scholarly journals CRISPR interference at the FAAH-OUT genomic region reduces FAAH expression

2019 ◽  
Author(s):  
Hajar Mikaeili ◽  
Charlix Yeung ◽  
Abdella M Habib ◽  
John N Wood ◽  
Andrei L Okorokov ◽  
...  
Keyword(s):  
Gene Editing ◽  
Clinical Symptoms ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Regulatory Elements ◽  
Human Cell Line ◽  
Genomic Region ◽  
Chromosome 1 ◽  
Bioactive Lipids ◽  
Post Operative Nausea

AbstractFAAH-OUT encodes a putative long non-coding RNA which is located next to the FAAH gene on human chromosome 1. Recently an ~8 kb microdeletion, that removes upstream regulatory elements and the first 2 exons of FAAH-OUT, was reported in a pain insensitive patient (PFS) with additional clinical symptoms including a happy, non-anxious disposition, fast wound healing, fear and memory deficits, and significant post-operative nausea and vomiting induced by morphine. PFS also carries a hypomorphic SNP in FAAH that significantly reduces the activity of the encoded fatty-acid amide hydrolase enzyme. The FAAH and FAAH-OUT mutations identified in PFS result in elevated levels of anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamine (OEA) measured in peripheral blood. These bioactive lipids, which are normally degraded by FAAH, have diverse biological functions including known roles in pain pathways.Here we report the first mechanistic insights into how the FAAH-OUT microdeletion affects FAAH function. Gene editing in a human cell line to mimic the FAAH-OUT microdeletion observed in PFS results in reduced expression of FAAH. Furthermore, CRISPRi experiments targeting the promoter region of FAAH-OUT or a short highly evolutionarily conserved element in the first intron of FAAH-OUT also result in reduced expression of FAAH. These experiments confirm the importance of FAAH-OUT and specific genomic elements within the ~8 kb microdeleted sequence to normal FAAH expression. Our results also highlight the potential of CRISPRi and gene editing strategies that target the FAAH-OUT region for the development of novel FAAH-based analgesic and anxiolytic therapies.

scholarly journals Endocannabinoid Levels in Ulcerative Colitis Patients Correlate With Clinical Parameters and Are Affected by Cannabis Consumption

2021 ◽  
Vol 12 ◽  
Author(s):  
Shelly Tartakover Matalon ◽  
Shahar Azar ◽  
David Meiri ◽  
Rivka Hadar ◽  
Alina Nemirovski ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Clinical Symptoms ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Endocannabinoid System ◽  
Beneficial Effects ◽  
Before And After ◽  
Bowel Diseases ◽  
Bowel Movements ◽  
Cannabis Consumption

BackgroundInflammatory bowel diseases (IBDs) are chronic, idiopathic, inflammatory, gastrointestinal disorders. The endocannabinoid system may have a role in the pathogenesis of IBD. We aimed to assess whether cannabis treatment influences endocannabinoids (eCBs) level and clinical symptoms of IBD patients.MethodsBlood samples and biopsies were taken from IBD patients treated by either cannabis or placebo for 8 weeks. Immunohistochemistry for N-acyl-phosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH) expression was done on colon biopsies, and sample levels of anandamide (AEA), eCB2-arachidonylglycerol (2-AG), arachidonic acid (AA), palmitoylethanolamine (PEA), and oleoylethanolamine (OEA) were measured in patient’s sera before and after cannabis treatment. Caco-2 cells were cultured with extracts of cannabis with/without tetrahydrocannabinol (THC) and their proteins extracted, and Western blotting for NAPE-PLD and FAAH expression was done.ResultsThirteen patients with Crohn’s disease (CD) and nine patients with ulcerative colitis (UC) were treated with cannabis. Seventeen patients with CD and 10 with UC served as placebo groups. In all CD patients, the levels of eCBs remained unaltered during the treatment period. In UC patients treated with placebo, but not in those treated with cannabis, the levels of PEA, AEA, and AA decreased significantly. The percent reduction in bowel movements was negatively correlated with changes observed in the circulating AEA and OEA, whereas improvement in quality of life was positively correlated with the levels of 2-AG. In the biopsies from UC patients, FAAH levels increased over the study period. In Caco-2 cells, both cannabis extracts increased NAPE-PLD levels but reduced FAAH expression levels.ConclusionOur study supports the notion that cannabis use affects eCB “tone” in UC patients and may have beneficial effects on disease symptoms in UC patients.

scholarly journals Heritability and family-based GWAS analyses of the N-acyl ethanolamine and ceramide plasma lipidome

2019 ◽  
Author(s):  
Kathryn A. McGurk ◽  
Simon G. Williams ◽  
Hui Guo ◽  
Hugh Watkins ◽  
Martin Farrall ◽  
...  
Keyword(s):  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Missense Variant ◽  
Bioactive Lipids ◽  
Gene Encoding ◽  
Disease Biomarkers ◽  
Lipid Species ◽  
A Subunit ◽  
Amide Hydrolase ◽  
Family Based

AbstractSignalling lipids of the N-acyl ethanolamine (NAE) and ceramide (CER) classes are emerging as novel cardiovascular disease biomarkers. We sought to establish the heritability of plasma NAEs (including the endocannabinoid anandamide) and CERs, and identify common DNA variants influencing the circulating concentrations of the heritable lipid species. Nine NAE and sixteen CER species were analysed in plasma samples from 999 members of 196 British Caucasian families, using targeted mass spectrometry (UPLC-MS/MS). Heritability was estimated and GWAS analyses were undertaken; all target lipids were significantly heritable (h2 = 36%-62%). A missense variant (rs324420) in the gene encoding the enzyme fatty acid amide hydrolase (FAAH), which degrades NAEs, associated at GWAS significance (P<2.15×10−8) with four NAEs (DHEA, PEA, LEA, VEA). The A allele of this SNP was associated with a 0.23 SD per-allele increase in plasma NAE species. Additionally, we found association between rs680379 in the SPTLC3 gene, which encodes a subunit of the rate limiting enzyme in CER biosynthesis, and a range of CER species (e.g. CER[N(24)S(19)]; P =4.82×10−27). We also observed three novel associations (CD83, SGPP1, FBXO28-DEGS1) influencing plasma CER traits, two of which (SGPP1 and DEGS1) implicate CER species in haematological phenotypes. NAE and CER are substantially heritable bioactive lipids, influenced by SNPs in key metabolic enzymes.

scholarly journals Endocannabinoid System as a Promising Therapeutic Target in Inflammatory Bowel Disease – A Systematic Review

2021 ◽  
Vol 12 ◽  
Author(s):  
Szymon Hryhorowicz ◽  
Marta Kaczmarek-Ryś ◽  
Aleksandra Zielińska ◽  
Rodney J. Scott ◽  
Ryszard Słomski ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Cannabinoid Receptors ◽  
Intestinal Inflammation ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Endocannabinoid System ◽  
Unknown Etiology ◽  
Bioactive Lipids ◽  
Inflammatory Bowel

Inflammatory bowel disease (IBD) is a general term used to describe a group of chronic inflammatory conditions of the gastrointestinal tract of unknown etiology, including two primary forms: Crohn’s disease (CD) and ulcerative colitis (UC). The endocannabinoid system (ECS) plays an important role in modulating many physiological processes including intestinal homeostasis, modulation of gastrointestinal motility, visceral sensation, or immunomodulation of inflammation in IBD. It consists of cannabinoid receptors (CB1 and CB2), transporters for cellular uptake of endocannabinoid ligands, endogenous bioactive lipids (Anandamide and 2-arachidonoylglycerol), and the enzymes responsible for their synthesis and degradation (fatty acid amide hydrolase and monoacylglycerol lipase), the manipulation of which through agonists and antagonists of the system, shows a potential therapeutic role for ECS in inflammatory bowel disease. This review summarizes the role of ECS components on intestinal inflammation, suggesting the advantages of cannabinoid-based therapies in inflammatory bowel disease.

scholarly journals High-Throughput Screening for the Discovery of Inhibitors of Fatty Acid Amide Hydrolase Using a Microsome-Based Fluorescent Assay

2006 ◽  
Vol 11 (5) ◽  
pp. 519-527 ◽  
Author(s):  
Yuren Wang ◽  
Fernando Ramirez ◽  
Girija Krishnamurthy ◽  
Adam Gilbert ◽  
Nina Kadakia ◽  
...  
Keyword(s):  
Fatty Acid ◽  
High Throughput ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Bioactive Lipids ◽  
Fluorescent Assay ◽  
Fatty Acid Amides ◽  
Amide Hydrolase ◽  
Faah Inhibitors ◽  
Fatty Acid Amide

Fatty acid amide hydrolase (FAAH) is a membrane-associated enzyme that catalyzes the hydrolysis of several endogenous bioactive lipids, including anandamide (AEA), N-palmitoylethanolamine (PEA), oleamide, and N-oleoylethanolamine (OEA). These fatty acid amides participate in many physiological activities such as analgesia, anxiety, sleep modulation, anti inflammatory responses, and appetite suppression. Because FAAH plays an essential role in controlling the tone and activity of these endogenous bioactive lipids, this enzyme has been implicated to be a drug target for the therapeutic management of pain, anxiety, and other disorders. In an effort to discover FAAH inhibitors, the authors have previously reported the development of a novel fluorescent assay using purified FAAH microsomes as an enzyme source and a fluorogenic substrate, arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA). Herein, the authors have adapted this assay to a high-throughput format and have screened a large library of small organic compounds, identifying a number of novel FAAH inhibitors. These data further verify that this fluorescent assay is sufficiently robust, efficient, and low-cost for the identification of FAAH inhibitory molecules and open this class of enzymes for therapeutic exploration.

scholarly journals Cooperative enzymatic control of N-acyl amino acids by PM20D1 and FAAH

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Joon T Kim ◽  
Stephanie M Terrell ◽  
Veronica L Li ◽  
Wei Wei ◽  
Curt R Fischer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Energy Homeostasis ◽  
Fatty Acid Amide Hydrolase ◽  
Acid Amide ◽  
Bioactive Lipids ◽  
Genetic Ablation ◽  
Amide Hydrolase ◽  
Enzymatic Pathways

The N-acyl amino acids are a family of bioactive lipids with pleiotropic physiologic functions, including in energy homeostasis. Their endogenous levels are regulated by an extracellular mammalian N-acyl amino acid synthase/hydrolase called PM20D1 (peptidase M20 domain containing 1). Using an activity-guided biochemical approach, we report the molecular identification of fatty acid amide hydrolase (FAAH) as a second intracellular N-acyl amino acid synthase/hydrolase. In vitro, FAAH exhibits a more restricted substrate scope compared to PM20D1. In mice, genetic ablation or selective pharmacological inhibition of FAAH bidirectionally dysregulates intracellular, but not circulating, N-acyl amino acids. Dual blockade of both PM20D1 and FAAH reveals a dramatic and non-additive biochemical engagement of these two enzymatic pathways. These data establish FAAH as a second intracellular pathway for N-acyl amino acid metabolism and underscore enzymatic division of labor as an enabling strategy for the regulation of a structurally diverse bioactive lipid family.

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