptors, or of inhibitors of endocannabinoid metabolism, as next-generation therapeutics will be discussed.     

Endocannabinoids measurement in human saliva as potential biomarker of obesity

Background

The discovery of the endocannabinoid system and of its role in the regulation of energy balance has significantly advanced our understanding of the physiopathological mechanisms leading to obesity and type 2 diabetes. New knowledge on the role of this system in humans has been acquired by measuring blood endocannabinoids. Here we explored endocannabinoids and related N-acylethanolamines in saliva and verified their changes in relation to body weight status and in response to a meal or to body weight loss.

Methodology/Principal Findings

Fasting plasma and salivary endocannabinoids and N-acylethanolamines were measured through liquid mass spectrometry in 12 normal weight and 12 obese, insulin-resistant subjects. Salivary endocannabinoids and N-acylethanolamines were evaluated in the same cohort before and after the consumption of a meal. Changes in salivary endocannabinoids and N-acylethanolamines after body weight loss were investigated in a second group of 12 obese subjects following a 12-weeks lifestyle intervention program. The levels of mRNAs coding for enzymes regulating the metabolism of endocannabinoids, N-acylethanolamines and of cannabinoid type 1 (CB₁) receptor, alongside endocannabinoids and N-acylethanolamines content, were assessed in human salivary glands.The endocannabinoids 2-arachidonoylglycerol (2-AG), N-arachidonoylethanolamide (anandamide, AEA), and the N-acylethanolamines (oleoylethanolamide, OEA and palmitoylethanolamide, PEA) were quantifiable in saliva and their levels were significantly higher in obese than in normal weight subjects. Fasting salivary AEA and OEA directly correlated with BMI, waist circumference and fasting insulin. Salivary endocannabinoids and N-acylethanolamines did not change in response to a meal. CB₁ receptors, ligands and enzymes were expressed in the salivary glands. Finally, a body weight loss of 5.3% obtained after a 12-weeks lifestyle program significantly decreased salivary AEA levels.

Conclusions/Significance

Endocannabinoids and N-acylethanolamines are quantifiable in saliva and their levels correlate with obesity but not with feeding status. Body weight loss significantly decreases salivary AEA, which might represent a useful biomarker in obesity.     

Sustained Endocannabinoid Signaling Compromises Decidual Function and Promotes Inflammation-induced Preterm Birth

Recent studies provide evidence that premature maternal decidual senescence resulting from heightened mTORC1 signaling is a cause of preterm birth (PTB). We show here that mice devoid of fatty acid amide hydrolase (FAAH) with elevated levels of N-arachidonyl ethanolamide (anandamide), a major endocannabinoid lipid mediator, were more susceptible to PTB upon lipopolysaccharide (LPS) challenge. Anandamide is degraded by FAAH and primarily works by activating two G-protein-coupled receptors CB1 and CB2, encoded by Cnr1 and Cnr2, respectively. We found that Faah^−/− decidual cells progressively underwent premature senescence as marked by increased senescence-associated β-galactosidase (SA-β-Gal) staining and γH2AX-positive decidual cells. Interestingly, increased endocannabinoid signaling activated MAPK p38, but not p42/44 or mTORC1 signaling, in Faah^−/− deciduae, and inhibition of p38 halted premature decidual senescence. We further showed that treatment of a long-acting anandamide in wild-type mice at midgestation triggered premature decidual senescence utilizing CB1, since administration of a CB1 antagonist greatly reduced the rate of PTB in Faah^−/− females exposed to LPS. These results provide evidence that endocannabinoid signaling is critical in regulating decidual senescence and parturition timing. This study identifies a previously unidentified pathway in decidual senescence, which is independent of mTORC1 signaling.     

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB₁ cannabinoid receptors (CB₁Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB₁R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB₁Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB₁Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes.     

Heparan Sulfate Inhibits Hematopoietic Stem and Progenitor Cell Migration and Engraftment in Mucopolysaccharidosis I

Mucopolysaccharidosis I Hurler (MPSI-H) is a pediatric lysosomal storage disease caused by genetic deficiencies in IDUA, coding for α-l-iduronidase. Idua^−/− mice share similar clinical pathology with patients, including the accumulation of the undegraded glycosaminoglycans (GAGs) heparan sulfate (HS), and dermatan sulfate (DS), progressive neurodegeneration, and dysostosis multiplex. Hematopoietic stem cell transplantation (HSCT) is the most effective treatment for Hurler patients, but reduced intensity conditioning is a risk factor in transplantation, suggesting an underlying defect in hematopoietic cell engraftment. HS is a co-receptor in the CXCL12/CXCR4 axis of hematopoietic stem and progenitor cell (HSPC) migration to the bone marrow (BM), but the effect of HS alterations on HSPC migration, or the functional role of HS in MPSI-H are unknown. We demonstrate defective WT HSPC engraftment and migration in Idua^−/− recipient BM, particularly under reduced intensity conditioning. Both intra- but especially extracellular Idua^−/− BM HS was significantly increased and abnormally sulfated. Soluble heparinase-sensitive GAGs from Idua^−/− BM and specifically 2-O-sulfated HS, elevated in Idua^−/− BM, both inhibited CXCL12-mediated WT HSPC transwell migration, while DS had no effect. Thus we have shown that excess overly sulfated extracellular HS binds, and sequesters CXCL12, limiting hematopoietic migration and providing a potential mechanism for the limited scope of HSCT in Hurler disease.     

The endocannabinoid 2-arachidonoylglycerol (2-AG) and metabolizing enzymes during rat fetoplacental development: A role in uterine remodelling

The main endocannabinoids (EC) identified in mammalian tissues are N-arachidonoylethanolamide (AEA, anandamide), and 2-arachidonoylglycerol (2-AG). AEA levels are critical in pregnancy, especially during implantation, decidualization, and placental development. As 2-AG functions in pregnancy are still largely undefined, we hypothesized that it may also have a role during fetoplacental development. We showed that 2-AG is not only present in the rat mesometrial decidua and plasma during fetoplacental development, but that both 2-AG synthesizing (diacylglycerol lipase) and degradation (monoacylglycerol lipase) enzymes are expressed by decidual cells. While lower concentrations of 2-AG induced apoptosis of rat primary decidual cells, via the CB1 receptor, higher concentrations induced a dramatic effect on cell morphology, cell viability and lactate dehydrogenase release, triggered through a mechanism independent of CB1. This study provides evidences that 2-AG fluctuation in maternal tissues throughout normal pregnancy is primarily regulated by its metabolizing enzymes. Together, these data supports the hypothesis that a deregulation of the endocannabinoid system through aberrant cannabinoid signalling may impact normal uterine remodelling process and consequently normal pregnancy.     

In vivo pharmacology of endocannabinoids and their metabolic inhibitors: Therapeutic implications in Parkinson's disease and abuse liability

This review focuses on the behavioral pharmacology of endogenous cannabinoids (endocannabinoids) and indirect-acting cannabinoid agonists that elevate endocannabinoid tone by inhibiting the activity of metabolic enzymes. Similarities and differences between prototype cannabinoid agonists, endocannabinoids and inhibitors of endocannabinoid metabolism are discussed in the context of endocannabinoid pharmacokinetics in vivo. The distribution and function of cannabinoid and non-CB₁/CB₂ receptors are also covered, with emphasis on their role in disorders characterized by dopamine dysfunction, such as drug abuse and Parkinson's disease. Finally, evidence is presented to suggest that FAAH inhibitors lack the abuse liability associated with CB₁ agonists, although they may modify the addictive properties of other drugs, such as alcohol.