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Diacylglycerol lipase ? manipulation reveals developmental roles for intercellular endocannabinoid signaling.

ABSTRACT: Endocannabinoids are small signaling lipids, with 2-arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB? cannabinoid receptors (CB?Rs) remains poorly understood. Here, we first show that cholinergic innervation of CA1 pyramidal cells of the hippocampus is sensitive to the genetic disruption of 2-AG signaling in DAGL? null mice. Next, we exploit a hybrid COS-7-cholinergic neuron co-culture system to demonstrate that heterologous DAGL? overexpression spherically excludes cholinergic growth cones from 2-AG-rich extracellular environments, and minimizes cell-cell contact in vitro. CB?R-mediated exclusion responses lasted 3 days, indicating sustained spherical 2-AG availability. Overall, these data suggest that extracellular 2-AG concentrations can be sufficient to activate CB?Rs along discrete spherical boundaries to modulate neuronal responsiveness.

SUBMITTER: Keimpema E

PROVIDER: S-EPMC3695556 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Diacylglycerol lipase α manipulation reveals developmental roles for intercellular endocannabinoid signaling.

Keimpema Erik E Alpár Alán A Howell Fiona F Malenczyk Katarzyna K Hobbs Carl C Hurd Yasmin L YL Watanabe Masahiko M Sakimura Kenji K Kano Masanobu M Doherty Patrick P Harkany Tibor T

Scientific reports 20130101

Endocannabinoids are small signaling lipids, with 2-arachidonoylglycerol (2-AG) implicated in modulating axonal growth and synaptic plasticity. The concept of short-range extracellular signaling by endocannabinoids is supported by the lack of trans-synaptic 2-AG signaling in mice lacking sn-1-diacylglycerol lipases (DAGLs), synthesizing 2-AG. Nevertheless, how far endocannabinoids can spread extracellularly to evoke physiological responses at CB₁ cannabinoid receptors (CB₁Rs) remains poorly unde ...[more]

PMID: 23806960

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Project description:IntroductionLipids 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 α and β (DAGLα, DAGLβ) 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.MethodsDAGLα and DAGLβ 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 DAGLβ transcripts to different cell types of the placenta. DAGLβ 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 µM] 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.ResultsWe demonstrate that mRNA expression of DAGLβ prevails in placental tissue, compared to DAGLα (p ≤ 0.0001) and that DAGLβ is mainly located to CK7 positive trophoblasts (p ≤ 0.0001). Although few DAGLα transcripts were identified, no active enzyme was detected applying in-gel or MS-based ABPP, which underlined that DAGLβ is the principal DAGL in the placenta. DAGLβ dependent substrate hydrolysis in placental membrane lysates was determined by the application of LEI-105 and DH376. Ex vivo pharmacological inhibition of DAGLβ 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.DiscussionOur results emphasize the role of DAGLβ 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.

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Diacylglycerol lipase ? manipulation reveals developmental roles for intercellular endocannabinoid signaling.

Publications

Diacylglycerol lipase α manipulation reveals developmental roles for intercellular endocannabinoid signaling.

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