Gene_locus Report for: human-DAGLA

In this report, we describe the kinetics characteristics of the diacylglycerol lipase-alpha (DGLalpha) located at the nuclear matrix of nuclei derived from adult cortical neurons. Thus, using high-resolution fluorescence microscopy, classical biochemical subcellular fractionation, and Western blot techniques, we demonstrate that the DGLalpha enzyme is located in the matrix of neuronal nuclei. Furthermore, by quantifying the 2-arachidonoylglycerol (2-AG) level by liquid chromatography and mass spectrometry when 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) was exogenously added as substrate, we describe the presence of a mechanism for 2-AG production through DGLalpha dependent biosynthesis with an apparent K(m) (K(m)(app)) of 180 microM and a V(max) of 1.3 pmol min(-1) microg(-1) protein. We also examined the presence of enzymes with hydrolytic and oxygenase activities that are able to use 2-AG as substrate, and described the localization and compartmentalization of the major 2-AG degradation enzymes, namely monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), alpha/beta-hydrolase domain 12 protein (ABHD12) and cyclooxygenase-2 (COX2). Of these, only ABHD12 exhibited the same distribution with respect to chromatin, lamin B1, SC-35 and NeuN as that described for DGLalpha. When 2-AG was exogenously added, we observed the production of arachidonic acid (AA), which was prevented by inhibitors (but not specific MGL or ABHD6 inhibitors) of the ABHD family. Overall, our results expand knowledge about the subcellular distribution of neuronal DGLalpha, and provide biochemical and morphological evidence to ensure that 2-AG is produced in the neuronal nuclear matrix. Thus, this work paves the way for proposing a working hypothesis about the role of 2-AG produced in neuronal nuclei.

INTRODUCTION: Lipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase alpha and beta (DAGLalpha, DAGLbeta) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. METHODS: DAGLalpha and DAGLbeta mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLbeta transcripts to different cell types of the placenta. DAGLbeta activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 microM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. RESULTS: We demonstrate that mRNA expression of DAGLbeta prevails in placental tissue, compared to DAGLalpha (p >= 0.0001) and that DAGLbeta is mainly located to CK7 positive trophoblasts (p >= 0.0001). Although few DAGLalpha transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLbeta is the principal DAGL in the placenta. DAGLbeta dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLbeta by DH376 led to reduced MAG tissue levels (p >= 0.01), including 2-AG (p>=0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. DISCUSSION: Our results emphasize the role of DAGLbeta activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.

As an important hydrolytic enzyme that yields 2-AG and free fatty acids, diacylglycerol lipase alpha (DAGLA) is involved in exacerbating malignant phenotypes and cancer progression, but the role of the DAGLA/2-AG axis in HCC progression remains unclear. Here, we found that the upregulation of components of the DAGLA/2-AG axis in HCC samples is correlated with tumour stage and patient prognosis. In vitro and in vivo experiments demonstrated that the DAGLA/2-AG axis promoted HCC progression by regulating cell proliferation, invasion and metastasis. Mechanistically, the DAGLA/2AG axis significantly inhibited LATS1 and YAP phosphorylation, promoted YAP nuclear translocation and activity, and ultimately led to TEAD2 upregulation and increased PHLDA2 expression, which could be enhanced by DAGLA/2AG-induced activation of the PI3K/AKT pathway. More importantly, DAGLA induced resistance to lenvatinib therapy during HCC treatment. Our study demonstrates that inhibiting the DAGLA/2-AG axis could be a novel therapeutic strategy to inhibit HCC progression and enhance the therapeutic effects of TKIs, which warrant further clinical studies.

In this report, we describe the kinetics characteristics of the diacylglycerol lipase-alpha (DGLalpha) located at the nuclear matrix of nuclei derived from adult cortical neurons. Thus, using high-resolution fluorescence microscopy, classical biochemical subcellular fractionation, and Western blot techniques, we demonstrate that the DGLalpha enzyme is located in the matrix of neuronal nuclei. Furthermore, by quantifying the 2-arachidonoylglycerol (2-AG) level by liquid chromatography and mass spectrometry when 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) was exogenously added as substrate, we describe the presence of a mechanism for 2-AG production through DGLalpha dependent biosynthesis with an apparent K(m) (K(m)(app)) of 180 microM and a V(max) of 1.3 pmol min(-1) microg(-1) protein. We also examined the presence of enzymes with hydrolytic and oxygenase activities that are able to use 2-AG as substrate, and described the localization and compartmentalization of the major 2-AG degradation enzymes, namely monoacylglycerol lipase (MGL), fatty acid amide hydrolase (FAAH), alpha/beta-hydrolase domain 12 protein (ABHD12) and cyclooxygenase-2 (COX2). Of these, only ABHD12 exhibited the same distribution with respect to chromatin, lamin B1, SC-35 and NeuN as that described for DGLalpha. When 2-AG was exogenously added, we observed the production of arachidonic acid (AA), which was prevented by inhibitors (but not specific MGL or ABHD6 inhibitors) of the ABHD family. Overall, our results expand knowledge about the subcellular distribution of neuronal DGLalpha, and provide biochemical and morphological evidence to ensure that 2-AG is produced in the neuronal nuclear matrix. Thus, this work paves the way for proposing a working hypothesis about the role of 2-AG produced in neuronal nuclei.

INTRODUCTION: Lipids and fatty acids are key components in metabolic processes of the human placenta, thereby contributing to the development of the fetus. Placental dyslipidemia and aberrant activity of lipases have been linked to diverse pregnancy associated complications, such as preeclampsia and preterm birth. The serine hydrolases, diacylglycerol lipase alpha and beta (DAGLalpha, DAGLbeta) catalyze the degradation of diacylglycerols, leading to the formation of monoacylglycerols (MAG), including one main endocannabinoid 2-arachidonoylglycerol (2-AG). The major role of DAGL in the biosynthesis of 2-AG is evident from various studies in mice but has not been investigated in the human placenta. Here, we report the use of the small molecule inhibitor DH376, in combination with the ex vivo placental perfusion system, activity-based protein profiling (ABPP) and lipidomics, to determine the impact of acute DAGL inhibition on placental lipid networks. METHODS: DAGLalpha and DAGLbeta mRNA expression was detected by RT-qPCR and in situ hybridization in term placentas. Immunohistochemistry staining for CK7, CD163 and VWF was applied to localize DAGLbeta transcripts to different cell types of the placenta. DAGLbeta activity was determined by in- gel and MS-based activity-based protein profiling (ABPP) and validated by addition of the enzyme inhibitors LEI-105 and DH376. Enzyme kinetics were measured by EnzChek lipase substrate assay. Ex vivo placental perfusion experiments were performed +/- DH376 [1 microM] and changes in tissue lipid and fatty acid profiles were measured by LC-MS. Additionally, free fatty acid levels of the maternal and fetal circulations were determined. RESULTS: We demonstrate that mRNA expression of DAGLbeta prevails in placental tissue, compared to DAGLalpha (p >= 0.0001) and that DAGLbeta is mainly located to CK7 positive trophoblasts (p >= 0.0001). Although few DAGLalpha transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLbeta is the principal DAGL in the placenta. DAGLbeta dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLbeta by DH376 led to reduced MAG tissue levels (p >= 0.01), including 2-AG (p>=0.0001). We further provide an activity landscape of serine hydrolases, showing a broad spectrum of metabolically active enzymes in the human placenta. DISCUSSION: Our results emphasize the role of DAGLbeta activity in the human placenta by determining the biosynthesis of 2-AG. Thus, this study highlights the special importance of intra-cellular lipases in lipid network regulation. Together, the activity of these specific enzymes may contribute to the lipid signaling at the maternal-fetal interface, with implications for function of the placenta in normal and compromised pregnancies.

The endogenous cannabinoid, 2-arachidonoylglycerol (2-AG), plays a key role in the regulation of anxiety- and stress-related behavioral phenotypes and may represent a novel target for the treatment of anxiety disorders. However, recent studies have suggested a more complex role for 2-AG signaling in the regulation of stress responsivity, including increases in acute fear responses after 2-AG augmentation under some conditions. Thus, 2-AG signaling within distinct brain regions and circuits could regulate anxiety-like behavior and stress responsivity in opposing manners. The ventral hippocampus (vHPC) is a critical region for emotional processing, anxiety-like behaviors, and stress responding. Here, we use a conditional knock-out of the 2-AG synthesis enzyme, diacylglycerol lipase alpha (DAGLalpha), to study the role of vHPC 2-AG signaling in the regulation of affective behavior. We show that vHPC DAGLalpha deletion decreases avoidance behaviors both basally and following an acute stress exposure. Genetic deletion of vHPC DAGLalpha also promotes stress resiliency, with no effect on fear acquisition, expression, or contextual fear generalization. Using slice electrophysiology, we demonstrate that vHPC DAGLalpha deletion shifts vHPC activity towards enhanced inhibition. Together, these data indicate endogenous 2-AG signaling in the vHPC promotes avoidance and increases stress reactivity, confirming the notion that 2-AG signaling within distinct brain regions may exert divergent effects on anxiety states and stress adaptability.

As an important hydrolytic enzyme that yields 2-AG and free fatty acids, diacylglycerol lipase alpha (DAGLA) is involved in exacerbating malignant phenotypes and cancer progression, but the role of the DAGLA/2-AG axis in HCC progression remains unclear. Here, we found that the upregulation of components of the DAGLA/2-AG axis in HCC samples is correlated with tumour stage and patient prognosis. In vitro and in vivo experiments demonstrated that the DAGLA/2-AG axis promoted HCC progression by regulating cell proliferation, invasion and metastasis. Mechanistically, the DAGLA/2AG axis significantly inhibited LATS1 and YAP phosphorylation, promoted YAP nuclear translocation and activity, and ultimately led to TEAD2 upregulation and increased PHLDA2 expression, which could be enhanced by DAGLA/2AG-induced activation of the PI3K/AKT pathway. More importantly, DAGLA induced resistance to lenvatinib therapy during HCC treatment. Our study demonstrates that inhibiting the DAGLA/2-AG axis could be a novel therapeutic strategy to inhibit HCC progression and enhance the therapeutic effects of TKIs, which warrant further clinical studies.

The endocannabinoid system is a highly conserved and ubiquitous signaling pathway with broad ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia, and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNAseq showed clear expression of the truncated transcript and no differences were found between mutant and wild type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique pediatric syndrome. Because enzymatic activity was preserved, the observed mis-localization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.

The endocannabinoid system modulates adult hippocampal neurogenesis by promoting the proliferation and survival of neural stem and progenitor cells (NSPCs). This is demonstrated by the disruption of adult neurogenesis under two experimental conditions: (1) NSPC-specific deletion of cannabinoid receptors and (2) constitutive deletion of the enzyme diacylglycerol lipase alpha (DAGLa) which produces the endocannabinoid 2-arachidonoylglycerol (2-AG). However, the specific cell types producing 2-AG relevant to neurogenesis remain unknown. Here we sought to identify the cellular source of endocannabinoids in the subgranular zone of the dentate gyrus (DG) in hippocampus, an important neurogenic niche. For this purpose, we used two complementary Cre-deleter mouse strains to delete Dagla either in neurons, or in astroglia and NSPCs. Surprisingly, neurogenesis was not altered in mice bearing a deletion of Dagla in neurons (Syn-Dagla KO), although neurons are the main source for the endocannabinoids in the brain. In contrast, a specific inducible deletion of Dagla in NPSCs and astrocytes (GLAST-CreERT2-Dagla KO) resulted in a strongly impaired neurogenesis with a 50% decrease in proliferation of newborn cells. These results identify Dagla in NSPCs in the DG or in astrocytes as a prominent regulator of adult hippocampal neurogenesis. We also show a reduction of Daglb expression in GLAST-CreERT2-Dagla KO mice, which may have contributed to the neurogenesis phenotype.

In order to understand the mechanism of the endocannabinoid (eCB) signal, which has so far been shown to work in oocyte genesis and maturation, it is critical to clarify detailed localization of the eCB synthesizing enzyme molecules as well as receptors for eCBs in oocytes in the ovary in situ. For this purpose, diacylglycerol lipase (DGL) alpha and beta are involved in the synthesis of an eCB 2-arachidonoylglycerol (2-AG). DGLalpha/beta and the cannabinoid receptor 1 (CB1) for 2-AG were shown to be localized to the primary oocytes of postnatal mice using immuno-light and electron microscopy. It was found that two types of localization existed: first, immunoreactivities for DGLalpha and beta were weakly detected throughout the ooplasm in light microscopy for which the intracellular membranes of vesicles forming tiny scattered aggregates were responsible. Secondly, DGLbeta-immunoreactivity was distinctly confined to the nuage of Balbiani bodies and small nuage-derivative structures; both amorphous materials and membranes of vesicles were responsible for their localization. On the other hand, the weak immunoreactivity for CB1 was localized in a pattern similar to the first one for DGLs, but not found in a pattern for the Balbiani nuage. Two routes of functional exertion of 2-AG synthesized by DGLs were suggested from the two types of localization: one was that the eCB synthesized at all the sites of DGLs is released from the oocytes and exerts paracrine or autocrine effects on adjacent intra-ovarian cells as well as the oocytes themselves. The other was that the eCB synthesized within the nuage was involved in the modulation of the posttranscriptional processing of oocytes. Owing to the failure in the detection of CB1 in the Balbiani nuage, however, the validity of the latter possibility remains to be elucidated.

Diacylglycerol lipase alpha (DAGLA), which catalyzes the hydrolysis of diacylglycerol to 2-arachidonoylglycerol and free fatty acid, is required for axonal growth during the brain development and for retrograde synaptic signaling at mature synapses. So far, no information was found regarding the possible role of DAGLA in human tumorigenesis. Thus, the current study sought to clarify the contribution of DAGLA in oral squamous cell carcinomas (OSCCs) and assess the clinical possibilities for OSCC treatment. Using real-time quantitative reverse transcription-polymerase chain reaction, immunoblotting, and immunohistochemistry, we found a significant up-regulation of DAGLA in OSCCs compared with normal cells and tissues both at mRNA and protein expression levels. Knockdown models in OSCC-derived cell lines for DAGLA (siDAGLA) and treatment with a lipase inhibitor (orlistat) showed several depressed cellular functions, including cellular proliferation and migratory activities through cell-cycle arrest at G1 phase. Furthermore, we found that DAGLA-positive OSCC samples were correlated highly with the primary tumoral size. We concluded that DAGLA may be a key determinant in tumoral progression and might be a therapeutic target for OSCCs.

The biosynthetic and catabolic enzymes of the endocannabinoids tightly regulate endocannabinoid-mediated activation of the cannabinoid CB1 receptor. Monitoring the activities of these endocannabinoid hydrolases in different brain regions is, therefore, key to gaining insight into spatiotemporal control of CB1 receptor-mediated physiology. We have employed a comparative chemical proteomics approach to quantitatively map the activity profile of endocannabinoid hydrolases in various mouse brain regions at the same time. To this end, we used two different activity-based probes: fluorophosphonate-biotin (FP-biotin), which quantifies FAAH, ABHD6, and MAG-lipase activity, and MB108, which detects DAGL-alpha, ABHD4, ABHD6, and ABHD12. In total, 32 serine hydrolases were evaluated in the frontal cortex, hippocampus, striatum, and cerebellum. Comparison of endocannabinoid hydrolase activity in the four brain regions revealed that FAAH activity was highest in the hippocampus, and MAGL activity was most pronounced in the frontal cortex, whereas DAGL-alpha was most active in the cerebellum. Comparison of the activity profiles with a global proteomics data set revealed pronounced differences. This could indicate that post-translational modification of the endocannabinoid hydrolases is important to regulate their activity. Next, the effect of genetic deletion of the CB1 receptor was studied. No difference in the enzymatic activity was found in the cerebellum, striatum, frontal cortex, and hippocampus of CB1 receptor knockout animals compared to wild type mice. Our results are in line with previous reports and indicate that the CB1 receptor exerts no regulatory control over the basal production and degradation of endocannabinoids and that genetic deletion of the CB1 receptor does not induce compensatory mechanisms in endocannabinoid hydrolase activity.

Diacylglycerol lipases (DAGLalpha and DAGLbeta) convert diacylglycerol to the endocannabinoid 2-arachidonoylglycerol. Our understanding of DAGL function has been hindered by a lack of chemical probes that can perturb these enzymes in vivo. Here, we report a set of centrally active DAGL inhibitors and a structurally related control probe and their use, in combination with chemical proteomics and lipidomics, to determine the impact of acute DAGL blockade on brain lipid networks in mice. Within 2 h, DAGL inhibition produced a striking reorganization of bioactive lipids, including elevations in DAGs and reductions in endocannabinoids and eicosanoids. We also found that DAGLalpha is a short half-life protein, and the inactivation of DAGLs disrupts cannabinoid receptor-dependent synaptic plasticity and impairs neuroinflammatory responses, including lipopolysaccharide-induced anapyrexia. These findings illuminate the highly interconnected and dynamic nature of lipid signaling pathways in the brain and the central role that DAGL enzymes play in regulating this network.

Diacylglycerol lipase alpha (DAGLalpha) hydrolyses DAG to generate the principal endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) in the central nervous system. DAGLalpha dependent cannabinoid (CB) signalling has been implicated in numerous processes including axonal growth and guidance, adult neurogenesis and retrograde signalling at the synapse. Recent studies have implicated DAGLalpha as an emerging drug target for several conditions including pain and obesity. Activity assays are critical to the drug discovery process; however, measurement of diacylglycerol lipase (DAGL) activity using its native substrate generally involves low-throughput MS techniques. Some relatively high-throughput membrane based assays utilizing surrogate substrates have been reported, but these do not take into account the rate-limiting effects often associated with the ability of a drug to cross the cell membrane. In the present study, we report the development of a live cell assay to measure DAGLalpha activity. Two previously reported DAGLalpha surrogate substrates, p-nitrophenyl butyrate (PNPB) and 6,8-difluoro-4-methylumbelliferyl octanoate (DiFMUO), were evaluated for their ability to detect DAGLalpha activity in live cell assays using a human cell line stably expressing the human DAGLalpha transgene. Following optimization, the small molecule chromogenic substrate PNPB proved to be superior by providing lower background activity along with a larger signal window between transfected and parental cells when compared with the fluorogenic substrate DiFMUO. The assay was further validated using established DAGL inhibitors. In summary, the live cell DAGLalpha assay reported here offers an economical and convenient format to screen for novel inhibitors as part of drug discovery programmes and compliments previously reported high-throughput membrane based DAGL assays.

Diacylglycerol lipase (DAGL)-alpha and -beta are enzymes responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Selective and reversible inhibitors are required to study the function of DAGLs in neuronal cells in an acute and temporal fashion, but they are currently lacking. Here, we describe the identification of a highly selective DAGL inhibitor using structure-guided and a chemoproteomics strategy to characterize the selectivity of the inhibitor in complex proteomes. Key to the success of this approach is the use of comparative and competitive activity-based proteome profiling (ABPP), in which broad-spectrum and tailor-made activity-based probes are combined to report on the inhibition of a protein family in its native environment. Competitive ABPP with broad-spectrum fluorophosphonate-based probes and specific beta-lactone-based probes led to the discovery of alpha-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-alpha/DAGL-beta inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metabolism including abhydrolase domain-containing protein 6, abhydrolase domain-containing protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concentration-dependently reduced 2-AG levels, but not anandamide levels, in Neuro2A cells. We show that cannabinoid CB1-receptor-mediated short-term synaptic plasticity in a mouse hippocampal slice model can be reduced by LEI105. Thus, we have developed a highly selective DAGL inhibitor and provide new pharmacological evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.

BACKGROUND AND PURPOSE: The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) alpha and beta is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects. EXPERIMENTAL APPROACH: Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice. KEY
RESULTS: Of the new compounds, O-7460 exhibited the highest potency (IC50 = 690 nM) against the human recombinant DAGLalpha, and selectivity (IC50 > 10 muM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations >/=10 muM, and showed that this compound has only one major 'off-target', that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB1 or CB2 cannabinoid receptors (Ki > 10 muM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 muM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0-12 mg.kg(-1) , i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight. CONCLUSIONS AND IMPLICATIONS: O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions. LINKED ARTICLES: This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http:\/\/dx.doi.org/10.1111/bph.2013.169.issue-4 & http:\/\/dx.doi.org/10.1111/bph.2012.167.issue-8.

Abstract The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase alpha (DGLalpha) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced 'on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGLalpha and stored in neurons, and that 2-AG is released from such 'pre-formed pools' without the participation of DGLalpha. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGLalpha or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca2+ elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca2+-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca2+ elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGLalpha rather than mobilization from presumptive pre-formed pools.

Acute stress reduces pain sensitivity by engaging an endocannabinoid signaling circuit in the midbrain. The neural mechanisms governing this process and molecular identity of the endocannabinoid substance(s) involved are unknown. We combined behavior, pharmacology, immunohistochemistry, RNA interference, quantitative RT-PCR, enzyme assays, and lipidomic analyses of endocannabinoid content to uncover the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in controlling pain sensitivity in vivo. Here, we show that footshock stress produces antinociception in rats by activating type 5 metabotropic glutamate receptors (mGlu(5)) in the dorsolateral periaqueductal gray (dlPAG) and mobilizing 2-AG. Stimulation of mGlu(5) in the dlPAG with DHPG [(S)-3,5-dihydroxyphenylglycine] triggered 2-AG formation and enhanced stress-dependent antinociception through a mechanism dependent upon both postsynaptic diacylglycerol lipase (DGL) activity, which releases 2-AG, and presynaptic CB(1) cannabinoid receptors. Pharmacological blockade of DGL activity in the dlPAG with RHC80267 [1,6-bis(cyclohexyloximinocarbonylamino)hexane] and (-)-tetrahydrolipstatin (THL), which inhibit activity of DGL-alpha and DGL-beta isoforms, suppressed stress-induced antinociception. Inhibition of DGL activity in the dlPAG with THL selectively decreased accumulation of 2-AG without altering levels of anandamide. The putative 2-AG-synthesizing enzyme DGL-alpha colocalized with mGlu(5) at postsynaptic sites of the dlPAG, whereas CB(1) was confined to presynaptic terminals, consistent with a role for 2-AG as a retrograde signaling messenger. Finally, virally mediated silencing of DGL-alpha, but not DGL-beta, transcription in the dlPAG mimicked effects of DGL inhibition in suppressing both endocannabinoid-mediated stress antinociception and 2-AG formation. The results indicate that activation of the postsynaptic mGlu(5)-DGL-alpha cascade triggers retrograde 2-AG signaling in vivo. This pathway is required for endocannabinoid-mediated stress-induced analgesia.

The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLalpha and DAGLbeta. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here we report a series of 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLbeta in living systems. Optimized inhibitors showed high selectivity for DAGLbeta over other serine hydrolases, including DAGLalpha ( approximately 60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb(-/-) mice, we show that DAGLbeta inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2. We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-alpha release. These findings indicate that DAGLbeta is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

Until recently, endocannabinoid (eCB) signalling was largely studied in the context of synaptic plasticity in the postnatal brain in the absence of detailed knowledge of the nature of the enzyme(s) responsible for the synthesis of the eCBs. However, the identification of two diacylglycerol lipases (DAGLalpha and DAGLbeta) responsible for the synthesis of 2-arachidonoylglycerol (2-AG) has increased the understanding of where this eCB is synthesised in relationship to the expression of cannabinoid receptor (CB)1 and CB2. Furthermore, the generation of knockout animals for each enzyme has allowed for the direct testing of their importance for established and emerging eCB functions. Based on this, we now know that DAGLalpha is enriched in dendritic spines that appose CB1-positive synaptic terminals, and that 2-AG functions as a retrograde signal controlling synaptic strength throughout the nervous system. Consequently, we have built on the principle that expression of eCB components dictates function to identify additional physiological functions of this signalling cassette. A number of studies have now provided support for DAGL-dependent eCB signalling playing important roles in brain development and in cellular plasticity in the adult nervous system. In this article, we will review evidence based on the localisation of the enzymes, as well as from genetic and pharmacological studies, that show DAGL-dependent eCB signalling to play an important role in axonal growth and guidance during development, in retrograde synaptic signalling at mature synapses, and in the control of adult neurogenesis in the hippocampus and subventricular zone.

Endocannabinoids are released from postsynaptic neurons and cause retrograde suppression of synaptic transmission. Anandamide and 2-arachidonoylglycerol (2-AG) are regarded as two major endocannabinoids. To determine to what extent 2-AG contributes to retrograde signaling, we generated and analyzed mutant mice lacking either of the two 2-AG synthesizing enzymes diacylglycerol lipase alpha (DGLalpha) and beta (DGLbeta). We found that endocannabinoid-mediated retrograde synaptic suppression was totally absent in the cerebellum, hippocampus, and striatum of DGLalpha knockout mice, whereas the retrograde suppression was intact in DGLbeta knockout brains. The basal 2-AG content was markedly reduced and stimulus-induced elevation of 2-AG was absent in DGLalpha knockout brains, whereas the 2-AG content was normal in DGLbeta knockout brains. Morphology of the brain and expression of molecules required for 2-AG production other than DGLs were normal in the two knockout mice. We conclude that 2-AG produced by DGLalpha, but not by DGLbeta, mediates retrograde suppression at central synapses.

Although inhibitors of the enzymatic hydrolysis of the endocannabinoid 2-arachidonoylglycerol are available, they are either rather weak in vitro (IC(50)>30 microM) or their selectivity towards other proteins of the endocannabinoid system has not been tested. Here we describe the synthesis and activity in vitro and in vivo of a tetrahydrolipstatin analogue, OMDM169, as a potent inhibitor of 2-AG hydrolysis, capable of enhancing 2-AG levels and of exerting analgesic activity via indirect activation of cannabinoid receptors. OMDM169 exhibited 0.13 microM10 microM) at human CB(1) and CB(2) receptors. However, OMDM169 shared with tetrahydrolipstatin the capability of inhibiting the human pancreatic lipase (IC(50)=0.6 microM). OMDM169 inhibited fatty acid amide hydrolase and diacylglycerol lipase only at higher concentrations (IC(50)=3.0 and 2.8 microM, respectively), and, accordingly, it increased by approximately 1.6-fold the levels of 2-AG, but not anandamide, in intact ionomycin-stimulated N18TG2 neuroblastoma cells. Acute intraperitoneal (i.p.) administration of OMDM169 to mice inhibited the second phase of the formalin-induced nocifensive response with an IC(50) of approximately 2.5 mg/kg, and concomitantly elevated 2-AG, but not anandamide, levels in the ipsilateral paw of formalin-treated mice. The antinociceptive effect of OMDM169 was antagonized by antagonists of CB(1) and CB(2) receptors, AM251 and AM630, respectively (1 mg/kg, i.p.). OMDM69 might represent a template for the development of selective and even more potent inhibitors of 2-AG hydrolysis.

Spinocerebellar ataxia type 20 (SCA20) has been linked to chromosome 11q12, but the underlying genetic defect has yet to be identified. We applied single-nucleotide polymorphism genotyping to detect structural alterations in the genomic DNA of patients with SCA20. We found a 260 kb duplication within the previously linked SCA20 region, which was confirmed by quantitative polymerase chain reaction and fiber fluorescence in situ hybridization, the latter also showing its direct orientation. The duplication spans 10 known and 2 unknown genes, and is present in all affected individuals in the single reported SCA20 pedigree. While the mechanism whereby this duplication may be pathogenic remains to be established, we speculate that the critical gene within the duplicated segment may be DAGLA, the product of which is normally present at the base of Purkinje cell dendritic spines and contributes to the modulation of parallel fiber-Purkinje cell synapses.

Enzymes for the biosynthesis and degradation of the endocannabinoid 2-arachidonoyl glycerol (2-AG) have been cloned and are the sn-1-selective-diacylglycerol lipases alpha and beta (DAGLalpha and beta) and the monoacylglycerol lipase (MAGL), respectively. Here, we used membranes from COS cells over-expressing recombinant human DAGLalpha to screen new synthetic substances as DAGLalpha inhibitors, and cytosolic fractions from wild-type COS cells to look for MAGL inhibitors. DAGLalpha and MAGL activities were assessed by using sn-1-[14C]-oleoyl-2-arachidonoyl-glycerol and 2-[3H]-arachidonoylglycerol as substrates, respectively. We screened known compounds as well as new phosphonate derivatives of oleic acid and fluoro-phosphinoyl esters of different length. Apart from the general lipase inhibitor tetrahydrolipstatin (orlistat) (IC50 approximately 60 nM), the most potent inhibitors of DAGLalpha were O-3640 [octadec-9-enoic acid-1-(fluoro-methyl-phosphoryloxymethyl)-propylester] (IC50 = 500 nM), and O-3841 [octadec-9-enoic acid 1-methoxymethyl-2-(fluoro-methyl-phosphinoyloxy)-ethyl ester] (IC50 = 160 nM). Apart from being almost inactive on MAGL, these two compounds showed high selectivity over rat liver triacylglycerol lipase, rat N-acylphosphatidyl-ethanolamine-selective phospholipase D (involved in anandamide biosynthesis), rat fatty acid amide hydrolase and human recombinant cannabinoid CB1 and CB2 receptors. Methylarachidonoyl-fluorophosphonate and the novel compound UP-101 [O-ethyl-O-p-nitro-phenyl oleylphosphonate] inhibited both DAGLalpha and MAGL with similar potencies (IC50 = 0.8-0.1 and 3.7-3.2 microM, respectively). Thus, we report the first potent and specific inhibitors of the biosynthesis of 2-AG that may be used as pharmacological tools to investigate the biological role of this endocannabinoid.

We describe a pedigree of Anglo-Celtic origin with a phenotypically unique form of dominantly inherited spinocerebellar ataxia (SCA) in 14 personally examined affected members. A remarkable observation is dentate nucleus calcification, producing a low signal on MRI sequences. Unusually for an SCA, dysarthria is typically the initial manifestation. Mild pyramidal signs and hypermetric saccades are noted in some. Its distinguishing clinical features, each present in a majority of affected persons, are palatal tremor, and a form of dysphonia resembling spasmodic dysphonia. Repeat expansion detection failed to identify either CAG/CTG or ATTCT/AGAAT repeat expansions segregating with the disease in this family. The testable SCA mutations have been excluded. On linkage analysis, the locus maps to chromosome 11, which rules out all the remaining mapped SCAs except for SCA5. While locus homogeneity with SCA5 is not formally excluded, we consider it rather unlikely on phenotypic grounds, and propose that this condition may represent an addition to the group of neurogenetic disorders subsumed under the rubric SCA. The International Nomenclature Committee has made a provisional assignment of 'SCA20', although firm designation will have to await a definite molecular distinction from SCA5.

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.

As an extension of our cDNA analysis for deducing the coding sequences of unidentified human genes, we have newly determined the sequences of 100 cDNA clones from a set of size-fractionated human brain cDNA libraries, and predicted the coding sequences of the corresponding genes, named KIAA0611 to KIAA0710. In vitro transcription-coupled translation assay was applied as the first screening to select cDNA clones which produce proteins with apparent molecular mass of 50 kDa and over. One hundred unidentified cDNA clones thus selected were then subjected to sequencing of entire inserts. The average size of the inserts and corresponding open reading frames was 4.9 kb and 2.8 kb (922 amino acid residues), respectively. Computer search of the sequences against the public databases indicated that predicted coding sequences of 87 genes were similar to those of known genes, 62% of which (54 genes) were categorized as proteins related to cell signaling/communication, cell structure/motility and nucleic acid management. The expression profiles in 10 human tissues of all the clones characterized in this study were examined by reverse transcription-coupled polymerase chain reaction and the chromosomal locations of the clones were determined by using human-rodent hybrid panels.