A collaboration investigating endocannabinoid signalling in brain and bone.

Investigations into the cellular and molecular mechanisms underlying the psychoactive effects of cannabis preparations have led to the discovery of the endocannabinoid system. Interest in the central nervous system effects was initially the main focus of the research, but it soon became evident that the endocannabinoid system affects virtually every organ. The research field has therefore experienced a tremendous growth over the last decade and is now truly interdisciplinary. This short review provides a personal account of an interdisciplinary collaboration between Itai Bab from the Hebrew University of Jerusalem and the author. It describes the discovery of the endocannabinoid system in bone and the analysis of its functions. I am summarising the role of CB1 signalling as a modulator of sympathetic inhibition of bone formation. Thus, activation of CB1 receptors on sympathetic nerve terminals in bone, presumably from endocannabinoids released from apposing osteoblasts, reduces the inhibition of bone formation of sympathetic norepinephrine. CB2 receptors on osteoblasts and osteoclasts also modulate the proliferation and functions of these cells. Thus, activation of CB2 stimulates bone formation and represses bone resorption, whereas the genetic disruption of CB2 results in an osteoporosis-like phenotype. This signalling mechanism is clinically relevant, as shown by the association of polymorphisms in the CB2 receptor gene, CNR2, with bone density and osteoporosis. Finally, the review provides a summary of the recently discovered role of endocannabinoid signalling in one elongation. This review will also discuss the benefits of interdisciplinary and international collaborations.

Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors.

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.

Contributions of endocannabinoid signaling to psychiatric disorders in humans: genetic and biochemical evidence.

The endocannabinoid signaling system is a widespread, neuromodulatory system in brain and is also widely utilized in the periphery to modulate metabolic functions and the immune system. Preclinical data demonstrate that endocannabinoid signaling is an important stress buffer and modulates emotional and cognitive functions. These data suggest the hypothesis that endocannabinoid signaling could be dysfunctional in a number of mental disorders. Genetic polymorphisms in the human genes for two important proteins of the endocannabinoid signaling system, the CB1 cannabinoid receptor (CB1R) and fatty acid amide hydrolase (FAAH), have been explored in the context of normal and pathological conditions. In the case of the gene for FAAH, the mechanistic relationships among the common genetic polymorphism, the expression of the FAAH protein, and its likely impact on endocannabinoid signaling are understood. However, multiple polymorphisms in the gene for the CB1R occur and are associated with human phenotypic differences without an understanding of the functional relationships among the gene, mRNA, protein, and protein function. The endocannabinoid ligands are found in the circulation, and several studies have identified changes in their concentrations under various conditions. These data are reviewed for the purpose of generating hypotheses and to encourage further studies in this very interesting and important area.

The genetic basis of the endocannabinoid system and drug addiction in humans.

The cannabinoid receptor (CNR1) and the fatty acid amide hydrolase (FAAH) genes are located on chromosomes 6 and 1 in the 6q15 and 1p33 cytogenetic bands, respectively. CNR1 encodes a seven-transmembrane domain protein of 472 amino acids, whereas FAAH encodes one transmembrane domain of 579 amino acids. Several mutations found in these genes lead to altered mRNA stability and transcription rate or a reduction of the activity of the encoded protein. Increasing evidence shows that these functional mutations are related to dependence upon cocaine, alcohol, marijuana, heroin, nicotine and other drugs. One of the most compelling associations is with the C385A single nucleotide polymorphism (SNP), which is found in the FAAH gene. For all of the genetic polymorphisms reviewed here, it is difficult to form overall conclusions due to the high diversity of population samples being studied, ethnicity, the use of volunteers, heterogeneity of the recruitment criteria and the drug addiction phenotype studied. Care should be taken when generalizing the results from different studies. However, many works have repeatedly associated polymorphisms in the CNR1 and FAAH genes with drug-related behaviours; this suggests that these genes should be examined in further genetic studies focusing on drug addiction and other psychiatric disorders.

Effect of capacitation on the endocannabinoid system of mouse sperm.

The presence of the elements of the endocannabinoid system (ECS) in sperm isolated from several species (from invertebrates to mammals, humans included) has supported the "evolutionary theory" that proposes endocannabinoids as check points in reproductive events like capacitation. In this study, we characterized the ECS elements at the mRNA, protein and functional levels in mouse sperm before and after capacitation. We found that the latter process increases the endogenous levels of the two major endocannabinoids (anandamide and 2-arachidonoylglycerol), through a decreased degradation and increased biosynthesis, respectively. Additionally, we found that the binding activity of cannabinoid receptors was not affected by sperm capacitation, whereas that of vanilloid receptor was reduced. Overall, our data demonstrate that mouse sperm have a fully functional ECS, and that capacitation alters the endogenous tone of the major endocannabinoids through distinct mechanisms.

Characterization of NTera2/D1 cells as a model system for the investigation of cannabinoid function in human neurons and astrocytes.

The limited availability and potential to culture primary human brain cells means that there is still a need for cell lines that reliably model human neurons and glial cells. The human-derived NTera2/D1 (NT2) cell line is a promising tool from which both neuronal (NT2N) and astrocytic (NT2A) cells can be derived in vitro. Here we have investigated the potential to use this cell model to investigate the endocannabinoid system in the CNS. Through immunocytochemical characterization with a range of neuronal and glial markers, we found that these cell lines differentiate into cells with immature neuronal and astrocytic phenotypes, respectively. By real-time PCR, immunocytochemistry, and functional inhibition of cAMP accumulation, the cannabinoid 1 receptors were identified only on NT2N cells, consistent with high levels of expression of this receptor in neuronal cells of the CNS. No evidence of cannabinoid 2 receptor expression was found on any of the NT2 cell types. Both the precursors and the differentiated NT2N and NT2A cells demonstrated mRNA expression for the key enzymes involved in endocannabinoid synthesis and degradation. This work establishes a cannabinergic phenotype in NT2N and NT2A cells, providing an alternative human derived renewable cell model for investigation of cannabinoid receptor function and endocannabinoid synthesis and metabolism in the CNS.

Genetic association between bipolar disorder and 524A>C (Leu133Ile) polymorphism of CNR2 gene, encoding for CB2 cannabinoid receptor.

INTRODUCTION: Several studies provided evidence that the endocannabinoid system (ECS) is involved in psychiatric diseases, like major depression, schizophrenia and bipolar disorder (BD), mainly focusing on CB1 cannabinoid receptor, and FAAH, the fatty acid amide hydrolase involved in endocannabinoid metabolism. In this study we investigated the possible association of BD with three missense SNPs, of the gene CNR2, encoding for CB2 cannabinoid receptor. METHODS: The possible association between BD and three CNR2 missense SNPs, namely rs2501432 (315A>G; Arg63Gln), rs41311993 (524C>A; Leu133Ile) and rs2229579 (1073C>T; Tyr316His), was investigated through a case-control study. Eighty patients and one hundred and sixty healthy subjects were recruited. Allele Specific Oligonucleotide (ASO)-PCR and restriction fragment length polymorphism (RFLP) methods were used for genotyping. RESULTS: A statistically significant association was found between BD and the CNR2 524C>A; Leu133Ile (P(χ(2)) = 0.001; OR = 4.74; 95% C.I. = 2.52-10.50) while no statistically significant difference between BD and control group was observed for the other two SNPs. CONCLUSION: Though further investigations are necessary to confirm this data, our results suggest that CB2 cannabinoid receptor may play a role in BD.

Early exposure to environmental enrichment alters the expression of genes of the endocannabinoid system.

Early environmental enrichment (EE) produces several changes in gene expression in the brain and confers protection against the behavioral, neurochemical and molecular effects of repeated administration of drugs of abuse. Because the endogenous cannabinoid system (ECS) is known to play an important role in the rewarding effects of drugs, we investigated whether the positive effects of early exposure to EE are associated with changes in the expression of genes encoding for proteins that belong to the ECS in C57 mice. Using in situ hybridization, we compared the expression of the cannabinoid receptor CB1, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MGL) enzymes in brain regions involved in drug addiction in mice reared in either EE or standard environments (SE) from weaning until adulthood. We found that EE increases CB1 mRNA levels in the hypothalamus and in the basolateral amygdala but decreased them in the basomedial amygdala. Similarly, we found that FAAH mRNA levels are higher in the hypothalamus and the basolateral amygdala of EE mice compared to SE mice, with no change in the basomedial amygdala. In contrast, MGL mRNA levels were not affected by EE in any of the areas analyzed. The regional selectivity of EE-induced changes may indicate that early exposure to EE induces changes in the ECS that could result in reduced responses to stress, as confirmed in EE mice in a novelty-induced suppression of feeding test, and, ultimately, in resistance to addiction.

Investigations of the human endocannabinoid system in two subcutaneous adipose tissue depots in lean subjects and in obese subjects before and after weight loss.

CONTEXT: Endocannabinoids (ECs) have a role in obesity by affecting appetite and through peripheral effects. Obesity is associated with a dysregulation of the endocannabinoid system (ECS). OBJECTIVE: We aimed to determine the ECS in subcutaneous adipose tissue (AT) in obese subject and investigate the influence of diet-induced weight loss on this system. DESIGN: The obese study participants underwent a 12 weeks diet regimen resulting in 10-12% weight loss. All study participants underwent fasting blood samples and AT biopsies from abdomen and gluteal region, the obese subjects both before and after weight loss. SETTING AND PARTICIPANTS: A total of 21 healthy obese individuals (10 men/11 women, age 39.5 ± 1.6 years, body mass index (BMI): 37.5 ± 0.8 kg m(-2)) and 21 age- and gender-matched lean subjects (BMI: 23.8 ± 0.4 kg m(-2)) were studied. MAIN OUTCOME MEASURES: The activity of ECS in AT was determined by measuring arachidonoyl glycerol (2-AG) and N-arachidonoylethanolamine/anandamide in AT by mass spectrometry and gene expressions of enzymes and receptors involved in the ECS. RESULTS: The EC, 2-AG was reduced in obese individuals in the gluteal AT depot (P<0.01). Moreover, 2-AG increased in both depots in the obese subjects following weight loss (P<0.05). The gene expression of the CB1 was either not affected by the obese state (in the gluteal AT depot) or reduced (in the abdominal depot, P<0.05) and significantly affected by weight loss. The expression of the degrading enzymes FAAH, FAAH2, MGL and MGL2 was differently affected by obesity, AT depot and weight loss. CONCLUSION: We found reduced levels of 2-AG in subcutaneous AT in obesity, which increased after weight loss. In abdominal AT, the low CB1 expression was normalised after weight loss, whereas in gluteal AT the CB1 expression was reduced after weight loss. These findings support the concept of a dysregulated ECS in AT in association with obesity.

Cannabinoid exposure during zebra finch sensorimotor vocal learning persistently alters expression of endocannabinoid signaling elements and acute agonist responsiveness.

BACKGROUND: Previously we have found that cannabinoid treatment of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. Such persistently altered behavior must be attributable to changes in physiological substrates responsible for song. We are currently working to identify the nature of such physiological changes, and to understand how they contribute to altered vocal learning. One possibility is that developmental agonist exposure results in altered expression of elements of endocannabinoid signaling systems. To test this hypothesis we have studied effects of the potent cannabinoid receptor agonist WIN55212-2 (WIN) on endocannabinoid levels and densities of CB1 immunostaining in zebra finch brain. RESULTS: We found that late postnatal WIN treatment caused a long-term global disregulation of both levels of the endocannabinoid, 2-arachidonyl glycerol (2-AG) and densities of CB1 immunostaining across brain regions, while repeated cannabinoid treatment in adults produced few long-term changes in the endogenous cannabinoid system. CONCLUSIONS: Our findings indicate that the zebra finch endocannabinoid system is particularly sensitive to exogenous agonist exposure during the critical period of song learning and provide insight into susceptible brain areas.

Characterization of the endocannabinoid system in mouse embryonic stem cells.

In this study, we have ascertained the presence and functionality in mouse embryonic stem cells (ESCs) of members of the endocannabinoid system that have been proposed as possible modulators of the survival and differentiation of various type of stem cells. We show that mouse ESCs, in addition to classical CB(1) and CB(2) cannabinoid receptors, express the transient receptor potential vanilloid receptor, at mRNA, protein, and binding levels. Remarkably, we demonstrate that ESCs have the mRNA, protein, and enzyme activity to synthesize and degrade the prominent endocannabinoids anandamide (through N-acyl-phosphatidylethanolamine-specific phospholipase D and fatty acid amide hydrolase) and 2-arachidonoylglycerol (through diacylglycerol lipase and monoacylglycerol lipase). In addition, both endocannabinoids were detected in ESCs that were also shown to constitutively release a fatty acid amide hydrolase-activating compound. Finally, we document that the stimulation of ESCs by methanandamide, a nonhydrolysable analog of anandamide, does not lead to overt alteration of the expression of Oct3/4, Nanog, and Cdx2, genes that are involved in early cell fate in the preimplantation embryo and stemness, or of the expression patterns of Brachyury and Hnf4, genes that are used as late markers of lineage differentiation capability of ESC-derived embryoid bodies. Similarly ineffective on the expression of the tested stemness genes was 2-arachidonoylglycerol. Taken together, these results confirm and extend the notion that ESCs express several functional members of the endocannabinoid system, but they leave open the question about their role in stem cells as modulators of stemness and differentiation potential.

Effects of C358A missense polymorphism of the endocannabinoid degrading enzyme fatty acid amide hydrolase on weight loss and cardiovascular risk factors 1 year after biliopancreatic diversion surgery.

BACKGROUND: Bariatric surgery is the most effective long-term treatment of morbid obesity and also results in a reduction of obesity-associated co-morbidities. We investigated the role of the polymorphism (C358A) of the fatty acid amide hydrolase gene on the clinical outcomes 1 year after biliopancreatic diversion in morbidly obese patients. METHODS: A total of 67 morbidly obese patients (body mass index >40 kg/m(2)) underwent biliopancreatic diversion. Their weight, blood pressure, basal glucose, triglycerides, total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol were measured at the baseline visit and at each follow-up visit. The frequency of the metabolic co-morbidities was recorded at each visit. RESULTS: Of the 67 patients, 46 (68.7%) had genotype C358C (wild-type group) and 21 (10.3%) had genotype C358A (mutant-type group). In the wild- and mutant-type groups, the body mass index, weight, waist circumference, systolic blood pressure, and glucose, total cholesterol, low-density lipoprotein cholesterol, and triglyceride concentrations decreased, without statistical significance between the 2 groups. The initial percentage of weight loss at 9 months and 1 year of follow-up was greater in the mutant-type group (9 months, 22.1% versus 28.8%, P <.05; and 1 year, 28.3% versus 36.4%, P <.05). CONCLUSION: The allele A358 of fatty acid amide hydrolase was associated with a better initial percentage of excess weight loss 9 and 12 months after biliopancreatic diversion.

Endocannabinoid signaling directs differentiation of trophoblast cell lineages and placentation.

In most mammals, placentation is critical for fetal development and pregnancy success. Exposure to marijuana during pregnancy has adverse effects, but whether the placenta is a target of cannabinoid/endocannabinoid signaling is not known. Using mice as a model system, we found that the endocannabinoid system is present in the ectoplacental cone and spongiotrophoblast cells. We also observed that aberrant endocannabinoid signaling confers premature trophoblast stem cell differentiation, and defective trophoblast development and invasion. These defects are reflected in retarded fetal development and compromised pregnancy outcome. Because the endocannabinoid system is conserved in mice and humans, our study suggests that endocannabinoid signaling is critical to placentation and pregnancy success in humans and implicates its potential significance in stem cell biology.

Polymorphisms in the endocannabinoid receptor 1 in relation to fat mass distribution.

OBJECTIVE: Both animal and human studies have associated the endocannabinoid system with obesity and markers of metabolic dysfunction. Blockade of the cannabinoid receptor 1 (CB1) caused weight loss and reduction in waist size in both obese and type II diabetics. Recent studies on common variants of the CB1 receptor gene (CNR1) and the link to obesity have been conflicting. The aim of the present study was to evaluate whether selected common variants of the CNR1 are associated with measures of obesity and fat distribution. DESIGN AND METHODS: The single nucleotide polymorphisms (SNPs) rs806381, rs10485179 and rs1049353 were genotyped, and body fat and fat distribution were assessed by the use of dual-energy X-ray absorptiometry and magnetic resonance imaging in a population-based study comprising of 783 Danish men, aged 20-29 years. RESULTS: The rs806381 polymorphism was significantly associated with visceral fat mass (FM) only, whereas the rs1049353 was significantly and directly associated with visceral and intermuscular FM. None of the SNPs analysed were associated with total body FM or subcutaneous FM. CONCLUSION: The results point towards a link between common variants of the CNR1 and fat distribution in young men.

Role of genetic variation in the cannabinoid type 1 receptor gene (CNR1) in the pathophysiology of human obesity.

AIMS: The endocannabinoid system may contribute to the association of visceral fat accumulation with metabolic diseases. Here we investigated the effects of genetic variation in the cannabinoid type 1 receptor gene (CNR1) on its mRNA expression in adipose tissue from visceral and subcutaneous depots and on the development of obesity. MATERIALS & METHODS: CNR1 was sequenced in 48 nonrelated German Caucasians to detect genetic variation. Five representative variants including HapMap tagging SNPs (rs12720071, rs806368, rs806370, rs1049353 and rs806369) were genotyped for subsequent association studies in two independent cohorts (total n = 2774) with detailed metabolic testing: subjects from the Leipzig Study (n = 1857) and a self-contained population of Sorbs from Germany (n = 917). RESULTS: In a case-control study of lean (BMI <25 kg/m(2)) versus obese (BMI >30 kg/m(2)) subjects, rs806368 was found to be nominally associated with obesity in the Sorbian cohort (adjusted p < 0.05), but not in the Leipzig cohort. Also, several SNPs (rs806368, rs806370 and rs12720071) were nominally associated with serum leptin levels (p < 0.05 after adjusting for age, sex and BMI). However, none of these associations remained significant after accounting for multiple testing. Furthermore, none of the SNPs were related to CNR1 mRNA expression in visceral and subcutaneous fat. CONCLUSION: The data suggest that common genetic variation in the CNR1 gene does not influence mRNA expression in adipose tissue nor does it play a significant role in the pathophysiology of obesity in German and Sorbian populations.

Variations in critical morphine biosynthesis genes and their potential to influence human health.

Endogenous morphine has been detected in human tissues from the vascular, immune and nervous systems. The genes/enzymes (CYP2D6, COMT and PNMT) that are involved in the biosynthesis of morphine have variations that affect their functionality. Some of these variations are the result of single nucleotide polymorphisms of DNA sequences. This review highlights some of the functional differences in the critical enzymes required for the biosynthesis of morphine that may affect human health. These variations have been shown to change the way animals react to stressors, perceive pain and behave. The presence of morphine signaling in almost all organ systems suggests that it is most likely playing a role in maintaining the health and promoting the normal functioning of these physiological systems.

Identification of the synthetic pathway producing the endocannabinoid that mediates the bulk of retrograde signaling in the brain.

Endocannabinoids serve as retrograde messengers that are released by postsynaptic cells to regulate neurotransmitter release from presynaptic boutons. A new study in this issue of Neuron by Tanimura et al. establishes that most endocannabinoid signaling in the brain is a consequence of the calcium-dependent or receptor-driven generation of the retrograde signal 2-arachidonoylglycerol (2-AG) by diacylglycerol lipase alpha (DGLalpha).

An endocannabinoid system is localized to the hypophysial pars tuberalis of Syrian hamsters and responds to photoperiodic changes.

The hypophysial pars tuberalis (PT), an important interface between neuroendocrine brain centers (hypothalamus, pineal organ) and the pars distalis (PD) of the hypophysis, plays a central role in regulating seasonal reproduction and prolactin release. However, the signaling molecules that transmit photoperiodic information from the PT to the PD and control prolactin release (the so-called "tuberalins") have not yet been identified, despite an intense search for more than three decades. Here, we demonstrate an endocannabinoid system in the PT of the Syrian hamster, a photoperiodic species. By means of in situ hybrization, the PT was found to express N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), sn-1-selective diacylglycerol lipases (DAGLalpha and DAGLbeta), and monoacylglycerol lipase (MAGL), enzymes involved in endocannabinoid synthesis and degradation. The expression of NAPE-PLD, FAAH, and DAGLalpha was confirmed by immunohistochemistry. Expression and protein levels of DAGLs controlling the synthesis of 2-arachidonoyl glycerol (2-AG), a major endocannabinoid, were upregulated in the PT of Syrian hamsters kept under long-day conditions. Consequently, 2-AG levels were increased in the PT of these hamsters. A primary target of 2-AG, the cannabinoid receptor 1 (CB1), was expressed in the PD. Double-immunolabeling revealed that most of the CB1-immunoreactive cells in the PD were folliculostellate cells that were also immunoreactive for S-100 protein. Thus, the PT comprises an endocannabinoid system, and 2-AG may act as a photoperiodic messenger from the PT to the PD for the regulation of hypophysial hormonal secretion.