Palmitoylethanolamide counteracts substance P-induced mast cell activation in vitro by stimulating diacylglycerol lipase activity.

BACKGROUND: Palmitoylethanolamide (PEA) is a pleiotropic endogenous lipid mediator currently used as a "dietary food for special medical purposes" against neuropathic pain and neuro-inflammatory conditions. Several mechanisms underlie PEA actions, among which the "entourage" effect, consisting of PEA potentiation of endocannabinoid signaling at either cannabinoid receptors or transient receptor potential vanilloid type-1 (TRPV1) channels. Here, we report novel molecular mechanisms through which PEA controls mast cell degranulation and substance P (SP)-induced histamine release in rat basophilic leukemia (RBL-2H3) cells, a mast cell model. METHODS: RBL-2H3 cells stimulated with SP were treated with PEA in the presence and absence of a cannabinoid type-2 (CB2) receptor antagonist (AM630), or a diacylglycerol lipase (DAGL) enzyme inhibitor (OMDM188) to inhibit the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The release of histamine was measured by ELISA and ß-hexosaminidase release and toluidine blue staining were used as indices of degranulation. 2-AG levels were measured by LC-MS. The mRNA expression of proposed PEA targets (Cnr1, Cnr2, Trpv1, Ppara and Gpr55), and of PEA and endocannabinoid biosynthetic (Napepld, Dagla and Daglb) and catabolic (Faah, Naaa and Mgl) enzymes were also measured. The effects of PEA on the activity of DAGL-α or -ß enzymes were assessed in COS-7 cells overexpressing the human recombinant enzyme or in RBL-2H3 cells, respectively. RESULTS: SP increased the number of degranulated RBL-2H3 cells and triggered the release of histamine. PEA counteracted these effects in a manner antagonized by AM630. PEA concomitantly increased the levels of 2-AG in SP-stimulated RBL-2H3 cells, and this effect was reversed by OMDM188. PEA significantly stimulated DAGL-α and -ß activity and, consequently, 2-AG biosynthesis in cell-free systems. Co-treatment with PEA and 2-AG at per se ineffective concentrations downmodulated SP-induced release of histamine and degranulation, and this effect was reversed by OMDM188. CONCLUSIONS: Activation of CB2 underlies the inhibitory effects on SP-induced RBL-2H3 cell degranulation by PEA alone. We demonstrate for the first time that the effects in RBL-2H3 cells of PEA are due to the stimulation of 2-AG biosynthesis by DAGLs.

Impaired brain endocannabinoid tone in the activity-based model of anorexia nervosa.

OBJECTIVE: Despite the growing knowledge on the functional relationship between an altered endocannabinoid (eCB) system and development of anorexia nervosa (AN), to date no studies have investigated the central eCB tone in the activity-based anorexia (ABA) model that reproduces key aspects of human AN. METHOD: We measured levels of two major eCBs, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), those of two eCB-related lipids, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and the cannabinoid type-1 receptor (CB1R) density in the brain of female ABA rats, focusing on areas involved in homeostatic and rewarding-related regulation of feeding behavior (i.e., prefrontal cortex, nucleus accumbens, caudato putamen, amygdala, hippocampus and hypothalamus). Analysis was carried out also at the end of recovery from the ABA condition. RESULTS: At the end of the ABA induction phase, 2-AG was significantly decreased in ABA rats in different brain areas but not in the caudato putamen. No changes were detected in AEA levels in any region, whereas the levels of OEA and PEA were decreased exclusively in the hippocampus and hypothalamus. Furthermore, CB1R density was decreased in the dentate gyrus of hippocampus and in the lateral hypothalamus. After recovery, both 2-AG levels and CB1R density were partially normalized in some areas. In contrast, AEA levels became markedly reduced in all the analyzed areas. DISCUSSION: These data demonstrate an altered brain eCB tone in ABA rats, further supporting the involvement of an impaired eCB system in AN pathophysiology that may contribute to the maintenance of some symptomatic aspects of the disease.

Different Routes to Inhibit Fatty Acid Amide Hydrolase: Do All Roads Lead to the Same Place?

There is robust evidence indicating that enhancing the endocannabinoid (eCB) tone has therapeutic potential in several brain disorders. The inhibition of eCBs degradation by fatty acid amide hydrolase (FAAH) blockade, is the best-known option to increase N-acyl-ethanolamines-(NAEs)-mediated signaling. Here, we investigated the hypothesis that intranasal delivery is an effective route for different FAAH inhibitors, such as URB597 and PF-04457845. URB597 and PF-04457845 were subchronically administered in C57BL/6 male mice every other day for 20 days for overall 10 drug treatment, and compared for their ability to inhibit FAAH activity by the way of three different routes of administration: intranasal (i.n.), intraperitoneal (i.p.) and oral (p.o.). Lastly, we compared the efficacy of the three routes in terms of URB597-induced increase of NAEs levels in liver and in different brain areas. Results: We show that PF-04457845 potently inhibits FAAH regardless the route selected, and that URB597 was less effective in the brain after p.o. administration while reached similar effects by i.n. and i.p. routes. Intranasal URB597 delivery always increased NAEs levels in brain areas, whereas a parallel increase was not observed in the liver. By showing the efficacy of intranasal FAAH inhibition, we provide evidence that nose-to-brain delivery is a suitable alternative to enhance brain eCB tone for the treatment of neurodegenerative disorders and improve patients' compliance.

Anti-inflammatory Properties of Cannabidiol, a Nonpsychotropic Cannabinoid, in Experimental Allergic Contact Dermatitis.

Phytocannabinoids modulate inflammatory responses by regulating the production of cytokines in several experimental models of inflammation. Cannabinoid type-2 (CB2) receptor activation was shown to reduce the production of the monocyte chemotactic protein-2 (MCP-2) chemokine in polyinosinic-polycytidylic acid [poly-(I:C)]-stimulated human keratinocyte (HaCaT) cells, an in vitro model of allergic contact dermatitis (ACD). We investigated if nonpsychotropic cannabinoids, such as cannabidiol (CBD), produced similar effects in this experimental model of ACD. HaCaT cells were stimulated with poly-(I:C), and the release of chemokines and cytokines was measured in the presence of CBD or other phytocannabinoids (such as cannabidiol acid, cannabidivarin, cannabidivarinic acid, cannabichromene, cannabigerol, cannabigerolic acid, cannabigevarin, tetrahydrocannabivarin, and tetrahydrocannabivarinic acid) and antagonists of CB1, CB2, or transient receptor potential vanilloid type-1 (TRPV1) receptors. HaCaT cell viability following phytocannabinoid treatment was also measured. The cellular levels of endocannabinoids [anandamide (AEA), 2-arachidonoylglycerol] and related molecules (palmitoylethanolamide, oleoylethanolamide) were quantified in poly-(I:C)-stimulated HaCaT cells treated with CBD. We show that in poly-(I:C)-stimulated HaCaT cells, CBD elevates the levels of AEA and dose-dependently inhibits poly-(I:C)-induced release of MCP-2, interleukin-6 (IL-6), IL-8, and tumor necrosis factor-α in a manner reversed by CB2 and TRPV1 antagonists 6-iodopravadoline (AM630) and 5'-iodio-resiniferatoxin (I-RTX), respectively, with no cytotoxic effect. This is the first demonstration of the anti-inflammatory properties of CBD in an experimental model of ACD.

Characterization of endocannabinoids and related acylethanolamides in the synovial fluid of dogs with osteoarthritis: a pilot study.

BACKGROUND: Cannabis-based drugs have been shown to be effective in inflammatory diseases. A number of endocannabinoids including N- arachidonoylethanolamide (anandamide, AEA) and 2-arachidonyl glycerol (2-AG) with activity at the cannabinoid receptors (CBR) CBR1 and CBR2, have been identified. Other structurally related endogenous fatty acid compounds such as oleoylethanolamide (OEA) and palmitoyl ethanolamide (PEA) have been identified in biological tissues. These compounds do not bind to CBR but might be involved in facilitating the actions of directly acting endocannabinoids and thus are commonly termed "entourage" compounds due to their ability to modulate the endocannabinoid system. The aim of this study was to evaluate the presence of endocannabinoids and entourage compounds in the synovial fluid of dogs with osteoarthritis subjected to arthrotomy of the knee joint. Cytokines and cytology were studied as well. RESULTS: AEA, 2-AG, OEA and PEA were all present in the synovial fluid of arthritic knees and in the contralateral joints; in addition, a significant increase of OEA and 2AG levels were noted in SF from OA knees when compared to the contralateral joints. CONCLUSION: The identification and quantification of endocannabinoids and entourage compounds levels in synovial fluids from dogs with OA of the knee is reported for the first time. Our data are instrumental for future studies involving a greater number of dogs. Cannabinoids represent an emerging and innovative pharmacological tool for the treatment of OA and further studies are warranted to evaluate the effectiveness of cannabinoids in veterinary medicine.

Diacerein is a potent and selective inhibitor of palmitoylethanolamide inactivation with analgesic activity in a rat model of acute inflammatory pain.

Palmitoylethanolamide (PEA) is produced by mammalian cells from its biosynthetic precursor, N-palmitoyl-phosphatidyl-ethanolamine, and inactivated by enzymatic hydrolysis to palmitic acid and ethanolamine. Apart from fatty acid amide hydrolase (FAAH), the N-acylethanolamine-hydrolyzing acid amidase (NAAA), a lysosomal enzyme, was also shown to catalyze the hydrolysis of PEA and to limit its analgesic and anti-inflammatory action. Here we report the finding of a new potential inhibitor of NAAA, EPT4900 (4,5-diacetyloxy-9,10-dioxo-anthracene-2-carboxylic acid, diacerein). EPT4900 exhibited a high inhibitory activity on human recombinant NAAA over-expressed in HEK293 cells (HEK-NAAA cells). EPT4900 selectively increased the levels of PEA in intact HEK-NAAA cells, and inhibited inflammation as well as hyperalgesia in rats treated with an intraplantar injection of carrageenan. This latter effect was accompanied by elevation of PEA endogenous levels in the paw skin.

A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

The dual blocker of FAAH/TRPV1 N-arachidonoylserotonin reverses the behavioral despair induced by stress in rats and modulates the HPA-axis.

In recent years, several studies have explored the involvement of the deregulation of the hypothalamus-pituitary-adrenal (HPA) axis in the pathophysiology of stress-related disorders. HPA hyper-activation as a consequence of acute/chronic stress has been found to play a major role in the neurobiological changes that are responsible for the onset of such states. Currently available medications for depression, one of the most relevant stress-related disorders, present several limitations, including a time lag for treatment response and low rates of efficacy. N-Arachidonoylserotonin (AA-5-HT), a dual blocker at fatty acid amide hydrolase (FAAH, the enzyme responsible for the inactivation of the endocannabinoid anandamide) and transient receptor potential vanilloid type-1 channel (TRPV1), produces anxiolytic-like effects in mice. The present study was designed to assess the capability of AA-5-HT to reverse the behavioral despair following exposure to stress in rats and the role of the HPA-axis. Behavioral tasks were performed, and corticosterone and endocannabinoid (anandamide and 2-arachidonoylglycerol) levels were measured in selected brain areas critically involved in the pathophysiology of stress-related disorders (medial PFC and hippocampus) under basal and stress conditions, and in response to treatment with AA-5-HT. Our data show that AA-5-HT reverses the rat behavioral despair in the forced swim test under stress conditions, and this effect is associated with the normalization of the HPA-axis deregulation that follows stress application and only in part with elevation of anandamide levels. Blockade of FAAH and TRPV1 may thus represent a novel target to design novel therapeutic strategies for the treatment of stress-related disorders.

Impact of omega-6 polyunsaturated fatty acid supplementation and γ-aminobutyric acid on astrogliogenesis through the endocannabinoid system.

Neural stem cells express cannabinoid CB1 and CB2 receptors and the enzymes for the biosynthesis and metabolism of endocannabinoids (eCBs). Here we have studied the role of neural stem cell-derived eCBs as autonomous regulatory factors during differentiation. First, we examined the effect of an indirect eCB precursor linoleic acid (LA), a major dietary omega-6 fatty acid, on the eCB system in neural stem/progenitor cells (NSPCs) cultured in DMEM/F12 supplemented with N2 (N2/DF) as monolayer cells. LA upregulated eCB system-related genes and 2-arachidonoylglycerol (2-AG), but not anandamide (AEA), levels. Glial fibrillary acidic protein (GFAP) was significantly higher under LA-enriched conditions, and this effect was inhibited by the cannabinoid receptor type-1 (CB1) antagonist AM251. Second, the levels of AEA and 2-AG, as well as of the mRNA of eCB system-related genes, were measured in NSPCs after γ-aminobutyric acid (GABA) treatment. GABA upregulated AEA levels significantly in LA-enriched cultures and increased the mRNA expression of the 2-AG-degrading enzyme monoacylglycerol lipase. These effects of GABA were reproduced under culture conditions using neurobasal media supplemented with B27, which is commonly used for neurosphere culture. GABA stimulated astroglial differentiation in this medium as indicated by increased GFAP levels. This effect was abolished by AM251, suggesting the involvement of AEA and CB1 in GABA-induced astrogliogenesis. This study highlights the importance of eCB biosynthesis and CB1 signalling in the autonomous regulation of NSPCs and the influence of the eCB system on astrogliogenesis induced by nutritional factors or neurotransmitters, such as LA and GABA.

Piperazinyl carbamate fatty acid amide hydrolase inhibitors and transient receptor potential channel modulators as "dual-target" analgesics.

We showed previously that inhibiting fatty acid amide hydrolase (FAAH), an endocannabinoid degrading enzyme, and transient receptor potential vanilloid type-1 (TRPV1) channels with the same molecule, the naturally occurring N-arachidonoyl-serotonin (AA-5-HT), produces more efficacious anti-nociceptive and anti-hyperalgesic actions than the targeting of FAAH or TRPV1 alone. We also reported the synthesis of some piperazinyl carbamates as "dual" FAAH inhibitors and either antagonists at TRPV1 or agonists/desensitizers of the transient receptor potential ankyrin type-1 (TRPA1) cannel, another target for analgesic drugs. We investigated here if two such compounds, the FAAH/TRPV1 blocker OMDM198 and the FAAH inhibitor/TRPA1 agonist, OMDM202, exert anti-nociceptive actions in the formalin test of pain in mice, and through what mechanism. Both compounds inhibited the second phase of the response to formalin, the effect being maximal at 3 mg/kg, i.p. Antagonism of CB1 or CB2 receptors with AM251 or AM630 (1 mg/kg, i.p.), respectively, reversed this effect. A TRPV1 agonist, palvanil (0.1 mg/kg, i.p.), also reversed the analgesic effect of OMDM198. OMDM202 action was also antagonized by a per se inactive dose of the selective TRPA1 blocker, AP-18 (0.05 mg/kg, i.p.), but not by a TRPV1 antagonist. AP-18 at higher doses (0.1-0.2 mg/kg) inhibited both the first and second phase of the formalin response. The effects of OMDM198 and OMDM202 were accompanied by elevation of anandamide levels in the spinal cord. OMDM198 (0.1-5.0 mg/kg, i.p.) also reversed carrageenan-induced oedema and thermal hyperalgesia in mice with efficacy similar to that of AA-5-HT. These data suggest that "dual" fatty acid amide hydrolase and transient receptor potential channel modulators should be clinically evaluated as novel analgesics.

First Evidence of the Protective Effects of 2-Pentadecyl-2-Oxazoline (PEA-OXA) in In Vitro Models of Acute Lung Injury.

Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disorder and a complication of SARS-CoV-2 infection. In patients with severe SARS-CoV-2 infection, the transition to ARDS is principally due to the occurrence of a cytokine storm and an exacerbated inflammatory response. The effectiveness of ultra-micronized palmitoylethanolamide (PEA-um) during the earliest stage of COVID-19 has already been suggested. In this study, we evaluated its protective effects as well as the effectiveness of its congener, 2-pentadecyl-2-oxazoline (PEA-OXA), using in vitro models of acute lung injury. In detail, human lung epithelial cells (A549) activated by polyinosinic-polycytidylic acid (poly-(I:C)) or Transforming Growth Factor-beta (TGF-ß) were treated with PEA-OXA or PEA. The release of IL-6 and the appearance of Epithelial-Mesenchymal Transition (EMT) were measured by ELISA and immunofluorescence assays, respectively. A possible mechanism of action for PEA-OXA and PEA was also investigated. Our results showed that both PEA-OXA and PEA were able to counteract poly-(I:C)-induced IL-6 release, as well as to revert TGF-ß-induced EMT. In addition, PEA was able to produce an "entourage" effect on the levels of the two endocannabinoids AEA and 2-AG, while PEA-OXA only increased PEA endogenous levels, in poly-(I:C)-stimulated A549 cells. These results evidence for the first time the superiority of PEA-OXA over PEA in exerting protective effects and point to PEA-OXA as a new promising candidate in the management of acute lung injury.

The endocannabinoidome mediator N-oleoylglycine is a novel protective agent against 1-methyl-4-phenyl-pyridinium-induced neurotoxicity.

N-oleoylglycine (OlGly) is a lipid mediator that belongs to the expanded version of the endocannabinoid (eCB) system, the endocannabinoidome (eCBome), which has recently gained increasing attention from the scientific community for its protective effects in a mouse model of mild traumatic brain injury. However, the effects of OlGly on cellular models of Parkinson's disease (PD) have not yet been investigated, whilst other lipoaminoacids have been reported to have beneficial effects. Moreover, the protective effects of OlGly seem to be mediated by direct activation of proliferator-activated receptor alpha (PPARα), which has already been investigated as a therapeutic target for PD. Therefore, this study aims to investigate the possible protective effects of OlGly in an in vitro model obtained by treating the neuroblastoma cell line, SH-SY5Y (both differentiated and not) with 1-methyl-4-phenyl-pyridinium (MPP+), which mimics some cellular aspects of a PD-like phenotype, in the presence or absence of the PPARα antagonist, GW6471. Our data show that MPP+ increases mRNA levels of PPARα in both non differentiated and differentiated cells. Using assays to assess cell metabolic activity, cell proliferation, and pro-inflammatory markers, we observed that OlGly (1 nM), both as treatment (1 h) and pre-treatment (4 h), is able to protect against neuronal damage induced by 24 h MPP+ exposure through PPARα. Moreover, using a targeted lipidomics approach, we demonstrate that OlGly exerts its effects also through the modulation of the eCBome. Finally, treatment with OlGly was able also to reduce increased IL-1ß induced by MPP+ in differentiated cells. In conclusion, our results suggest that OlGly could be a promising therapeutic agent for the treatment of MPP+-induced neurotoxicity.

The Focal Mechanical Vibration for Balance Improvement in Elderly - A Systematic Review.

BACKGROUND: Aging has been associated with the progressive depletion of lean mass, reductions in muscle strength and the coordination of the lower extremities, accompanied by decreased gait assurance and balance control. Also, less balance control favors falling which is the leading cause of injury among the elderly. The aim of this systematic review is to identify and evaluate existing evidence regarding the use of focused vibration (FV) to improve balance and reduce the risk of falling during the rehabilitation of elderly populations. METHODS: The PICO question is what are the effects of focal/segmental/local vibration training on the assessment of balance and the risk of falls among the elderly population? A thorough literature review was conducted between May 1, 2009, and June 30, 2019, for studies in English, randomized clinical trials, including crossover and prospective design studies with assessing balance and the risk of falls in elderly populations (age > 60 years). RESULTS: Eight articles (N = 8) satisfied the inclusion criteria and were considered, of which 6 are RTC, one cross-sectional study and one clinical study, for a total of 635 participants. A total of 6 different vibration devices were used, each of which was associated with different FV frequency and amplitude characteristics and different treatment protocols. CONCLUSION: In conclusion, FV can be effective in decreasing the risk of falls and improving the assessment of balance, but more evidence is necessary considering the limits of the studies; however, it does look an important promise during rehabilitative treatment.

Modulation of Endocannabinoid Tone in Osteoblastic Differentiation of MC3T3-E1 Cells and in Mouse Bone Tissue over Time.

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

A Glucuronic Acid-Palmitoylethanolamide Conjugate (GLUPEA) Is an Innovative Drug Delivery System and a Potential Bioregulator.

Palmitoylethanolamide (PEA) is an endogenous anti-inflammatory lipid mediator and a widely used nutraceutical. In this study, we designed, realized, and tested a drug-carrier conjugate between PEA (the active drug) and glucuronic acid (the carrier). The conjugate, named GLUPEA, was characterized for its capability of increasing PEA levels and exerting anti-inflammatory activity both in vitro and in vivo. GLUPEA treatment, compared to the same concentration of PEA, resulted in higher cellular amounts of PEA and the endocannabinoid 2-arachidonoyl glycerol (2-AG), and increased 2-AG-induced transient receptor potential vanilloid type 1 (TRPV1) channel desensitization to capsaicin. GLUPEA inhibited pro-inflammatory monocyte chemoattractant protein 2 (MCP-2) release from stimulated keratinocytes, and it was almost as efficacious as ultra-micronized PEA at reducing colitis in dinitrobenzene sulfonic acid (DNBS)-injected mice when using the same dose. GLUPEA is a novel pro-drug able to efficiently mimic the anti-inflammatory and endocannabinoid enhancing actions of PEA.

Maternal omega-3 intake differentially affects the endocannabinoid system in the progeny`s neocortex and hippocampus: Impact on synaptic markers.

Omega-3 (n-3) polyunsaturated fatty acids (PUFA) and the endocannabinoid system (ECS) modulate several functions through neurodevelopment including synaptic plasticity mechanisms. The interplay between n-3PUFA and the ECS during the early stages of development, however, is not fully understood. This study investigated the effects of maternal n-3PUFA supplementation (n-3Sup) or deficiency (n-3Def) on ECS and synaptic markers in postnatal offspring. Female rats were fed with a control, n-3Def, or n-3Sup diet from 15 days before mating and during pregnancy. The cerebral cortex and hippocampus of mothers and postnatal 1-2 days offspring were analyzed. In the mothers, a n-3 deficiency reduced CB1 receptor (CB1R) protein levels in the cortex and increased CB2 receptor (CB2R) in both cortex and hippocampus. In neonates, a maternal n-3 deficiency reduced the hippocampal CB1R amount while it increased CB2R. Additionally, total GFAP isoform expression was increased in both cortex and hippocampus in neonates of the n-3Def group. Otherwise, maternal n-3 supplementation increased the levels of n-3-derived endocannabinoids, DHEA and EPEA, in the cortex and hippocampus and reduced 2-arachidonoyl-glycerol (2-AG) concentrations in the cortex of the offspring. Furthermore, maternal n-3 supplementation also increased PKA phosphorylation in the cortex and ERK phosphorylation in the hippocampus. Synaptophysin immunocontent in both regions was also increased. In vitro assays showed that the increase of synaptophysin in the n-3Sup group was independent of CB1R activation. The findings show that variations in maternal dietary omega-3 PUFA levels may impact differently on the ECS and molecular markers in the cerebral cortex and hippocampus of the progeny.

miRNeye: a microRNA expression atlas of the mouse eye.

BACKGROUND: MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. RESULTS: In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina. CONCLUSIONS: This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures.

Differential alterations of the concentrations of endocannabinoids and related lipids in the subcutaneous adipose tissue of obese diabetic patients.

BACKGROUND: The endocannabinoids, anandamide and 2-AG, are produced by adipocytes, where they stimulate lipogenesis via cannabinoid CB1 receptors and are under the negative control of leptin and insulin. Endocannabinoid levels are elevated in the blood of obese individuals and nonobese type 2 diabetes patients. To date, no study has evaluated endocannabinoid levels in subcutaneous adipose tissue (SAT) of subjects with both obesity and type 2 diabetes (OBT2D), characterised by similar adiposity and whole body insulin resistance and lower plasma leptin levels as compared to non-diabetic obese subjects (OB). DESIGN AND METHODS: The levels of anandamide and 2-AG, and of the anandamide-related PPARalpha ligands, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), in the SAT obtained by abdominal needle biopsy in 10 OBT2D, 11 OB, and 8 non-diabetic normal-weight (NW) subjects, were measured by liquid chromatography-mass spectrometry. All subjects underwent a hyperinsulinaemic euglycaemic clamp. RESULTS: As compared to NW, anandamide, OEA and PEA levels in the SAT were 2-4.4-fold elevated (p < 0.05), and 2-AG levels 2.3-fold reduced (p < .05), in OBT2D but not in OB subjects. Anandamide, OEA and PEA correlated positively (p < .05) with SAT leptin mRNA and free fatty acid during hyperinsulinaemic clamp, and negatively with SAT LPL activity and plasma HDL-cholesterol, which were all specifically altered in OBT2D subjects. CONCLUSIONS: The observed alterations emphasize, for the first time in humans, the potential different role and regulation of adipose tissue anandamide (and its congeners) and 2-AG in obesity and type 2 diabetes.

Modeling Acquired Resistance to the Second-Generation Androgen Receptor Antagonist Enzalutamide in the TRAMP Model of Prostate Cancer.

Enzalutamide (MDV3100) is a potent second-generation androgen receptor antagonist approved for the treatment of castration-resistant prostate cancer (CRPC) in chemotherapy-naïve as well as in patients previously exposed to chemotherapy. However, resistance to enzalutamide and enzalutamide withdrawal syndrome have been reported. Thus, reliable and integrated preclinical models are required to elucidate the mechanisms of resistance and to assess therapeutic settings that may delay or prevent the onset of resistance. In this study, the prostate cancer multistage murine model TRAMP and TRAMP-derived cells have been used to extensively characterize in vitro and in vivo the response and resistance to enzalutamide. The therapeutic profile as well as the resistance onset were characterized and a multiscale stochastic mathematical model was proposed to link the in vitro and in vivo evolution of prostate cancer. The model showed that all therapeutic strategies that use enzalutamide result in the onset of resistance. The model also showed that combination therapies can delay the onset of resistance to enzalutamide, and in the best scenario, can eliminate the disease. These results set the basis for the exploitation of this "TRAMP-based platform" to test novel therapeutic approaches and build further mathematical models of combination therapies to treat prostate cancer and CRPC.Significance: Merging mathematical modeling with experimental data, this study presents the "TRAMP-based platform" as a novel experimental tool to study the in vitro and in vivo evolution of prostate cancer resistance to enzalutamide.

Protective Effects of N-Oleoylglycine in a Mouse Model of Mild Traumatic Brain Injury.

Traumatic brain injury (TBI) is one of the main causes of death in young people for which currently no efficacious treatment exists. Recently, we have reported that mice with mild-TBI with a specific injury in the insula showed elevated levels of a little investigated N-acyl amino acid, N-oleoylglycine (OlGly). N-acyl amino acids have recently experienced an increased interest because of their important biological activities. They belong to the endocannabinoidome family of lipids with structural similarities with the endocannabinoids (eCBs). The aim of this study was to test the neuroprotective and antihyperalgesic actions of OlGly in a model of mouse mild-TBI (mTBI) and its effect on levels of eCBs and N-acylethanolamines at the end of treatment. Following mTBI, mice were administered a daily injection of OlGly (10-50-100 mg/kg i.p.) for 14 days. Treatment with OlGly normalized motor impairment and behavior in the light/dark box test, ameliorated TBI-induced thermal hyperalgesia and mechanical allodynia, and normalized aggressiveness and depression. Moreover, levels of eCBs and some N-acylethanolamines underwent significant changes 60 days after TBI, especially in the prefrontal cortex and hypothalamus, and OlGly reversed some of these changes. In conclusion, our findings reveal that OlGly ameliorates the behavioral alterations associated with mTBI in mice, while concomitantly modulating eCB and eCB-like mediator tone.