The etiology of bipolar disorder (BD) is still poorly understood, involving genetic and epigenetic mechanisms as well as environmental contributions. This study aimed to investigate the degree of DNA methylation at the promoter region of the brain-derived neurotrophic factor (BDNF) gene, as one of the candidate genes associated with major psychoses, in peripheral blood mononuclear cells isolated from 94 patients with BD (BD I ¼ 49, BD II ¼ 45) and 52 healthy controls. A significant BDNF gene expression downregulation was observed in BD II 0.53±0.11%; Po0.05), but not in BD I (1.13±0.19%) patients compared with controls (CONT: 1±0.2%). Consistently, an hypermethylation of the BDNF promoter region was specifically found in BD II patients (CONT: 24.0±2.1%; BDI: 20.4±1.7%; BDII: 33.3 ± 3.5%, Po0.05). Of note, higher levels of DNA methylation were observed in BD subjects on pharmacological treatment with mood stabilizers plus antidepressants (34.6 ± 4.2%, predominantly BD II) compared with those exclusively on mood-stabilizing agents (21.7 ± 1.8%; Po0.01, predominantly BD I). Moreover, among the different pharmacological therapies, lithium (20.1 ± 3.8%, Po0.05) and valproate (23.6 ± 2.9%, Po0.05) were associated with a significant reduction of DNA methylation compared with other drugs (35.6 ± 4.6%). Present findings suggest selective changes in DNA methylation of BDNF promoter in subjects with BD type II and highlight the importance of epigenetic factors in mediating the onset and/or susceptibility to BD, providing new insight into the mechanisms of gene expression. Moreover, they shed light on possible mechanisms of action of mood-stabilizing compounds vs antidepressants in the treatment of BD, pointing out that BDNF regulation might be a key target for their effects.

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Gilbert syndrome is a mild hereditary unconjugated hyperbilirubinemia caused by mutations in the bilirubin UDP-glucuronosyltransferase gene (UGT1A1). The mutation, A(TA)7TAA, is thought to be the sole cause of the syndrome in Caucasians, but an enhancer polymorphism (T-3279G) that lowers transcriptional activity has recently been reported. We have tested the linkage of the two mutations in 11 Caucasians and 12 Japanese patients who were homozygous for A(TA)7TAA. All 23 patients were also homozygous for T-3279G, indicating that T-3279G and A(TA)7TAA were linked. The decrease in transcription caused by both mutations together may be essential to the syndrome.

BACKGROUND AND PURPOSEEndocannabinoid signalling has been shown to have a role in the control of epidermal physiology, whereby anandamide is able to regulate the expression of skin differentiation genes through DNA methylation. Here, we investigated the possible epigenetic regulation of these genes by several phytocannabinoids, plant-derived cannabinoids that have the potential to be novel therapeutics for various human diseases. EXPERIMENTAL APPROACHThe effects of cannabidiol, cannabigerol and cannabidivarin on the expression of skin differentiation genes keratins 1 and 10, involucrin and transglutaminase 5, as well as on DNA methylation of keratin 10 gene, were investigated in human keratinocytes (HaCaT cells). The effects of these phytocannabinoids on global DNA methylation and the activity and expression of four major DNA methyltransferases (DNMT1, 3a, 3b and 3L) were also examined. KEY RESULTSCannabidiol and cannabigerol significantly reduced the expression of all the genes tested in differentiated HaCaT cells, by increasing DNA methylation of keratin 10 gene, but cannabidivarin was ineffective. Remarkably, cannabidiol reduced keratin 10 mRNA through a type-1 cannabinoid (CB1) receptor-dependent mechanism, whereas cannabigerol did not affect either CB1 or CB2 receptors of HaCaT cells. In addition, cannabidiol, but not cannabigerol, increased global DNA methylation levels by selectively enhancing DNMT1 expression, without affecting DNMT 3a, 3b or 3L. CONCLUSIONS AND IMPLICATIONSThese findings show that the phytocannabinoids cannabidiol and cannabigerol are transcriptional repressors that can control cell proliferation and differentiation. This indicates that they (especially cannabidiol) have the potential to be lead compounds for the development of novel therapeutics for skin diseases. AbbreviationsAEA, anandamide; 2-AG, 2-arachidonoylglycerol; CB1, cannabinoid receptor type I; CB2, cannabinoid receptor type II; CBD, cannabidiol; CBDV, cannabidivarin; CBG, cannabigerol; CPZ, capsazepine; DAGL, diacylglycerol lipase; DNMT, DNA methyltransferase; eCBs, endocannabinoids; ECS, endocannabinoid system; FAAH, fatty acid amide hydrolase; K1, keratin 1; K10, keratin 10; NAPE-PLD, N-acyl-phosphatidylethanolamines-specific phospholipase D; NHEK, normal human epidermal keratinocytes; MAGL, monoacylglycerol lipase; SR141716, N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole carboxamide; SR144528, N-[(1)-endo-1,3,3-trimethy-1-bicyclo [2.2.1]-heptan-2-yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide; TGase 5, transglutaminase 5; THC, Δ 9-tetrahydrocannbinol; TPA, 12-O-tetradecanoylphorbol 13-acetate; TRPV1, transient receptor potential vanilloid 1 IntroductionEndocannabinoids (eCBs) are lipid mediators derived from membrane precursors and are involved in multiple regulatory functions, both in health and disease (Di Marzo and Petrosino, 2007). The two most important eCBs are N-arachidonylethanolamine ('anandamide', AEA) and 2-arachidonoylglycerol (2-AG) that elicit their a...

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In view of the need for easily accessible biomarkers, we evaluated in ADHD children the epigenetic status of the 5'-untranslated region (UTR) in the SLC6A3 gene, coding for human dopamine transporter (DAT). We analysed buccal swabs and sera from 30 children who met DSM-IV-TR criteria for ADHD, assigned to treatment according to severity. Methylation levels at six-selected CpG sites (among which, a CGGCGGCGG and a CGCG motif), alone or in combination with serum titers in auto-antibodies against dopamine transporter (DAT aAbs), were analysed for correlation with CGAS scores (by clinicians) and Conners' scales (by parents), collected at recruitment and after 6 weeks. In addition, we characterized the DAT genotype, i.e., the variable number tandem repeat (VNTR) polymorphisms at the 3'-UTR of the gene. DAT methylation levels were greatly reduced in ADHD patients compared to control, healthy children. Within patients carrying at least one DAT 9 allele (DAT 9/x), methylation at positions CpG2 and/or CpG6 correlated with recovery, as evident from delta-CGAS scores as well as delta Conners' scales ('inattentive' and 'hyperactive' subscales). Moreover, hypermethylation at CpG1 position denoted severity, specifically for those patients carrying a DAT 10/10 genotype. Intriguingly, high serum DAT-aAbs titers appeared to corroborate indications from high CpG1 versus high CpG2/CpG6 levels, likewise denoting severity versus recovery in DAT 10/10 versus 9/x patients, respectively. These profiles suggest that DAT 5'UTR epigenetics plus serum aAbs can serve as suitable biomarkers, to confirm ADHD diagnosis and/or to predict the efficacy of treatment.

An increasing perspective conceptualizes obesity and overeating as disorders related to addictive-like processes that could share common neurobiological mechanisms. In the present study, we aimed at validating an animal model of eating addictive-like behavior in mice, based on the DSM-5 substance use disorder criteria, using operant conditioning maintained by highly palatable chocolate-flavored pellets. For this purpose, we evaluated persistence of food-seeking during a period of non-availability of food, motivation for food, and perseverance of responding when the reward was associated with a punishment. This model has allowed identifying extreme subpopulations of mice related to addictive-like behavior. We investigated in these subpopulations the epigenetic and proteomic changes. A significant decrease in DNA methylation of CNR1 gene promoter was revealed in the prefrontal cortex of addict-like mice, which was associated with an upregulation of CB1 protein expression in the same brain area. The pharmacological blockade (rimonabant 3 mg/kg; i.p.) of CB1 receptor during the late training period reduced the percentage of mice that accomplished addiction criteria, which is in agreement with the reduced performance of CB1 knockout mice in this operant training. Proteomic studies have identified proteins differentially expressed in mice vulnerable or not to addictive-like behavior in the hippocampus, striatum, and prefrontal cortex. These changes included proteins involved in impulsivity-like behavior, synaptic plasticity, and cannabinoid signaling modulation, such as alpha-synuclein, phosphatase 1-alpha, doublecortin-like kinase 2, and diacylglycerol kinase zeta, and were validated by immunoblotting. This model provides an excellent tool to investigate the neurobiological substrate underlying the vulnerability to develop eating addictive-like behavior.

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