Genetic variation in endocannabinoid signaling: Anxiety, depression, and threat- and reward-related brain functioning during the transition into adolescence.

BACKGROUND: The endocannabinoid system modulates neural activity throughout the lifespan. In adults, neuroimaging studies link a common genetic variant in fatty acid amide hydrolase (FAAH C385A)-an enzyme that regulates endocannabinoid signaling-to reduced risk of anxiety and depression, and altered threat- and reward-related neural activity. However, limited research has investigated these associations during the transition into adolescence, a period of substantial neurodevelopment and increased psychopathology risk. METHODS: This study included FAAH genotype and longitudinal neuroimaging and neurobehavioral data from 4811 youth (46% female; 9-11 years at Baseline, 11-13 years at Year 2) from the Adolescent Brain Cognitive DevelopmentSM Study. Linear mixed models examined the effects of FAAH and the FAAH x time interaction on anxiety and depressive symptoms, amygdala reactivity to threatening faces, and nucleus accumbens (NAcc) response to happy faces during the emotional n-back task. RESULTS: A significant main effect of FAAH on depressive symptoms was observed, such that depressive symptoms were lower across both timepoints in those with the AA genotype compared to both AC and CC genotypes (p's<0.05). There were no significant FAAH x time interactions for anxiety, depression, or neural responses (p's>0.05). Additionally, there were no main effects of FAAH on anxiety or neural responses (p's>0.05). CONCLUSIONS: Our findings add to emerging evidence linking the FAAH C385A variant to lower risk of psychopathology, and extend these findings to a developmental sample. In particular, we found lower depressive symptoms in FAAH AA genotypes compared to AC and CC genotypes. Future research is needed to characterize the role of the FAAH variant and the eCB system more broadly in neurodevelopment and psychiatric risk.

Selective alterations of endocannabinoid system genes expression in obsessive compulsive disorder.

Obsessive Compulsive Disorder (OCD) is listed as one of the top 10 most disabling neuropsychiatric conditions in the world. The neurobiology of OCD has not been completely understood and efforts are needed in order to develop new treatments. Beside the classical neurotransmitter systems and signalling pathways implicated in OCD, the possible involvement of the endocannabinoid system (ECS) has emerged in pathophysiology of OCD. We report here selective downregulation of the genes coding for enzymes allowing the synthesis of the endocannabinoids. We found reduced DAGLα and NAPE-PLD in blood samples of individuals with OCD (when compared to healthy controls) as well as in the amygdala complex and prefrontal cortex of dopamine transporter (DAT) heterozygous rats, manifesting compulsive behaviours. Also mRNA levels of the genes coding for cannabinoid receptors type 1 and type 2 resulted downregulated, respectively in the rat amygdala and in human blood. Moreover, NAPE-PLD changes in gene expression resulted to be associated with an increase in DNA methylation at gene promoter, and the modulation of this gene in OCD appears to be correlated to the progression of the disease. Finally, the alterations observed in ECS genes expression appears to be correlated with the modulation in oxytocin receptor gene expression, consistently with what recently reported. Overall, we confirm here a role for ECS in OCD at both preclinical and clinical level. Many potential biomarkers are suggested among its components, in particular NAPE-PLD, that might be of help for a prompt and clear diagnosis.

Association between the FAAH C385A variant (rs324420) and obesity-related traits: a systematic review.

BACKGROUND: Overweight and obesity are the consequence of a sustained positive energy balance. Twin studies show high heritability rates pointing to genetics as one of the principal risk factors. By 2022, genomic studies led to the identification of almost 300 obesity-associated variants that could help to fill the gap of the high heritability rates. The endocannabinoid system is a critical regulator of metabolism for its effects on the central nervous system and peripheral tissues. Fatty acid amide hydrolase (FAAH) is a key enzyme in the inactivation of one of the two endocannabinoids, anandamide, and of its congeners. The rs324420 variant within the FAAH gene is a nucleotide missense change at position 385 from cytosine to adenine, resulting in a non-synonymous amino acid substitution from proline to threonine in the FAAH enzyme. This change increases sensitivity to proteolytic degradation, leading to reduced FAAH levels and increased levels of anandamide, associated with obesity-related traits. However, association studies of this variant with metabolic parameters have found conflicting results. This work aims to perform a systematic review of the existing literature on the association of the rs324420 variant in the FAAH gene with obesity and its related traits. METHODS: A literature search was conducted in PubMed, Web of Science, and Scopus. A total of 645 eligible studies were identified for the review. RESULTS/CONCLUSIONS: After the identification, duplicate elimination, title and abstract screening, and full-text evaluation, 28 studies were included, involving 28 183 individuals. We show some evidence of associations between the presence of the variant allele and higher body mass index, waist circumference, fat mass, and waist-to-hip ratio levels and alterations in glucose and lipid homeostasis. However, this evidence should be taken with caution, as many included studies did not report a significant difference between genotypes. These discordant results could be explained mainly by the pleiotropy of the endocannabinoid system, the increase of other anandamide-like mediators metabolized by FAAH, and the influence of gene-environment interactions. More research is necessary to study the endocannabinoidomic profiles and their association with metabolic diseases.

Cross-disorder GWAS meta-analysis of endocannabinoid DNA variations in major depressive disorder, bipolar disorder, attention deficit hyperactivity disorder, autism spectrum disorder, and schizophrenia.

The endocannabinoid system (ECS) is implicated in multiple mental disorders. In this study, we explored DNA variations in the ECS across major depressive disorder (MDD), bipolar disorder, attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia by performing a cross-disorder genome-wide association study (GWAS) meta-analysis. We obtained six datasets from the Psychiatric Genomics Consortium containing GWAS summary statistics from European cohorts (284,023 cases and 508,515 controls). Effective sample size weighted meta-analysis was performed for 2241 single nucleotide polymorphisms (SNPs) pertaining to gene bodies of 33 endocannabinoid genes using METAL, where an overall z-statistic is calculated for each marker based on a weighted sum of individual statistics. Heterogeneity was examined with I2 and X2 tests. MAGMA gene-based analysis was also performed. We identified nine SNPs significantly associated with a change in risk of having a mental disorder. The lead SNP was rs12805732 (Gene: Diacylglycerol Lipase Alpha; DAGLA). Four SNPs had substantial heterogeneity (I2>60 %). DAGLA had the strongest association with disease risk in gene-based analysis. Our findings suggest that the ECS may be a shared pathway in mental disorders. Future studies validating these findings would contribute to the identification of biomarkers of disease risk across multiple mental disorders.

Functional Variation in the FAAH Gene Is Directly Associated with Subjective Well-Being and Indirectly Associated with Problematic Alcohol Use.

Fatty acid amide hydrolase (FAAH) is an enzyme that degrades anandamide, an endocannabinoid that modulates mesolimbic dopamine release and, consequently, influences states of well-being. Despite these known interactions, the specific role of FAAH in subjective well-being remains underexplored. Since well-being is a dynamic trait that can fluctuate over time, we hypothesized that we could provide deeper insights into the link between FAAH and well-being using longitudinal data. To this end, we analyzed well-being data collected three years apart using the WHO (Ten) Well-Being Index and genotyped a functional polymorphism in the FAAH gene (rs324420, Pro129Thr) in a sample of 2822 individuals. We found that the A-allele of rs324420, which results in reduced FAAH activity and elevated anandamide levels, was associated with lower well-being scores at both time points (Wave I, B: -0.52, p = 0.007; Wave II, B: -0.41, p = 0.03, adjusted for age and sex). A subsequent phenome-wide association study (PheWAS) affirmed our well-being findings in the UK Biobank (N = 126,132, alternative C-allele associated with elevated happiness, p = 0.008) and revealed an additional association with alcohol dependence. In our cohort, using lagged longitudinal mediation analyses, we uncovered evidence of an indirect association between rs324420 and problematic alcohol use (AUDIT-P) through the pathway of lower well-being (indirect effect Boot: 0.015, 95% CI [0.003, 0.030], adjusted for AUDIT in Wave I). We propose that chronically elevated anandamide levels might influence disruptions in the endocannabinoid system-a biological contributor to well-being-which could, in turn, contribute to increased alcohol intake, though multiple factors may be at play. Further genetic studies and mediation analyses are needed to validate and extend these findings.

The peripheral endocannabinoid system and its association with biomarkers of inflammation in untreated patients with multiple sclerosis.

BACKGROUND AND PURPOSE: The endocannabinoid system (ECS) has been found altered in patients with multiple sclerosis (MS). However, whether the ECS alteration is present in the early stage of MS remains unknown. First, we aimed to compare the ECS profile between newly diagnosed MS patients and healthy controls (HCs). Next, we explored the association of the ECS, biomarkers of inflammation, and clinical parameters in newly diagnosed MS patients. METHODS: Whole blood gene expression of ECS components and levels of endocannabinoids in plasma were measured by real-time quantitative polymerase chain reaction and ultra-high-pressure liquid chromatography-mass spectrometry, respectively, in 66 untreated MS patients and 46 HCs. RESULTS: No differences were found in the gene expression or plasma levels of the selected ECS components between newly diagnosed MS patients and HCs. Interferon-γ, encoded by the gene IFNG, correlated positively (ρ = 0.60) with the expression of G protein-coupled receptor 55 (GPR55), and interleukin1ß (IL1B) correlated negatively (ρ = -0.50) with cannabinoid receptor 2 (CNR2) in HCs. CONCLUSIONS: We found no alteration in the peripheral ECS between untreated patients with MS and HC. Furthermore, our results indicate that the ECS has a minor overall involvement in the early stage of MS on inflammatory markers and clinical parameters when compared with HCs.

The study of rs324420 (C385A) polymorphism of the FAAH gene of the endocannabinoid system in patients with epilepsy and ADHD.

The endocannabinoid (eCB) system regulates many physiological functions in the central nervous system. Fatty acid amide hydrolase (FAAH) is an essential enzyme in the eCB system, degrading anandamide. Single nucleotide polymorphism (SNP) rs324420 is a common genetic polymorphism of the FAAH gene and has been associated with susceptibility to neurological conditions. This study examined whether the SNP rs324420 (C385A) is associated with epilepsy and attention deficit hyperactivity disorder (ADHD). This study consists of two case-control parts. The first part comprises 250 epilepsy subjects and 250 healthy individuals as controls. The second one comprises 157 cases with ADHD and 136 healthy individuals as controls. Genotyping was carried out using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Interestingly, the FAAH C384A genotype (OR 1.755, 95 % CI 1.124-2.742, p = 0.013) and allele (OR 1.462, 95 % CI 1.006-2.124, p = 0.046) distribution showed an association with generalized epilepsy. On the other hand, this SNP was not associated with the risk of ADHD. To our knowledge, there was no study on the association between rs324420 (C385A) polymorphism and the risks of ADHD or epilepsy. This study provided the first evidence of an association between generalized epilepsy and rs324420 (C385A) of FAAH. Larger sample sizes and functional studies are warranted to explore the clinical utility of FAAH genotyping as a possible marker for increased generalized epilepsy risk.

Bioinformatics of the Endocannabinoid System: Study of DNA Methylation at Rat Cnr1 Gene Promoter.

In this chapter, we will describe the bioinformatic tools that allow verifying the presence of CpG islands in a gene promoter region. We will also describe the tools needed to identify consensus motifs for specific transcription factors, focusing on the study of rat type-1 cannabinoid receptor gene (R_Cnr1) as a case study.

Endocannabinoid-dependent formation of columnar axonal projection in the mouse cerebral cortex.

Columnar structure is one of the most fundamental morphological features of the cerebral cortex and is thought to be the basis of information processing in higher animals. Yet, how such a topographically precise structure is formed is largely unknown. Formation of columnar projection of layer 4 (L4) axons is preceded by thalamocortical formation, in which type 1 cannabinoid receptors (CB1R) play an important role in shaping barrel-specific targeted projection by operating spike timing-dependent plasticity during development (Itami et al., J. Neurosci. 36, 7039-7054 [2016]; Kimura & Itami, J. Neurosci. 39, 3784-3791 [2019]). Right after the formation of thalamocortical projections, CB1Rs start to function at L4 axon terminals (Itami & Kimura, J. Neurosci. 32, 15000-15011 [2012]), which coincides with the timing of columnar shaping of L4 axons. Here, we show that the endocannabinoid 2-arachidonoylglycerol (2-AG) plays a crucial role in columnar shaping. We found that L4 axon projections were less organized until P12 and then became columnar after CB1Rs became functional. By contrast, the columnar organization of L4 axons was collapsed in mice genetically lacking diacylglycerol lipase α, the major enzyme for 2-AG synthesis. Intraperitoneally administered CB1R agonists shortened axon length, whereas knockout of CB1R in L4 neurons impaired columnar projection of their axons. Our results suggest that endocannabinoid signaling is crucial for shaping columnar axonal projection in the cerebral cortex.

Gene expression of the endocannabinoid system in endometrium through menstrual cycle.

Endocannabinoids mediate cellular functions and their activity is controlled by a complex system of enzymes, membrane receptors and transport molecules. Endocannabinoids are present in endometrium, a cyclical regenerative tissue requiring tightly regulated cellular mechanisms for maturation. The objective of this study was to investigate the gene expression of key elements involved in the endocannabinoid system across the menstrual cycle. RNA was isolated from endometrial tissue and genome-wide gene expression datasets were generated using RNA-sequencing. An a priori set of 70 genes associated with endocannabinoid system were selected from published literature. Gene expression across the menstrual cycle was analyzed using a moderated t test, corrected for multiple testing with Bonferroni's method. A total of 40 of the 70 genes were present in > 90% of the samples, and significant differential gene expression identified for 29 genes. We identified 4 distinct regulation patterns for synthesizing enzymes, as well as a distinct regulation pattern for degradations and transporting enzymes. This study charts the expression of endometrial endocannabinoid system genes across the menstrual cycle. Altered expression of genes that control endocannabinoid may allow fine control over endocannabinoid concentrations and their influence on cellular function, maturation and differentiation as the endometrium matures through the menstrual cycle.

Exploring the endocannabinoidome in genetically obese (ob/ob) and diabetic (db/db) mice: Links with inflammation and gut microbiota.

BACKGROUND: Obesity and type 2 diabetes are two interrelated metabolic disorders characterized by insulin resistance and a mild chronic inflammatory state. We previously observed that leptin (ob/ob) and leptin receptor (db/db) knockout mice display a distinct inflammatory tone in the liver and adipose tissue. The present study aimed at investigating whether alterations in these tissues of the molecules belonging to the endocannabinoidome (eCBome), an extension of the endocannabinoid (eCB) signaling system, whose functions are important in the context of metabolic disorders and inflammation, could reflect their different inflammatory phenotypes. RESULTS: The basal eCBome lipid and gene expression profiles, measured by targeted lipidomics and qPCR transcriptomics, respectively, in the liver and subcutaneous or visceral adipose tissues, highlighted a differentially altered eCBome tone, which may explain the impaired hepatic function and more pronounced liver inflammation remarked in the ob/ob mice, as well as the more pronounced inflammatory state observed in the subcutaneous adipose tissue of db/db mice. In particular, the levels of linoleic acid-derived endocannabinoid-like molecules, of one of their 12-lipoxygenase metabolites and of Trpv2 expression, were always altered in tissues exhibiting the highest inflammation. Correlation studies suggested the possible interactions with some gut microbiota bacterial taxa, whose respective absolute abundances were significantly different between ob/ob and the db/db mice. CONCLUSIONS: The present findings emphasize the possibility that bioactive lipids and the respective receptors and enzymes belonging to the eCBome may sustain the tissue-dependent inflammatory state that characterizes obesity and diabetes, possibly in relation with gut microbiome alterations.

Genetic variation in endocannabinoid signaling is associated with differential network-level functional connectivity in youth.

The endocannabinoid system is an important regulator of emotional responses such as fear, and a number of studies have implicated endocannabinoid signaling in anxiety. The fatty acid amide hydrolase (FAAH) C385A polymorphism, which is associated with enhanced endocannabinoid signaling in the brain, has been identified across species as a potential protective factor from anxiety. In particular, adults with the variant FAAH 385A allele have greater fronto-amygdala connectivity and lower anxiety symptoms. Whether broader network-level differences in connectivity exist, and when during development this neural phenotype emerges, remains unknown and represents an important next step in understanding how the FAAH C385A polymorphism impacts neurodevelopment and risk for anxiety disorders. Here, we leveraged data from 3,109 participants in the nationwide Adolescent Brain Cognitive Development Study℠ (10.04 ± 0.62 years old; 44.23% female, 55.77% male) and a cross-validated, data-driven approach to examine associations between genetic variation and large-scale resting-state brain networks. Our findings revealed a distributed brain network, comprising functional connections that were both significantly greater (95% CI for p values = [<0.001, <0.001]) and lesser (95% CI for p values = [0.006, <0.001]) in A-allele carriers relative to non-carriers. Furthermore, there was a significant interaction between genotype and the summarized connectivity of functional connections that were greater in A-allele carriers, such that non-carriers with connectivity more similar to A-allele carriers (i.e., greater connectivity) had lower anxiety symptoms (ß = -0.041, p = 0.030). These findings provide novel evidence of network-level changes in neural connectivity associated with genetic variation in endocannabinoid signaling and suggest that genotype-associated neural differences may emerge at a younger age than genotype-associated differences in anxiety.

Endocannabinoid System and Tumour Microenvironment: New Intertwined Connections for Anticancer Approaches.

The tumour microenvironment (TME) is now recognised as a hallmark of cancer, since tumour:stroma crosstalk supports the key steps of tumour growth and progression. The dynamic co-evolution of the tumour and stromal compartments may alter the surrounding microenvironment, including the composition in metabolites and signalling mediators. A growing number of evidence reports the involvement of the endocannabinoid system (ECS) in cancer. ECS is composed by a complex network of ligands, receptors, and enzymes, which act in synergy and contribute to several physiological but also pathological processes. Several in vitro and in vivo evidence show that ECS deregulation in cancer cells affects proliferation, migration, invasion, apoptosis, and metastatic potential. Although it is still an evolving research, recent experimental evidence also suggests that ECS can modulate the functional behaviour of several components of the TME, above all the immune cells, endothelial cells and stromal components. However, the role of ECS in the tumour:stroma interplay remains unclear and research in this area is particularly intriguing. This review aims to shed light on the latest relevant findings of the tumour response to ECS modulation, encouraging a more in-depth analysis in this field. Novel discoveries could be promising for novel anti-tumour approaches, targeting the microenvironmental components and the supportive tumour:stroma crosstalk, thereby hindering tumour development.

The Novel Monoacylglycerol Lipase Inhibitor MJN110 Suppresses Neuroinflammation, Normalizes Synaptic Composition and Improves Behavioral Performance in the Repetitive Traumatic Brain Injury Mouse Model.

Modulation of the endocannabinoid system has emerged as an effective approach for the treatment of many neurodegenerative and neuropsychological diseases. However, the underlying mechanisms are still uncertain. Using a repetitive mild traumatic brain injury (mTBI) mouse model, we found that there was an impairment in locomotor function and working memory within two weeks post-injury, and that treatment with MJN110, a novel inhibitor of the principal 2-arachidononyl glycerol (2-AG) hydrolytic enzyme monoacylglycerol lipase dose-dependently ameliorated those behavioral changes. Spatial learning and memory deficits examined by Morris water maze between three and four weeks post-TBI were also reversed in the drug treated animals. Administration of MJN110 selectively elevated the levels of 2-AG and reduced the production of arachidonic acid (AA) and prostaglandin E2 (PGE2) in the TBI mouse brain. The increased production of proinflammatory cytokines, accumulation of astrocytes and microglia in the TBI mouse ipsilateral cerebral cortex and hippocampus were significantly reduced by MJN110 treatment. Neuronal cell death was also attenuated in the drug treated animals. MJN110 treatment normalized the expression of the NMDA receptor subunits NR2A and NR2B, the AMPA receptor subunits GluR1 and GluR2, and the GABAA receptor subunits α1, ß2,3 and γ2, which were all reduced at 1, 2 and 4 weeks post-injury. The reduced inflammatory response and restored glutamate and GABA receptor expression likely contribute to the improved motor function, learning and memory in the MJN110 treated animals. The therapeutic effects of MJN110 were partially mediated by activation of CB1 and CB2 cannabinoid receptors and were eliminated when it was co-administered with DO34, a novel inhibitor of the 2-AG biosynthetic enzymes. Our results suggest that augmentation of the endogenous levels of 2-AG can be therapeutically useful in the treatment of TBI by suppressing neuroinflammation and maintaining the balance between excitatory and inhibitory neurotransmission.

Effects of Nutrients on Platelet Function: A Modifiable Link between Metabolic Syndrome and Neurodegeneration?

Metabolic syndrome increases the risk of vascular dementia and other neurodegenerative disorders. Recent studies underline that platelets play an important role in linking peripheral with central metabolic and inflammatory mechanisms. In this narrative review, we address the activation of platelets in metabolic syndrome, their effects on neuronal processes and the role of the mediators (e.g., serotonin, platelet-derived growth factor). Emerging evidence shows that nutritional compounds and their metabolites modulate these interactions-specifically, long chain fatty acids, endocannabinoids and phenolic compounds. We reviewed the role of activated platelets in neurovascular processes and nutritional compounds in platelet activation.

Genetic and environmental influences on serum oxylipins, endocannabinoids, bile acids and steroids.

Lipid bioactivity is a result of direct action and the action of lipid mediators including oxylipins, endocannabinoids, bile acids and steroids. Understanding the factors contributing to biological variation in lipid mediators may inform future approaches to understand and treat complex metabolic diseases. This research aims to determine the contribution of genetic and environmental influences on lipid mediators involved in the regulation of inflammation and energy metabolism. This study recruited 138 monozygotic (MZ) and dizygotic (DZ) twins aged 18-65 years and measured serum oxylipins, endocannabinoids, bile acids and steroids using liquid chromatography mass-spectrometry (LC-MS). In this classic twin design, the similarities and differences between MZ and DZ twins are modelled to estimate the contribution of genetic and environmental influences to variation in lipid mediators. Heritable lipid mediators included the 12-lipoxygenase products 12-hydroxyeicosatetraenoic acid [0.70 (95% CI: 0.12,0.82)], 12-hydroxyeicosatetraenoic acid [0.73 (95% CI: 0.30,0.83)] and 14­hydroxy-docosahexaenoic acid [0.51 (95% CI: 0.07,0.71)], along with the endocannabinoid docosahexaenoy-lethanolamide [0.52 (95% CI: 0.15,0.72)]. For others such as 13-hydroxyoctadecatrienoic acid and lithocholic acid the contribution of environment to variation was stronger. With increased understanding of lipid mediator functions in health, it is important to understand the factors contributing to their variance. This study provides a comprehensive analysis of lipid mediators and extends pre-existing knowledge of the genetic and environmental influences on the human lipidome.

Cannabinoid Receptors Overexpression in a Rat Model of Irritable Bowel Syndrome (IBS) after Treatment with a Ketogenic Diet.

The administration of a ketogenic diet (KD) has been considered therapeutic in subjects with irritable bowel syndrome (IBS). This study aimed to investigate the molecular mechanisms by which a low-carbohydrate diet, such as KD, can improve gastrointestinal symptoms and functions in an animal model of IBS by evaluating possible changes in intestinal tissue expression of endocannabinoid receptors. In rats fed a KD, we detected a significant restoration of cell damage to the intestinal crypt base, a histological feature of IBS condition, and upregulation of CB1 and CB2 receptors. The diet also affected glucose metabolism and intestinal membrane permeability, with an overexpression of the glucose transporter GLUT1 and tight junction proteins in treated rats. The present data suggest that CB receptors represent one of the molecular pathways through which the KD works and support possible cannabinoid-mediated protection at the intestinal level in the IBS rats after dietary treatment.

Genetic analyses of the endocannabinoid pathway in association with affective phenotypic variants.

BACKGORUND: Increasing experimental data confirm the crucial role of the endocannabinoid (eCB) system in the regulation of stress response and emotional processes. Despite of the fact, that genetically determined vulnerability for stress is a widely accepted concept in the pathomechanism of affective disorders, replicable human genetic results with interaction analyses of early life trauma and eCB genes are rare. The aim of this study is to test the associations between genetic variants of the eCB pathway, childhood trauma and affective phenotypes. METHODS: We selected 18,897 SNPs in the eCB pathway of a GWAS dataset in two general population cohorts (BP sample N = 837; MN sample N = 988). Association analyses were performed on the anxious and depressive subscales of the Brief Symptom Inventory (BSI-ANX and BSI-DEP, respectively). Childhood trauma was assessed by the Childhood Adversity Questionnaire (CAQ). Association analyses were performed in the R 2.0. statistical program using the SNPassoc package. REULTS: Genetic effect was more robust in the BP sample than in the MN sample. The most comprehensive results showed that SNPs in the CACNA1C gene associated with depressive phenotype in interaction with CAQ in both BP (p = 1.2 × 10-4) and MN samples (p = 1.6 × 10-4). Direct association analyses (without interaction) provided significant associations between SNPs in different genesets of the two study populations. SNPs in KCNJ3 and GNB5 genes on the BSI-DEP (p = 6.1 × 10-5; p = 7.1 × 10-4) and GNG12 gene on the BSI-ANX (p = 7.4 × 10-6) in the BP sample, while GABAergic, ADCY1 and HTR2A gene variants can be outlined from results of MN sample with less strong p-values. CONCLUSION: Our results confirmed the prominent role of CACNA1C gene in the pathogenic effect of early life stress in the development of affective vulnerability in two different study populations using GxE interaction analysis. CACNA1C gene, as it encodes for L-type voltage-gated calcium channel, contributes to neuronal excitability, plasticity and neurogenesis being a crucial effector of both eCB signaling and the BDNF-CREB pathway as well. Our findings suggest that childhood trauma related depression may have more robust genetically determined basis than without early life stress.

The contributions of the endocannabinoid system and stress on the neural processing of reward stimuli.

The brain's endocannabinoid system plays a crucial role in reward processes by mediating appetitive learning and encoding the reinforcing properties of substances. Evidence also suggests that endocannabinoids are an important constituent of neuronal substrates involved in emotional responses to stress. Thus, it is critical to understand how the endocannabinoid system and stress may affect reward processes given their importance in substance use disorders. We examined the relationship between factors that regulate endocannabinoid system signaling (i.e., cannabinoid receptor genes and prolonged cannabis exposure) and stress on fMRI BOLD response to reward cues using multivariate statistical analysis. We found that proxies for endocannabinoid system signaling (i.e., endocannabinoid genes and chronic exposure to cannabis) and stress have differential effects on neural response to cannabis cues. Specifically, a single nucleotide polymorphism (SNP) variant in the cannabinoid receptor 1 (CNR1) gene, early life stress, and current perceived stress modulated reward responsivity in long-term, heavy cannabis users, while a variant in the fatty acid amide hydrolase (FAAH) gene and current perceived stress modulated cue-elicited response in non-using controls. These associations were related to distinct neural responses to cannabis-related cues compared to natural reward cues. Understanding the contributions of endocannabinoid system factors and stress that lead to downstream effects on neural mechanisms underlying sensitivity to rewards, such as cannabis, will contribute towards a better understanding of endocannabinoid-targeted therapies as well as individual risks for cannabis use disorder.

Three of a Kind: Control of the Expression of Liver-Expressed Antimicrobial Peptide 2 (LEAP2) by the Endocannabinoidome and the Gut Microbiome.

The endocannabinoidome (expanded endocannabinoid system, eCBome)-gut microbiome (mBIome) axis plays a fundamental role in the control of energy intake and processing. The liver-expressed antimicrobial peptide 2 (LEAP2) is a recently identified molecule acting as an antagonist of the ghrelin receptor and hence a potential effector of energy metabolism, also at the level of the gastrointestinal system. Here we investigated the role of the eCBome-gut mBIome axis in the control of the expression of LEAP2 in the liver and, particularly, the intestine. We confirm that the small intestine is a strong contributor to the circulating levels of LEAP2 in mice, and show that: (1) intestinal Leap2 expression is profoundly altered in the liver and small intestine of 13 week-old germ-free (GF) male mice, which also exhibit strong alterations in eCBome signaling; fecal microbiota transfer (FMT) from conventionally raised to GF mice completely restored normal Leap2 expression after 7 days from this procedure; in 13 week-old female GF mice no significant change was observed; (2) Leap2 expression in organoids prepared from the mouse duodenum is elevated by the endocannabinoid noladin ether, whereas in human Caco-2/15 epithelial intestinal cells it is elevated by PPARγ activation by rosiglitazone; (3) Leap2 expression is elevated in the ileum of mice with either high-fat diet-or genetic leptin signaling deficiency-(i.e., ob/ob and db/db mice) induced obesity. Based on these results, we propose that LEAP2 originating from the small intestine may represent a player in eCBome- and/or gut mBIome-mediated effects on food intake and energy metabolism.