Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Repeated binge-like eating episodes in female rats alter adenosine A2A and dopamine D2 receptor genes regulation in the brain reward system.

OBJECTIVE: Binge-eating disorder is an eating disorder characterized by recurrent binge-eating episodes, during which individuals consume excessive amounts of highly palatable food (HPF) in a short time. This study investigates the intricate relationship between repeated binge-eating episode and the transcriptional regulation of two key genes, adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R), in selected brain regions of rats. METHOD: Binge-like eating behavior on HPF was induced through the combination of food restrictions and frustration stress (15 min exposure to HPF without access to it) in female rats, compared to control rats subjected to only restriction or only stress or none of these two conditions. After chronic binge-eating episodes, nucleic acids were extracted from different brain regions, and gene expression levels were assessed through real-time quantitative PCR. The methylation pattern on genes' promoters was investigated using pyrosequencing. RESULTS: The analysis revealed A2AAR upregulation in the amygdala and in the ventral tegmental area (VTA), and D2R downregulation in the nucleus accumbens in binge-eating rats. Concurrently, site-specific DNA methylation alterations at gene promoters were identified in the VTA for A2AAR and in the amygdala and caudate putamen for D2R. DISCUSSION: The alterations on A2AAR and D2R genes regulation highlight the significance of epigenetic mechanisms in the etiology of binge-eating behavior, and underscore the potential for targeted therapeutic interventions, to prevent the development of this maladaptive feeding behavior. These findings provide valuable insights for future research in the field of eating disorders. PUBLIC SIGNIFICANCE: Using an animal model with face, construct, and predictive validity, in which cycles of food restriction and frustration stress evoke binge-eating behavior, we highlight the significance of epigenetic mechanisms on adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R) genes regulation. They could represent new potential targets for the pharmacological management of eating disorders characterized by this maladaptive feeding behavior.

Endocannabinoid System Regulation in Female Rats with Recurrent Episodes of Binge Eating.

Recurrent Binge Eating (BE) episodes characterize several eating disorders. Here, we attempted to reassemble a condition closer to BE disorder, and we analyzed whether recurrent episodes might evoke molecular alterations in the hypothalamus of rats. The hypothalamus is a brain region which is sensitive to stress and relevant in motivated behaviors, such as food intake. A well-characterized animal model of BE, in which a history of intermittent food restriction and stress induce binge-like palatable food consumption, was used to analyze the transcriptional regulation of the endocannabinoid system (ECS). We detected, in rats showing the BE behavior, an up-regulated gene expression of cannabinoid type-1 receptor (CB1), sn-1-specific diacylglycerol lipase, as well as fatty acid amide hydrolase (Faah) and monoacylglycerol lipase. A selective reduction in DNA methylation was also observed at the promoter of Faah, which is consistent with the changes in the gene expression. Moreover, BE behavior in rats was associated with an increase in anandamide (AEA) levels. Our findings support the relevant role of the ECS in the regulation of food intake in rats subjected to repeated BE episodes, and, in particular, on AEA signaling, acting via CB1 and FAAH modulation. Notably, the epigenetic regulation of the Faah gene might suggest this enzyme as a possible target for developing new therapeutical approaches.

On the Role of Central Type-1 Cannabinoid Receptor Gene Regulation in Food Intake and Eating Behaviors.

Different neuromodulatory systems are involved in long-term energy balance and body weight and, among these, evidence shows that the endocannabinoid system, in particular the activation of type-1 cannabinoid receptor, plays a key role. We here review current literature focusing on the role of the gene encoding type-1 cannabinoid receptors in the CNS and on the modulation of its expression by food intake and specific eating behaviors. We point out the importance to further investigate how environmental cues might have a role in the development of obesity as well as eating disorders through the transcriptional regulation of this gene in order to prevent or to treat these pathologies.

Epigenetic regulation of the cannabinoid receptor CB1 in an activity-based rat model of anorexia nervosa.

OBJECTIVE: Both environmental and genetic factors are known to contribute to the development of anorexia nervosa (AN), but the exact etiology remains poorly understood. Herein, we studied the transcriptional regulation of the endocannabinoid system, an interesting target for body weight maintenance and the control of food intake and energy balance. METHOD: We used two well-characterized animal models of AN: (a) the activity-based anorexia (ABA) model in which rats, housed with running wheels and subjected to daily food restriction, show reductions in body weight and increase in physical activity; (b) the genetic anx/anx mouse displaying the core features of AN: low food intake and emaciation. RESULTS: Among the evaluated endocannabinoid system components, we observed a selective and significant down-regulation of the gene encoding for the type 1 cannabinoid receptor (Cnr1) in ABA rats' hypothalamus and nucleus accumbens and, in the latter area, a consistent, significant and correlated increase in DNA methylation at the gene promoter. No changes were evident in the anx/anx mice except for a down-regulation of Cnr1, in the prefrontal cortex. DISCUSSION: Our findings support a possible role for Cnr1 in the ABA animal model of AN. In particular, its regulation in the nucleus accumbens appears to be triggered by environmental cues due to the consistent epigenetic modulation of the promoter. These data warrant further studies on Cnr1 regulation as a possible target for treatment of AN.

Transcriptional regulation of the endocannabinoid system in a rat model of binge-eating behavior reveals a selective modulation of the hypothalamic fatty acid amide hydrolase gene.

OBJECTIVE: Binge-eating episodes are recurrent and are defining features of several eating disorders. Thus binge-eating episodes might influence eating disorder development of which exact underlying mechanisms are still largely unknown. METHODS: Here we focused on the transcriptional regulation of the endocannabinoid system, a potent regulator of feeding behavior, in relevant rat brain regions, using a rat model in which a history of intermittent food restriction and a frustration stress induce binge-like palatable food consumption. RESULTS: We observed a selective down-regulation of fatty acid amide hydrolase (faah) gene expression in the hypothalamus of rats showing the binge-eating behavior with a consistent reduction in histone 3 acetylation at lysine 4 of the gene promoter. No relevant changes were detected for any other endocannabinoid system components in any brain regions under study, as well as for the other epigenetic mechanisms investigated (DNA methylation and histone 3 lysine 27 methylation) at the faah gene promoter. DISCUSSION: Our findings suggest that faah transcriptional regulation is a potential biomarker of binge-eating episodes, with a relevant role in the homeostatic regulation of food intake.

Gene expression profiling in adipose tissue of Sprague Dawley rats identifies olfactory receptor 984 as a potential obesity treatment target.

The aim of the study was to identify and functionally characterize novel candidate gene/s involved in the development of resistance to diet-induced obesity in rats. In a high-fat-diet (HFD) study of rats, we found subgroups which either developed resistance to HFD-induced obesity (DR) or showed an obesity-prone phenotype (DIO). Gene expression analysis in 10 samples (5 DIO vs 5 DR) was performed. The most promising gene, OR6C3 (orthologous with rat Olr984 and mouse Olfr788) was measured by qRT-PCR in paired samples of human visceral (Vis) and subcutaneous (SC) adipose tissue (AT) (n = 225) and in sub-fractions of adipocytes and cells of stromal vascular fraction. Gene expression analyses showed Olr984 with significantly reduced mRNA expression in DR rats. In the Vis AT of human samples we found an up-regulation of OR6C3 compared to SC AT, independent of gender, glucose tolerance or type 2 diabetes. We observed significantly lower levels of SC AT OR6C3 mRNA in subjects with obesity compared to those with normal-weight or overweight. OR6C3 is more expressed in SVF than in adipocytes. Olr984 could be a novel candidate gene related to diet-induced obesity in rats. Variation in human AT mRNA expression is related to obesity parameters and glucose homeostasis and linked to the regulatory role of insulin on the Olr984.

Potential for diagnosis versus therapy monitoring of attention deficit hyperactivity disorder: a new epigenetic biomarker interacting with both genotype and auto-immunity.

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.

Estrogenic suppression of binge-like eating elicited by cyclic food restriction and frustrative-nonreward stress in female rats.

Because binge eating and emotional eating vary through the menstrual cycle in human females, we investigated cyclic changes in binge-like eating in female rats and their control by estrogens. Binge-like eating was elicited by three cycles of 4 days of food restriction and 4 days of free feeding followed by a single frustrative nonreward-stress episode (15 min visual and olfactory exposure to a familiar palatable food) immediately before presentation of the palatable food. Intact rats showed binge-like eating during the diestrous and proestrous phases of the ovarian cycle, but not during the estrous (periovulatory) phase. Ovariectomized (OVX) rats not treated with estradiol (E2) displayed binge-like eating, whereas E2-treated OVX rats did not. The procedure did not increase signs of anxiety in an open-field test. OVX rats not treated with E2 that were subjected to food restriction and sacrificed immediately after frustrative nonreward had increased numbers of cells expressing phosphorylated extracellular signal-regulated kinases (ERK) in the central nucleus of the amygdala (CeA), paraventricular nucleus of hypothalamus (PVN), and dorsal and ventral bed nuclei of the stria terminalis (BNST) compared with nonrestricted or E2-treated rats. These data suggest that this female rat model is appropriate for mechanistic studies of some aspects of menstrual-cycle effects on emotional and binge eating in human females, that anxiety is not a sufficient cause of binge-like eating, and that the PVN, CeA, and BNST may contribute to information processing underlying binge-like eating.

Epigenetic regulation of nociceptin/orphanin FQ and corticotropin-releasing factor system genes in frustration stress-induced binge-like palatable food consumption.

Evidence suggests that binge eating may be caused by a unique interaction between dieting and stress. We developed a binge-eating model in which female rats with a history of intermittent food restriction show binge-like palatable food consumption after a 15-minute exposure to the sight of the palatable food (frustration stress). The aim of the present study was to investigate the regulation of the stress neurohormone corticotropin-releasing factor (CRF) system and of the nociceptin/orphanin FQ (N/OFQ) system genes in selective rat brain regions, using our animal model. Food restriction by itself seems to be responsible in the hypothalamus for the downregulation on messenger RNA levels of CRF-1 receptor, N/OFQ and its receptor (NOP). For the latter, this alteration might be due to selective histone modification changes. Instead, CRF gene appears to be upregulated in the hypothalamus as well as in the ventral tegmental area only when rats are food restricted and exposed to frustration stress, and, of relevance, these changes appear to be due to a reduction in DNA methylation at gene promoters. Moreover, also CRF-1 receptor gene resulted to be differentially regulated in these two brain regions. Epigenetic changes may be viewed as adaptive mechanisms to environmental perturbations concurring to facilitate food consumption in adverse conditions, that is, in this study, under food restriction and stressful conditions. Our data on N/OFQ and CRF signaling provide insight on the use of this binge-eating model for the study of epigenetic modifications in controlled genetic and environmental backgrounds.

Dopamine and Serotonin Transporter Genes Regulation in Highly Sensitive Individuals during Stressful Conditions: A Focus on Genetics and Epigenetics.

Background: Coping with stress is essential for mental well-being and can be critical for highly sensitive individuals, characterized by a deeper perception and processing of stimuli. So far, the molecular bases characterizing high-sensitivity traits have not been completely investigated and gene × environment interactions might play a key role in making some people more susceptible than others. Methods: In this study, 104 young adult university students, subjects that might face overwhelming experiences more than others, were evaluated for the genetics and epigenetics of dopamine (DAT1) and serotonin (SERT) transporter genes, in addition to the expression of miR-132, miR-491, miR-16, and miR-135. Results: We found an increase in DNA methylation at one specific CpG site at DAT1 5'UTR in highly sensitive students reporting high levels of perceived stress when compared to those less sensitive and/or less stressed. Moreover, considering DAT1 VNTR at 3'UTR, we observed that this effect was even more pronounced in university students having the 9/9 genotype when compared to those with the 9/10 genotype. These data are corroborated by the higher levels of miR-491, targeting DAT1, in highly sensitive subjects with high levels of perceived stress. SERT gene DNA methylation at one specific CpG site was reported to instead be higher in subjects reporting lower perceived stress when compared to more stressed subjects. Consistently, miR-135 expression, regulating SERT, was lower in subjects with higher perceived stress. Conclusions: We here suggest that the correlation of DAT1 and SERT genetic and epigenetic data with the analysis of stress and sensitivity might be useful to suggest possible biomarkers to monitor mental health wellness in vulnerable subjects.

Endocannabinoids stimulate human melanogenesis via type-1 cannabinoid receptor.

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 µM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 µM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

The faah gene is the first direct target of estrogen in the testis: role of histone demethylase LSD1.

Estrogen (E(2)) regulates spermatogenesis, yet its direct target genes have not been identified in the testis. Here, we cloned the proximal 5' flanking region of the mouse fatty acid amide hydrolase (faah) gene upstream of the luciferase reporter gene, and demonstrated its promoter activity and E(2) inducibility in primary mouse Sertoli cells. Specific mutations in the E(2) response elements (ERE) of the faah gene showed that two proximal ERE sequences (ERE2/3) are essential for E(2)-induced transcription, and chromatin immunoprecipitation experiments showed that E(2) induced estrogen receptor ß binding at ERE2/3 sites in the faah promoter in vivo. Moreover, the histone demethylase LSD1 was found to be associated with ERE2/3 sites and to play a role in mediating E(2) induction of FAAH expression. E(2) induced epigenetic modifications at the faah proximal promoter compatible with transcriptional activation by remarkably decreasing methylation of both DNA at CpG site and histone H3 at lysine 9. Finally, FAAH silencing abolished E(2) protection against apoptosis induced by the FAAH substrate anandamide. Taken together, our results identify FAAH as the first direct target of E(2).

Assessing Gene Expression of the Endocannabinoid System Components by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction.

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), a major development in PCR technology, is a powerful and sensitive gene analysis technique that has revolutionized the field of gene expression assays. In this chapter, we describe in detail RNA extraction, reverse transcription (RT), and relative quantification of genes forming the endocannabinoid system in different experimental models. In particular, we here provide specific and sensitive assays to be used to assess gene expression of the endocannabinoid system components in mouse, rat, or human samples.

DNA Methylation Analysis of Cnr1 Gene Promoter.

DNA methylation pattern could be considered a biomarker to be exploited for the study and management of several human diseases. In this chapter, detailed protocols are provided for two experimental approaches used for quantitative methylation analysis of bisulfite converted DNA: methylation-specific PCR (MSP) and pyrosequencing.

Methylation Dynamics on 5'-UTR of DAT1 Gene as a Bio-Marker to Recognize Therapy Success in ADHD Children.

Attention-deficit/hyperactivity disorder (ADHD), a neuropsychiatric condition characterized by inattention, hyperactivity, and impulsivity, afflicts 5% of children worldwide. Each ADHD patient presents with individual cognitive and motivational peculiarities. Furthermore, choice of appropriate therapy is still up to clinicians, who express somewhat qualitative advice on whether a child is being successfully cured or not: it would be more appropriate to use an objective biomarker to indicate whether a treatment led to benefits or not. The aim of our work is to search for such clinical biomarkers. We recruited 60 ADHD kids; psychopathological scales were administered at recruitment and after six weeks of therapy. Out of such a cohort of ADHD children, we rigorously extracted two specific subgroups; regardless of the initial severity of their disease, we compared those who obtained the largest improvement (ΔCGAS > 5) vs. those who were still characterized by a severe condition (CGAS < 40). After such a therapy, methylation levels of DNA extracted from buccal swabs were measured in the 5'-UTR of the DAT1 gene. CpGs 3 and 5 displayed, in relation to the other CpGs, a particular symmetrical pattern; for "improving" ADHD children, they were methylated together with CpG 2 and CpG 6; instead, for "severe" ADHD children, they accompanied a methylated CpG 1. These specific patterns of methylation could be used as objective molecular biomarkers of successful cures, establishing if a certain therapy is akin to a given patient (personalized medicine). Present data support the use of post-therapy molecular data obtained with non-invasive techniques.

A possible role for G-quadruplexes formation and DNA methylation at IMOOD gene promoter in Obsessive Compulsive Disorder.

Obsessive Compulsive Disorder (OCD) is a mental health condition still classified and diagnosed with subjective interview-based assessments and which molecular clues have not completely been elucidated. We have recently identified a new regulator of anxiety and OCD-like behavior called Immuno-moodulin (IMOOD) and, here, we report that IMOOD gene promoter is differentially methylated in OCD subjects when compared to genomic material collected from healthy controls and this alteration is significantly correlated with the increased expression of the gene in OCD. We also demonstrated that IMOOD promoter can form G-quadruplexes and we suggest that, in homeostatic conditions, these structures could evoke DNA-methylation silencing the gene, whereas in pathological conditions, like OCD, could induce gene expression making the promoter more accessible to transcriptional factors. We here thus further suggest IMOOD as a new biomarker for OCD and also hypothesize new mechanisms of gene regulation.

Methylation patterns within 5'-UTR of DAT1 gene as a function of allelic 3'-UTR variants and their maternal or paternal origin: May these affect the psychopathological phenotypes in children? An explorative study.

Psychopathological symptoms such as depression/anxiety vs attention or aggression problems, in children, have been associated to altered expression of the DAT1/SLC6A3 gene. Inheriting specific 9- or 10-repeat VNTR alleles could modify the pattern of methylation in the CpGs islands at the 5'-UTR of the DAT1 gene. Through accurate recruitment at primary schools, we ended up with four subgroups of children: 9/9 and 10/10 homozygous; 9/10 heterozygous born from 9/10 mothers and 10/10 fathers (called heM); 9/10 heterozygous born from 10/10 mothers and 9/10 fathers (called heF). (Epi)genetical changes were found to be in relation to internalizing and externalizing symptoms: compared to other genotypes, our 9/9 children exhibited mainly internalizing symptoms, while 10/10 genotype was previously associated with ADHD severity. We found that 10/10 children bear 5'-UTR motifs showing a CpGs 1-2-3-5 unity with anticorrelated CpG 6, while 9/9 children showed rather a demethylated CpG 1 linked to demethylated CpG 6. We found two different patterns between heMs and heFs: a feature of heM children is in CpGs 1-3 methylated pattern with CpGs 2, 5 and 6 demethylated together, supporting a "split" unitary destiny. Within the heF children, the status for CpGs 3 + 6 remained opposite, yet pattern of (de)methylation was not well defined. The prevailing one between inherited parental alleles may somewhat influence the motif destiny of heterozigous children. Present work aimed to identify novel epigenetic biomarkers, to be exploited as fairly indicators of children's psychopathological vulnerability.

Regulation of oxytocin receptor gene expression in obsessive-compulsive disorder: a possible role for the microbiota-host epigenetic axis.

BACKGROUND: Obsessive-compulsive disorder (OCD) is a prevalent and severe clinical condition. Robust evidence suggests a gene-environment interplay in its etiopathogenesis, yet the underlying molecular clues remain only partially understood. In order to further deepen our understanding of OCD, it is essential to ascertain how genes interact with environmental risk factors, a cross-talk that is thought to be mediated by epigenetic mechanisms. The human microbiota may be a key player, because bacterial metabolites can act as epigenetic modulators. We analyzed, in the blood and saliva of OCD subjects and healthy controls, the transcriptional regulation of the oxytocin receptor gene and, in saliva, also the different levels of major phyla. We also investigated the same molecular mechanisms in specific brain regions of socially isolated rats showing stereotyped behaviors reminiscent of OCD as well as short chain fatty acid levels in the feces of rats. RESULTS: Higher levels of oxytocin receptor gene DNA methylation, inversely correlated with gene expression, were observed in the blood as well as saliva of OCD subjects when compared to controls. Moreover, Actinobacteria also resulted higher in OCD and directly correlated with oxytocin receptor gene epigenetic alterations. The same pattern of changes was present in the prefrontal cortex of socially-isolated rats, where also altered levels of fecal butyrate were observed at the beginning of the isolation procedure. CONCLUSIONS: This is the first demonstration of an interplay between microbiota modulation and epigenetic regulation of gene expression in OCD, opening new avenues for the understanding of disease trajectories and for the development of new therapeutic strategies.

Endocannabinoid signaling and epidermal differentiation.

Endocannabinoids represent a class of endogenous lipid mediators, that are involved in various biological processes, both centrally and peripherally. The prototype member of this group of compounds, anandamide, regulates cell growth, differentiation and death; this holds true also in the skin, that is the largest organ of the body constantly exposed to physical, chemical, bacterial and fungal challenges. The epidermis is a keratinized multistratified epithelium that functions as a barrier to protect the organism from dehydration, mechanical trauma, and microbial insults, and epidermal differentiation represents one of the best characterized mechanisms of cell specialization. In this review, we shall summarize current knowledge about the main members of the so-called "endocannabinoid system (ECS)", in order to put in a better perspective the manifold roles that they play in skin pathophysiology. In particular, we shall discuss some aspects of the molecular regulation by endocannabinoids of proliferation and terminal differentiation ("cornification") of mammalian epidermis, showing that ECS is finely regulated by, and can interfere with, the differentiation program. In addition, we shall review evidence demonstrating that disruption of this fine regulation might cause different skin diseases, such as acne, seborrhoea, allergic dermatitis, itch, psoriasis and hair follicle regression (catagen), making of ECS an attractive target for therapeutic intervention.