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ArticlesJanuary 20, 2017

Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases
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Cite this: ACS Chem. Biol. 2017, 12, 3, 852–861

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https://doi.org/10.1021/acschembio.6b01052

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Abstract

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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-α, 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-α 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.

Subjects

The endocannabinoid system consists of the cannabinoid type 1 and 2 (CB₁ and CB₂) receptors, lipid messengers termed endocannabinoids, and the hydrolytic enzymes responsible for the biosynthesis and catabolism of these lipid signaling molecules. 2-Arachidonoylglycerol (2-AG) and anandamide (AEA) are the two main endocannabinoids. (1) The CB₁ receptor has a relatively high expression in the central nervous system, while the CB₂ receptor is more abundant in immune cells. The CB₁ receptor is among the most abundant G-protein coupled receptors in the brain and modulates a wide variety of signaling events, including inhibition of adenylate cyclase activity, stimulation of ERK activation, closure of voltage-sensitive Ca²⁺ channels, and opening of K⁺ channels. (2, 3) Activation of the CB₁ receptor is associated with multiple physiological processes, such as energy balance, learning and memory, pain sensation, and neuro-inflammation. (4-7) Unlike classical polar neurotransmitters, which are stored in presynaptic vesicles, 2-AG and AEA are synthesized “on-demand” from postsynaptic membranes and act as retrograde messengers activating presynaptic CB₁ receptors, thereby modulating neurotransmitter release. This implies that the biosynthetic and catabolic machinery of the endocannabinoids tightly regulates CB₁ receptor activation. (8)

Several biosynthetic and catabolic endocannabinoid hydrolases control 2-AG and AEA levels (Figure 1). Diacylglycerol lipase-α and -β (DAGL-α and DAGL-β) are the main enzymes producing 2-AG. They display a tissue specific distribution and studies using mice with congenital deletion of DAGL-α or DAGL-β have identified DAGL-α as the primary enzyme responsible for the biosynthesis of 2-AG in the brain. (9, 10) α,β-Hydrolase-domain-containing protein 6 and 12 (ABHD6 and ABHD12) and monoacylglycerol lipase (MAGL) inactivate 2-AG by hydrolysis to give arachidonic acid (AA) and glycerol. MAGL is responsible for the bulk hydrolysis of 2-AG, while ABHD6 and ABHD12 play a more distinct role in specific cell populations. (11)

Figure 1. Biosynthetic and catabolic pathways of 2-AG and AEA.

The biosynthetic pathways toward AEA appear to be more complex compared with those of 2-AG. (4, 6)N-arachidonoyl-phosphatidylethanolamine (NArPE) is a key intermediate in AEA biosynthesis. NArPE can be converted via multiple phospholipase-dependent pathways to AEA: (a) hydrolysis by N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD), a metallo-β-lactamase, producing AEA and a phosphatidic acid in one step (12) and (b) phospholipase A₂ (PLA₂) and α,β-hydrolase domain-containing protein 4 (ABHD4) mediated conversion to lyso-NArPE, followed by the action of an unknown lysophospholipase D (PLD). Of note, lyso-NArPE can also be converted in a two-step sequence by ABHD4 to glycerophospho-AEA (GP-AEA) and subsequently hydrolyzed to AEA by glycerophosphodiesterase 1 or 4 (GDE1 or GDE4); (13, 14) (c) in macrophages, NArPE serves as a substrate for an unidentified phospholipase C yielding phospho-AEA. Hydrolysis of the phosphate group by the phosphatases, PTPN22 or SHIP1, provides AEA. (15, 16) The enzymatic inactivation of AEA is less complex and is primarily mediated by fatty acid amide hydrolase (FAAH), which hydrolyses AEA toward arachidonic acid and ethanolamine. (4, 17)

The ability to monitor the activity of the different endocannabinoid hydrolases in various brain regions is key to gaining insight in spatiotemporal control of CB₁ receptor activation and its physiological role. The existence of a feedback mechanism in anterograde neurotransmitter systems through presynaptic “autoreceptors” to control the release of the neurotransmitters has led to the hypothesis that such an autoregulatory mechanism could also be present in retrograde systems, such as the endocannabinoid system. (18-20) Previous studies have investigated the effect of CB₁ receptor modulation on basal endocannabinoid levels. Several studies observed changes in AEA and/or 2-AG levels, (21-25) whereas others did not. (18, 26) Interestingly, Maccarrone et al. found that the endocannabinoid system of CB₁ knockout mice adapted with age by upregulating AEA catabolism. (23, 24) Recently, Belluomo et al. reported that the MAGL inhibitor JZL-195 reduced 2-AG accumulation rates in the frontal cortex of mice lacking the CB₁ receptor in glutamatergic neurons and an increase of 2-AG accumulation under the same conditions in mice with congenital deletion of the CB₁ receptor in astrocytes. (18) In addition, chronic elevation of 2-AG levels by genetic deletion of MAGL or repeated administration of JZL184 led to CB₁ receptor desensitization. (27) These observations suggest the existence of crosstalk between the CB₁ receptor and the endocannabinoid regulatory machinery. The pre- and postsynaptic autoregulatory mechanisms controlling endocannabinoid levels are, however, poorly understood.

Changes in bulk endocannabinoid levels do not reveal which biosynthetic or catabolic pathways are responsible for the regulation of CB₁ activity. (18, 26) To this end, the activity of each hydrolytic enzyme should be studied. Over the years, a brain region and cell type resolved map of the molecular distribution of endocannabinoid hydrolases has been generated by in situ hybridization and global proteomics. (28-30) These studies provided a detailed understanding of the molecular composition of the endocannabinoid system in different brain regions at the mRNA and protein level. However, actual enzymatic activity does not always correlate with mRNA and protein levels in specific brain regions due to post-transcriptional and post-translational processes. (31, 32) The activity of DAGL-α is, for example, regulated by CaMKII-mediated phosphorylation, and MAGL activity can be modulated by sulfenylation of specific cysteines. (33, 34) Consequently, it is important to measure actual enzyme activity in the various brain regions.

Conventional enzyme activity assays rely on radiolabeled substrates and LC/MS-based methods. (35) These assays are expensive and time-consuming and measure the activity of one enzyme at a time. Others and we have recently applied comparative activity-based protein profiling (ABPP) and chemoproteomics to measure serine hydrolase activity in complex proteomes. (36-39) ABPP is a technique pioneered by Cravatt et al., which relies on active site directed chemical probes that react, in a mechanism based fashion, with the catalytic nucleophile of targeted enzymes. The probe establishes a covalent bond with the catalytically active protein and therefore reports on the abundance of active enzymes. Activity-based probes (ABPs) generally have a fluorescent or biotin reporter group for visualization and identification, respectively. (36) Many enzymes regulating brain endocannabinoid levels, including MAGL, DAGL, FAAH, ABHD4, ABHD6, and ABHD12, belong to the serine hydrolase family; therefore they have conserved structural features, which allow for their targeting by specific ABPs, such as FP-TAMRA and MB064. Here, we have used ABPP to map the relative activity of endocannabinoid hydrolases in different brain regions, such as cerebellum, frontal cortex, hippocampus, and striatum. We applied this method to investigate the existence of an autoregulatory feedback mechanism of the CB₁ receptor on the endocannabinoid hydrolase activity by comparing the brains of CB₁ receptor knockout mice versus wild-type mice.

Results and Discussion

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Gel-based Brain Region Comparison

Previously, a β-lactone (MB064) and fluorophosphonate (FP-TAMRA) were applied as tools to study the selectivity of the DAGL inhibitors LEI104, LEI105, and DH376. (38, 40) The use of two ABPs extends the range of enzymes, because the probes have orthogonal warheads and different recognition elements, leading to a different interaction profile. Here, both ABPs were used to map brain region dependent activity of endocannabinoid hydrolases by comparative ABPP. Global assessment of serine hydrolase activity across four different brain regions was performed using a gel-based assay. Membrane and soluble proteomes from mouse cerebellum, frontal cortex, hippocampus, or striatum were incubated with MB064 (250 nM) or FP-TAMRA (500 nM) and resolved on SDS-PAGE, and the in-gel fluorescence was analyzed. Coomassie staining was used as a protein loading control. Using MB064, nine intense bands were identified in the membrane fractions of the mouse brain regions, and the identity of endocannabinoid hydrolase bands could be established using reference inhibitors and KO tissue as previously reported. (38, 40)

In this manner, the bands at 120 kDa, 45 kDa, and 35 kDa were ascribed to DAGL-α, ABHD12, and ABHD6, respectively. FP-TAMRA labeled FAAH (64 kDa), MAGL (35 and 38 kDa), and ABHD6 (35 kDa). All identified endocannabinoid proteins were present in the four brain regions, but several hydrolases demonstrated pronounced region-dependent activity (Figure 2). For example, DAGL-α activity was ∼3 times higher in the cerebellum compared to the frontal cortex, hippocampus, and striatum (Figure 2A,B). Interestingly, the fluorescent protein band corresponding to DAGL-α in the cerebellum was shifted toward a higher molecular weight compared to the hippocampus, striatum, and frontal cortex. This could indicate that DAGL-α carries a post-translational modification. The activity of ABHD12 was similar in all four brain regions, while the activity of ABHD6 was ∼25% higher in the cerebellum compared to the frontal cortex, hippocampus, and the striatum. The fluorophosphonate-based ABP revealed the activity of FAAH to be ∼2 times higher in the frontal cortex and the hippocampus compared to the cerebellum and striatum. The signals of MAGL and ABHD6 as measured with this activity-based probe are overlapping and could not be quantified independently (Figure 2C).

Figure 2. Gel-based mapping of brain region dependent hydrolase activity. (A) Activity-based protein profiling with MB064 in four mouse brain regions. (B) Quantification of endocannabinoid hydrolase activity as determined with MB064 (mean ± SD; n = 3). (C) Activity-based protein profiling with FP-TAMRA in four mouse brain regions. (D) Quantification of FAAH activity as measured with ABP FP-TAMRA (mean ± SD; n = 3). Statistical analysis was performed by means of two-way ANOVA with Tukey multiple comparisons test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; C = cerebellum, F = frontal cortex, H = hippocampus, S = striatum). Integrated band intensity is corrected for protein loading (coomassie).

Chemoproteomic Brain Region Comparison

To analyze the relative activities in depth, ABPP was coupled to high resolution mass spectrometry. This methodology enables direct identification of the enzymes and provides a more accurate quantification by avoiding band overlap and yields a broader range of hydrolases due to a higher sensitivity. Membrane and soluble proteomes from cerebellum, hippocampus, frontal cortex, or striatum were separately incubated with MB108 or FP-biotin, biotinylated versions of MB064 and FP-TAMRA, respectively. Targeted enzymes were enriched by avidin chromatography, followed by on-bead digestion using sequencing grade trypsin. Tryptic peptides from different brain regions were isotopically labeled by reductive dimethylation with deuterated or nondeuterated formaldehyde. After 1:1 mixing of the differentially labeled brain regions, samples were measured by high resolution MS/MS and analyzed using Maxquant software (a schematic overview of the chemoproteomic workflow is given in Figure 3A). (41)

Figure 3. Brain region dependent chemoproteomic mapping of relative hydrolase activity. (A) Schematic representation of the chemoproteomic workflow. (B) Heat map of relative enzyme activity as measured by a fluorophosphonate (FP)-based activity-based probe conjugated to a biotin reporter tag (FP-biotin) and a β-lactone-based activity-based probe conjugated to a biotin reporter tag (MB108). Data are calculated from the mean ratios of the comparison between cerebellum and striatum, frontal cortex and striatum, and cerebellum and hippocampus. Each comparison was performed in three biological replicates. The relative enzyme activity in the brain region in which the serine hydrolase displayed highest activity was set to 100%. (C) Correlation graph for enzymes detected by both MB108 and FP-biotin. (D) Endocannabinoid regulating enzymes show significant difference between the studied brain regions (mean ± SEM; n = 3). Statistical analysis was performed by means of two-way ANOVA with Tukey multiple comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001) (C = cerebellum, F = frontal cortex, H = hippocampus, S = striatum).

Using this chemoproteomic methodology, the relative activity of 33 different serine hydrolases was quantified in all brain regions, including DAGL-α, FAAH, ABHD12, MAGL, ABHD6, and ABHD4 (see Figure 3 for a heat map). Eleven enzymes reacted with both probes. A high correlation was found between the quantified enzymatic activities for each probe (Pearson’s correlation of 0.86 (P < 0.0001)), indicating that probe-independent protein activity was measured (Figure 3). The fluorophosphonate-based ABP targeted 14 unique proteins, including MAGL and FAAH, whereas the β-lactone-based ABP MB108 targeted seven unique proteins including ABHD4, ABHD12, and DAGL-α. ABHD4 showed an equal activity across all brain regions, while FAAH had the highest activity in the hippocampus and frontal cortex. The activity of DAGL-α in the cerebellum was ∼2-fold higher compared to the striatum, hippocampus, and frontal cortex. Interestingly, MAGL activity was lowest in the cerebellum. The results from the chemoproteomic analysis were in line with the gel-based ABPP method, except for ABHD6, which is likely to be caused by band overlap in the gel-based assay. Further detailed analysis showed that overall relative enzymatic activities followed the same trends as observed for the protein abundance derived from a global proteomics data set published by Sharma et al. (29) (Supporting Figure 1). However, several pronounced differences were detected. These different intensity profiles for activity and protein abundance may suggest that protein activity is regulated by post-translational modifications. For example, the activity of ABHD12 was equally distributed over the four brain regions, while ABHD12 abundance in the hippocampus was twice as high as in the cerebellum, frontal cortex and striatum. These different profiles might be explained by a down regulation of ABHD12 activity in the hippocampus. DAGL-α activity was highest in the cerebellum, while its relative activity in the frontal cortex was 47.2 ± 8.6%, 64.6 ± 9.0% in the hippocampus and 36.3 ± 5.4% in the striatum. In contrast, global proteomics data showed the highest abundance of DAGL-α in the hippocampus, while less than 30% was found in the cerebellum, frontal cortex, and striatum. To investigate this apparent discrepancy, we have performed a Western blot analysis to check the DAGL-α protein levels in the four brain regions of our mice. We observed relatively high DAGL-α abundance in the cerebellum (Supporting Figure 2), which matched the relative activity as measured with our ABPP method.

Many other serine hydrolases were detected next to the endocannabinoid hydrolases (Figure 3). Marked differences in activity between the brain regions were observed for some of these enzymes. For example, acetylcholinesterase (ACHE), which is responsible for acetylcholine hydrolysis, showed over 10-fold activity in the striatum compared to the other three brain regions. ABHD16A, which is a phosphatidylserine lipase producing lyso-phosphatidyl-serine, (42) demonstrated ∼2-fold increased activity in the cerebellum compared to other brain regions. Platelet-activating factor acetylhydrolase (PLA2G7), which cleaves the sn-2 acetyl of acetyl-glyceryl-ether-phosphorylcholine, showed an approximate 3-fold higher activity in the cerebellum compared to the other brain regions. (43)

Comparison of CB₁^+/+ and CB₁^–/– Brain Regions

Next, the regulatory control of the CB₁ receptor over basal production and degradation of endocannabinoids was investigated. To this end, the enzymatic activity of hydrolases involved in endocannabinoid biosynthesis and degradation in the cerebellum, hippocampus, frontal cortex, and striatum of CB₁ receptor knockout mice was compared to their wild-type counterparts. Fluorescent scanning of gels from ABPP experiments using MB064 and FP-TAMRA did not reveal any difference in labeling patterns in the brain regions (Figure 4).

Figure 4. Gel based CB1^+/+ vs CB1^–/– comparison. Study of enzyme activity in CB1^+/+ and CB1^–/– mouse brain regions as measured by two activity based probes (MB064 and TAMRA-FP, n = 3). No difference in enzyme activity between CB1^–/– and CB1^+/+ was observed.

To investigate in depth the effect of CB₁ receptor deletion on serine hydrolase activity, the chemoproteomic assay in the four mouse brain regions was performed. The relative activities of 36 different hydrolases were detected and quantified (Figure 5). No difference in the enzymatic activity of the 2-AG biosynthetic enzyme (DAGL-α) and the 2-AG catabolic enzymes ABHD6, ABHD12, and MAGL was observed in the cerebellum, frontal cortex, hippocampus, or striatum. Nor were any changes observed in ABHD4 and FAAH activities.

Figure 5. Comparison of enzyme activity in CB1^+/+ and CB1^–/– mouse brain regions. Log₂ ratio of enzyme activity in CB1^–/– brain regions compared to CB1^+/+ (mean ± SD). Activity is measured by activity-based proteomics using FP-biotin (10 μM) and MB108 (10 μM). Combined data from both activity based probes (n = 4 for each brain region). Statistical analysis by means of one-way ANOVA, each CB1 WT/KO ratio was compared to a Log₂ ratio of 0; subsequently the p values were subjected to Benjamini Hochberg correction. Setting the false discovery rate at 10%.

Discussion

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The enzymes responsible for endocannabinoid biosynthesis and degradation tightly regulate endocannabinoid CB₁ receptor activation. A fluorescence and chemoproteomic ABPP assay was employed to quantify the activity of hydrolases in the cerebellum, hippocampus, striatum, and frontal cortex to gain more insight in the spatiotemporal control of CB₁ receptor-mediated physiology. ABPP relies on the mechanism-based formation of a covalent bond between the ABP and the catalytic nucleophile of its target enzyme. Therefore, the abundance of only active enzymes is monitored by ABPP. The gel-based fluorescence assay has a relatively short experimental time (∼3h) and has the ability to retrieve information on differences in protein migration on SDS-PAGE caused by, e.g., phosphorylation, glycosylation, or proteolysis. The chemoproteomic methodology is complementary and directly reports on the identity and activity of a large number of enzymes in parallel in their native environment with all post-translational modifications in place.

Previous studies have indicated the importance of analyzing different brain regions when studying the endocannabinoid system. Endocannabinoid levels have been shown to vary between brain regions. Moreover, the CB₁ receptor and the endocannabinoid regulatory machinery do not present a homogeneous expression across brain regions. (1, 26, 29, 44) These variations are confirmed in the comparative ABPP approach using cerebellum, hippocampus, frontal cortex, and striatum. Significant differences were found in the enzyme activity of DAGL-α, MAGL, ABHD6, and FAAH between different brain regions. For instance, the highest FAAH activity was observed in the hippocampus, which is in line with previous reports indicating that the hippocampus has the highest FAAH expression levels, highest AEA levels, and turnover rate compared to other brain regions. (18, 21, 26) These observations suggest that AEA has an important physiological role in the hippocampus, such as providing “on demand” CB₁ receptor-dependent neuroprotection against excitotoxicity. (45) Next to its role as an endocannabinoid, AEA functions also as an endogenous agonist of the TRPV1 ion channel. (46) It could be envisioned that FAAH not only terminates CB₁ receptor signaling but also limits TRPV1 signaling by AEA in the hippocampus. In this respect, it would be interesting to investigate whether TRPV1 mediates crosstalk between AEA and 2-AG biosynthesis as was previously observed in the striatum. (47) Of note, FAAH does not only inactivate AEA but uses a wide range of long chain fatty acid amides as substrates. The physiological role of these endogenous signaling lipids is poorly understood, but the hippocampus would be an excellent brain region in which to investigate their biological role.

DAGL-α activity was ∼2 fold higher in the cerebellum compared to the frontal cortex, striatum, and hippocampus. The activity of MAGL in the cerebellum was only 23.3 ± 11% of the total MAGL activity in the frontal cortex. In addition, no relative compensatory activity of ABHD6 and ABHD12 was observed in the cerebellum (Figure 2). Yet, 2-AG levels are not substantially higher in the cerebellum. (21, 26, 48) This may suggest that 2-AG levels and CB₁ receptor activity are controlled by MAGL, rather than by 2-AG biosynthesis, or that other catabolic pathways, such as oxidative catabolism, could play a role in this brain region.

Comparison of the activity-based proteomics data with protein expression values from the literature showed an overall correlation. For several enzymes (e.g., ABHD16A, ABHD10, ABHD12), however, the activity profile did not match the protein abundance profile as reported in a global proteomics data set. (29) This may indicate that the activity of these proteins is regulated by post-translational modifications or could result from differences in the protein expression due to variation in age and mouse species analyzed in the various studies (as observed with DAGL-α).

Previous studies have investigated the effect of CB₁ receptor modulation by pharmacological or genetic means on endocannabinoid levels. (18, 21-26) Here, this question was revisited, and the ABPP-methodology was employed to study the effect of genetic deletion of the CB₁ receptor on endocannabinoid hydrolase activity in various brain regions. No significant differences in the activity of endocannabinoid biosynthetic and catabolic enzymes between CB₁ KO and WT brain regions were observed. These results are in line with previous reports. (26, 18) Yet, it cannot be excluded that specific CB₁ receptor populations might increase production and/or degradation while others might decrease it, leading to a lack of change when removing all these receptor populations. In support of this hypothesis is the observation that within the frontal cortex the turnover of 2-AG is decreased and increased in mutant mice lacking CB₁ receptors on principal neurons (possibly glutamatergic) and on astrocytes, respectively. (18)

Of note, the applied technique has several limitations. The total enzyme activity is measured by an end point measurement using a covalent inhibitor. Therefore, small changes in the K_M as a result of post-translational modifications are difficult to monitor by ABPP. Additionally, although the proteomics assay is capable of detecting a wide array of hydrolases that play an important role in regulation of endocannabinoid signaling, this methodology is not compatible with enzymes that do not form a covalent intermediate with their substrate, such as the β-metallo-lactamase NAPE-PLD. Importantly, other mechanisms that regulate endocannabinoid signaling, such as the putative endocannabinoid transporter proteins and oxidative metabolism of endocannabinoids toward eicosanoids, are not taken into account by the ABPP-method.

In conclusion, we have employed an ABPP method that can measure the activity of six different enzymes with endocannabinoid hydrolase activity in their native setting in a single experiment. This methodology was used to map endocannabinoid hydrolase activity in the cerebellum, striatum, frontal cortex, and cerebellum. This revealed brain region specific differences in endocannabinoid hydrolase activity. The method was applied to study the effect of genetic deletion of the CB₁ receptor in these brain regions. The results indicate that the CB1 does not exert regulatory control over the basal production and degradation of endocannabinoids and that genetic deletion of the CB₁ receptor does not induce any compensatory mechanism in endocannabinoid hydrolase activity.

Methods

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Animals

The experiments were conducted in strict compliance with European directives and French laws on animal experimentation (authorization number C33 12024 to F.C. from the French Ministry of Agriculture). The experiments were conducted on brains of male CB₁ WT and KO mice that were sacrificed at the age of 8–9 weeks. The mice were bred at the NeuroCentre INSERM U862.

Preparation of Mouse Tissue Proteome

The mouse brain regions, hippocampus, striatum, cerebellum, and frontal cortex, were slowly thawed on ice. The thawed mouse brain regions were dounce homogenized in cold (4 °C) pH 7.2 lysis buffer A (20 mM HEPES at pH 7.2, 2 mM DTT, 1 mM MgCl₂, 25 U/mL Benzonase) and incubated for 5 min on ice. The suspension was centrifuged (2500g, 3 min, 4 °C) to remove debris. The supernatant was collected and subjected to ultracentrifugation (100.000g, 45 min, 4 °C, Beckman Coulter, Type Ti70 rotor). This yielded the membrane fraction as a pellet and the cytosolic fraction in the supernatant. The supernatant was collected, and the membrane fraction was suspended in storage buffer (20 mM HEPES at pH 7.2, 2 mM DTT). The total protein concentration was determined with Quick Start Bradford assay (Biorad) or Qubit protein assay (Invitrogen). Membranes and supernatant fractions were both diluted to a total protein concentration of 0.5 mg mL^–1 and were used directly or flash frozen in liquid nitrogen and stored in aliquots at −80 °C until use.

Activity-based Protein Profiling

Mouse hippocampus, striatum, cerebellum, or frontal cortex (0.5 mg mL^–1) were incubated with activity-based probe MB064 (250 nM) or TAMRA-FP (500 nM) for 20 min. At room temperature, Laemlli buffer was added to quench the protein activity, and the mixture was allowed to stand at room temperature for 30 min before the samples were loaded and resolved on SDS page gel (10% acrylamide). The gels were scanned using a ChemiDoc MP system (Cy3 settings, 605/50 filter) and analyzed using Image lab 4.1. After fluorescent scanning, the gels were stained with a coomassie staining solution. After destaining, the gels were scanned, and protein loading was quantified using image lab 4.1. Gel fluorescence intensities were corrected for protein abundance (loading control).

Proteomics

Proteomics experiments were performed in a similar fashion to those reported previously. (38) A detailed protocol is given in the Supporting Information.

Statistical Analysis

Statistical analysis for mouse brain WT versus CB₁ knockout screen was performed. For proteins identified by both probes, the normalized ratios from Maxquant were combined for further analysis. Using GraphPad Prism 7.0 software, the binary logarithm of each ratio was compared to 0 with one-way ANOVA. The resulting p values were subjected to a Benjamini–Hochberg correction, setting the false discovery rate at 10% (q = 0.1). Briefly, the p values of all quantifiable hits were ordered from lowest to highest, and the Benjamini–Hochberg statistic was calculated as q * (position in the list) divided by the number of tests. Subsequently, the proteins for which the p value is smaller than the BH statistic are controlled for an FDR of q* 10%.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b01052.

Supporting Table 1 (XLSX)
Supporting Table 2 (XLSX)
Supporting Table 3 (XLSX)
Supporting Table 4 (XLSX)
Supporting Methods, Supporting Figures 1 and 2, Supporting Tables 1 and 2 (PDF)

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Author Information

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Corresponding Author
- Mario van der Stelt - Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands; Email: [email protected]
Authors
- Marc P. Baggelaar - Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands; http://orcid.org/0000-0002-9784-6250
- Annelot C. M. van Esbroeck - Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
- Eva J. van Rooden - Department of Molecular Physiology, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
- Bogdan I. Florea - Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
- Herman S. Overkleeft - Department of Bio-organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands
- Giovanni Marsicano - Plateforme de Chimie Analytique, NeuroCentre INSERM U862, Bordeaux, France
- Francis Chaouloff - Plateforme de Chimie Analytique, NeuroCentre INSERM U862, Bordeaux, France
Author Contributions
Contributed equally
Notes
The authors declare no competing financial interest.

References

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Dalton, George D.; Howlett, Allyn C.
British Journal of Pharmacology (2012), 165 (8), 2497-2511CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
Signalling networks that regulate the progression of cannabinoid CB1 receptor-mediated extracellular signal-regulated kinase (ERK) activation in neurons are poorly understood. We investigated the cellular mechanisms involved in CB1 receptor-stimulated ERK phosphorylation in a neuronal cell model. Murine N18TG2 neuronal cells were used to analyze the effect of specific protein kinase and phosphatase inhibitors on CB1 receptor-stimulated ERK phosphorylation. The LI-COR In Cell Western assay and immunoblotting were used to measure ERK phosphorylation. Results showed that the time-course of CB1 receptor-stimulated ERK activation occurs in three phases that are regulated by distinct cellular mechanisms in N18TG2 cells. Phase I (0-5 min) maximal ERK phosphorylation is mediated by CB1 receptor-stimulated ligand-independent transactivation of multiple receptor tyrosine kinases (RTKs). Phase I requires Gi/o βγ subunit-stimulated phosphatidylinositol 3-kinase activation and Src kinase activation and is modulated by inhibition of cAMP-activated protein kinase A (PKA) levels. Src kinase activation is regulated by the protein tyrosine phosphatases 1B and Shp1. The Phase II (5-10 min) rapid decline in ERK phosphorylation involves PKA inhibition and serine/threonine phosphatase PP1/PP2A activation. The Phase III (>10 min) plateau in ERK phosphorylation is mediated by CB1 receptor-stimulated, ligand-independent, transactivation of multiple RTKs. Thus, the complex expression of CB1 receptor-stimulated ERK activation provides cellular selectivity, modulation of sensitivity to agonists, and coincidence detection with RTK signaling. RTK and PKA pathways may provide routes to novel CB1-based therapeutic interventions in the treatment of addictive disorders or neurodegenerative diseases.
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Howlett, A. C., Barth, F., Bonner, T. I., Cabral, G., Casellas, P., Devane, W. A., Felder, C. C., Herkenham, M., Mackie, K., Martin, B. R., Mechoulam, R., and Pertwee, R. G. (2002) International Union of Pharmacology. XXVII. Classification of cannabinoid receptors Pharmacol. Rev. 54, 161– 202 DOI: 10.1124/pr.54.2.161
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International Union of Pharmacology. XXVII. Classification of cannabinoid receptors
Howlett, A. C.; Barth, F.; Bonner, T. I.; Cabral, G.; Casellas, P.; Devane, W. A.; Felder, C. C.; Herkenham, M.; Mackie, K.; Martin, B. R.; Mechoulam, R.; Pertwee, R. G.
Pharmacological Reviews (2002), 54 (2), 161-202CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
A review. Two types of cannabinoid receptor have been discovered so far, CB1 (2.1: CBD:1:CB1:), cloned in 1990, and CB2 (2.1:CBD:2:CB2:), cloned in 1993. Distinction between these receptors is based on differences in their predicted amino acid sequence, signaling mechanisms, tissue distribution, and sensitivity to certain potent agonists and antagonists that show marked selectivity for one or the other receptor type. Cannabinoid receptors CB1 and CB2 exhibit 48% amino acid sequence identity. Both receptor types are coupled through G proteins to adenylyl cyclase and mitogen-activated protein kinase. CB1 receptors are also coupled through G proteins to several types of calcium and potassium channels. These receptors exist primarily on central and peripheral neurons, one of their functions being to inhibit neurotransmitter release. Indeed, endogenous CB1 agonists probably serve as retrograde synaptic messengers. CB2 receptors are present mainly on immune cells. Such cells also express CB1 receptors, albeit to a lesser extent, with both receptor types exerting a broad spectrum of immune effects that includes modulation of cytokine release. Of several endogenous agonists for cannabinoid receptors identified thus far, the most notable are arachidonoylethanolamide, 2-arachidonoylglycerol, and 2-arachidonylglyceryl ether. It is unclear whether these eicosanoid mols. are the only, or primary, endogenous agonists. Hence, the authors consider it premature to rename cannabinoid receptors after an endogenous agonist as is recommended by the International Union of Pharmacol. Committee on Receptor Nomenclature and Drug Classification. Although pharmacol. evidence for the existence of addnl. types of cannabinoid receptor is emerging, other kinds of supporting evidence are still lacking.
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Ahn, K., McKinney, M. K., and Cravatt, B. F. (2008) Enzymatic pathways that regulate endocannabinoid signaling in the nervous system Chem. Rev. 108, 1687– 1707 DOI: 10.1021/cr0782067
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Enzymatic pathways that regulate endocannabinoid signaling in the nervous system
Ahn, Kay; McKinney, Michele K.; Cravatt, Benjamin F.
Chemical Reviews (Washington, DC, United States) (2008), 108 (5), 1687-1707CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
A review. Recent advances in the understanding of the compn. and regulation of endocannabinoid metabolic pathways, esp. with respect to the nervous system, are reviewed. The current state of the understanding of the 4 major pathways for endocannabinoid metab. are discussed. These include (1) anandamide degrdn.; (2) anandamide biosynthesis; (3) 2-arachidonoylglycerol (2-AG) degrdn.; and (4) 2-AG biosynthesis. Some of these pathways, such as anandamide degrdn., are relatively well characterized, at least in terms of the participating enzymes, esp. fatty acid amide hydrolase (FAAH), and the advent of specific research tools to probe their function in vivo. For the other pathways, candidate enzymes have been identified, but the specific roles that these proteins play in regulating endocannabinoid metab. in vivo remains to be elucidated.
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Di Marzo, V. (2011) Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight Nat. Neurosci. 14, 9– 15 DOI: 10.1038/nn.2720
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Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight
Di Marzo, Vincenzo
Nature Neuroscience (2011), 14 (1), 9-15CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
A review. Studies of the endocannabinoid system in the CNS have been mostly focused on endocannabinoid receptors and inactivating mechanisms. Until recently, very little was known about the role of biosynthetic enzymes in endocannabinoid signaling. New data from the recent development of pharmacol. and genetic tools for the study of these enzymes point to their fundamental role in detg. where and when endocannabinoids function, and raise the possibility of new intriguing and previously unsuspected concepts in the general strategy of endocannabinoid signaling. However, even with these new tools, the cross-talk between anandamide and 2-arachidonoylglycerol biosynthesis makes it difficult to dissect one from the other, and data will need to be interpreted with this in mind.
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Piomelli, D. (2014) More surprises lying ahead. The endocannabinoids keep us guessing Neuropharmacology 76 (B) 228– 234 DOI: 10.1016/j.neuropharm.2013.07.026
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Pacher, P., Batkai, S., and Kunos, G. (2006) The endocannabinoid system as an emerging target of pharmacotherapy Pharmacol. Rev. 58, 389– 462 DOI: 10.1124/pr.58.3.2
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The endocannabinoid system as an emerging target of pharmacotherapy
Pacher, Pal; Batkai, Sandor; Kunos, George
Pharmacological Reviews (2006), 58 (3), 389-462CODEN: PAREAQ; ISSN:0031-6997. (American Society for Pharmacology and Experimental Therapeutics)
A review. The recent identification of cannabinoid receptors and their endogenous lipid ligands has triggered an exponential growth of studies exploring the endocannabinoid system and its regulatory functions in health and disease. Such studies have been greatly facilitated by the introduction of selective cannabinoid receptor antagonists and inhibitors of endocannabinoid metab. and transport, as well as mice deficient in cannabinoid receptors or the endocannabinoid-degrading enzyme fatty acid amidohydrolase. In the past decade, the endocannabinoid system has been implicated in a growing no. of physiol. functions, both in the central and peripheral nervous systems and in peripheral organs. More importantly, modulating the activity of the endocannabinoid system turned out to hold therapeutic promise in a wide range of disparate diseases and pathol. conditions, ranging from mood and anxiety disorders, movement disorders such as Parkinson's and Huntington's disease, neuropathic pain, multiple sclerosis and spinal cord injury, to cancer, atherosclerosis, myocardial infarction, stroke, hypertension, glaucoma, obesity/metabolic syndrome, and osteoporosis, to name just a few. An impediment to the development of cannabinoid medications has been the socially unacceptable psychoactive properties of plant-derived or synthetic agonists, mediated by CB1 receptors. However, this problem does not arise when the therapeutic aim is achieved by treatment with a CB1 receptor antagonist, such as in obesity, and may also be absent when the action of endocannabinoids is enhanced indirectly through blocking their metab. or transport. The use of selective CB2 receptor agonists, which lack psychoactive properties, could represent another promising avenue for certain conditions. The abuse potential of plant-derived cannabinoids may also be limited through the use of prepns. with controlled compn. and the careful selection of does and route of administration. The growing no. of preclin. studies and clin. trials with compds. that modulate the endocannabinoid system will probably result in novel therapeutic approaches in a no. of diseases for which current treatments do not fully address the patients' need. Here, we provide a comprehensive overview on the current state of knowledge of the endocannabinoid system as a target of pharmacotherapy.
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Di Marzo, V., Bifulco, M., and De Petrocellis, L. (2004) The endocannabinoid system and its therapeutic exploitation Nat. Rev. Drug Discovery 3, 771– 784 DOI: 10.1038/nrd1495
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The endocannabinoid system and its therapeutic exploitation
Di Marzo, Vincenzo; Bifulco, Maurizio; De Petrocellis, Luciano
Nature Reviews Drug Discovery (2004), 3 (9), 771-784CODEN: NRDDAG; ISSN:1474-1776. (Nature Publishing Group)
A review with refs. The term 'endocannabinoid' - originally coined in the mid-1990s after the discovery of membrane receptors for the psychoactive principle in Cannabis, Δ9-tetrahydrocannabinol and their endogenous ligands - now indicates a whole signalling system that comprises cannabinoid receptors, endogenous ligands and enzymes for ligand biosynthesis and inactivation. This system seems to be involved in an ever-increasing no. of pathol. conditions. With novel products already being aimed at the pharmaceutical market little more than a decade since the discovery of cannabinoid receptors, the endocannabinoid system seems to hold even more promise for the future development of therapeutic drugs. We explore the conditions under which the potential of targeting the endocannabinoid system might be realized in the years to come.
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Gao, Y., Vasilyev, D. V., Goncalves, M. B., Howell, F. V., Hobbs, C., Reisenberg, M., Shen, R., Zhang, M. Y., Strassle, B. W., Lu, P., Mark, L., Piesla, M. J., Deng, K., Kouranova, E. V., Ring, R. H., Whiteside, G. T., Bates, B., Walsh, F. S., Williams, G., Pangalos, M. N., Samad, T. A., and Doherty, P. (2010) Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice J. Neurosci. 30, 2017– 2024 DOI: 10.1523/JNEUROSCI.5693-09.2010
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Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice
Gao, Ying; Vasilyev, Dmitry V.; Goncalves, Maria Beatriz; Howell, Fiona V.; Hobbs, Carl; Reisenberg, Melina; Shen, Ru; Zhang, Mei-Yi; Strassle, Brian W.; Lu, Peimin; Mark, Lilly; Piesla, Michael J.; Deng, Kangwen; Kouranova, Evguenia V.; Ring, Robert H.; Whiteside, Garth T.; Bates, Brian; Walsh, Frank S.; Williams, Gareth; Pangalos, Menelas N.; Samad, Tarek A.; Doherty, Patrick
Journal of Neuroscience (2010), 30 (6), 2017-2024CODEN: JNRSDS; ISSN:0270-6474. (Society for Neuroscience)
Endocannabinoids (eCBs) function as retrograde signaling mols. at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-α (DAGLα) and -β (DAGLβ) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their resp. contribution to this and to eCB signaling has not been tested. In the present study, we show ∼80% redns. in 2-AG levels in the brain and spinal cord in DAGLα-/- mice and a 50% redn. in the brain in DAGLβ-/- mice. In contrast, DAGLβ plays a more important role than DAGLα in regulating 2-AG levels in the liver, with a 90% redn. seen in DAGLβ-/- mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLα-/- animals and relatively unaffected in DAGLβ-/- animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLα-/- and/or DAGLβ-/- mice. These findings provide the first evidence that DAGLα is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.
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Tanimura, A., Yamazaki, M., Hashimotodani, Y., Uchigashima, M., Kawata, S., Abe, M., Kita, Y., Hashimoto, K., Shimizu, T., Watanabe, M., Sakimura, K., and Kano, M. (2010) The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase alpha Mediates Retrograde Suppression of Synaptic Transmission Neuron 65, 320– 327 DOI: 10.1016/j.neuron.2010.01.021
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Murataeva, N., Straiker, A., and Mackie, K. (2014) Parsing the players: 2-arachidonoylglycerol synthesis and degradation in the CNS Br. J. Pharmacol. 171, 1379– 1391 DOI: 10.1111/bph.12411
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Parsing the players: 2-arachidonoylglycerol synthesis and degradation in the CNS
Murataeva, N.; Straiker, A.; Mackie, K.
British Journal of Pharmacology (2014), 171 (6), 1379-1391CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
A review. The endogenous cannabinoid signalling system, composed of endogenous cannabinoids, cannabinoid receptors and the enzymes that synthesize and degrade the endogenous cannabinoids, is much more complex than initially conceptualized. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid and plays a major role in CNS development and synaptic plasticity. Over the past decade, many key players in 2-AG synthesis and degrdn. have been identified and characterized. Most 2-AG is synthesized from membrane phospholipids via sequential activation of a phospholipase Cβ and a diacylglycerol lipase, although other pathways may contribute in specialized settings. 2-AG breakdown is more complicated with at least eight different enzymes participating. These enzymes can either degrade 2-AG into its components, arachidonic acid and glycerol, or transform 2-AG into highly bioactive signal mols. The implications of the precise temporal and spatial control of the expression and function of these pleiotropic metabolizing enzymes have only recently come to be appreciated. In this review, we will focus on the primary organization of the synthetic and degradative pathways of 2-AG and then discuss more recent findings and their implications, with an eye towards the biol. and therapeutic implications of manipulating 2-AG synthesis and metab.
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Di Marzo, V., Fontana, A., Cadas, H., Schinelli, S., Cimino, G., Schwartz, J. C., and Piomelli, D. (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons Nature 372, 686– 691 DOI: 10.1038/372686a0
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Sun, Y. X., Tsuboi, K., Okamoto, Y., Tonai, T., Murakami, M., Kudo, I., and Ueda, N. (2004) Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D Biochem. J. 380, 749– 756 DOI: 10.1042/bj20040031
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There is no corresponding record for this reference.
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Tsuboi, K., Okamoto, Y., Rahman, I. A., Uyama, T., Inoue, T., Tokumura, A., and Ueda, N. (2015) Glycerophosphodiesterase GDE4 as a novel lysophospholipase D: a possible involvement in bioactive N-acylethanolamine biosynthesis Biochim. Biophys. Acta, Mol. Cell Biol. Lipids 1851, 537– 548 DOI: 10.1016/j.bbalip.2015.01.002
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Glycerophosphodiesterase GDE4 as a novel lysophospholipase D: a possible involvement in bioactive N-acylethanolamine biosynthesis
Tsuboi, Kazuhito; Okamoto, Yoko; Rahman, Iffat Ara Sonia; Uyama, Toru; Inoue, Tomohito; Tokumura, Akira; Ueda, Natsuo
Biochimica et Biophysica Acta, Molecular and Cell Biology of Lipids (2015), 1851 (5), 537-548CODEN: BBMLFG; ISSN:1388-1981. (Elsevier B. V.)
Bioactive N-acylethanolamines include anti-inflammatory palmitoylethanolamide, anorexic oleoylethanolamide, and an endocannabinoid arachidonoylethanolamide (anandamide). In animal tissues, these mols. are biosynthesized from N-acylethanolamine phospholipids directly by phospholipase D-type enzyme or through multi-step routes via N-acylethanolamine lysophospholipids. We previously found that mouse brain has a lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines and that this activity could be partially attributed to glycerophosphodiesterase (GDE) 1. In the present study, we examd. catalytic properties of GDE4, another member of the GDE family. When overexpressed in HEK293 cells, murine GDE4 mostly resided in the membrane fraction. Purified GDE4 showed lysoPLD activity toward various lysophospholipids, including N-acylethanolamine lysophospholipids as well as lysophosphatidylethanolamine and lysophosphatidylcholine. When HEK293 cells were metabolically labeled with N-[14C]palmitoylethanolamine lysophospholipid, the transient expression of GDE4 increased the [14C]palmitoylethanolamide level, while the knockdown of endogenous GDE4 decreased this level. These results suggested that GDE4 functions as an N-acylethanolamine-generating lysoPLD in living cells. Moreover, the expression of GDE4 increased most species of lysophosphatidic acid (LPA), which can be produced from various lysophospholipids by the lysoPLD activity of GDE4. GDE4 mRNA was widely distributed among mouse tissues including brain, stomach, ileum, colon, and testis. In conclusion, GDE4 may act as a lysoPLD, which is involved in the generation of N-acylethanolamines and LPA.
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Liu, J., Wang, L., Harvey-White, J., Huang, B. X., Kim, H. Y., Luquet, S., Palmiter, R. D., Krystal, G., Rai, R., Mahadevan, A., Razdan, R. K., and Kunos, G. (2008) Multiple pathways involved in the biosynthesis of anandamide Neuropharmacology 54, 1– 7 DOI: 10.1016/j.neuropharm.2007.05.020
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Multiple pathways involved in the biosynthesis of anandamide
Liu Jie; Wang Lei; Harvey-White Judith; Huang Bill X; Kim Hee-Yong; Luquet Serge; Palmiter Richard D; Krystal Gerald; Rai Ravi; Mahadevan Anu; Razdan Raj K; Kunos George
Neuropharmacology (2008), 54 (1), 1-7 ISSN:0028-3908.
Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.
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Rahman, I. A. S., Tsuboi, K., Uyama, T., and Ueda, N. (2014) New players in the fatty acyl ethanolamide metabolism Pharmacol. Res. 86, 1– 10 DOI: 10.1016/j.phrs.2014.04.001
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New players in the fatty acyl ethanolamide metabolism
Rahman, Iffat Ara Sonia; Tsuboi, Kazuhito; Uyama, Toru; Ueda, Natsuo
Pharmacological Research (2014), 86 (), 1-10CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
A review. Fatty acyl ethanolamides represent a class of endogenous bioactive lipid mols. and are generally referred to as N-acylethanolamines (NAEs). NAEs include palmitoylethanolamide (anti-inflammatory and analgesic substance), oleoylethanolamide (anorexic substance), and anandamide (endocannabinoid). The endogenous levels of NAEs are mainly regulated by enzymes responsible for their biosynthesis and degrdn. In mammalian tissues, the major biosynthetic pathway starts from glycerophospholipids and is composed of two enzyme reactions. The first step is N-acylation of ethanolamine phospholipids catalyzed by Ca2+-dependent N-acyltransferase and the second step is the release of NAEs from N-acylated ethanolamine phospholipids by N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD). As for the degrdn. of NAEs, fatty acid amide hydrolase plays the central role. However, recent studies strongly suggest the involvement of other enzymes in the NAE metab. These enzymes include members of the HRAS-like suppressor family (also called phospholipase A/acyltransferase family), which were originally discovered as tumor suppressors but can function as Ca2+-independent NAPE-forming N-acyltransferases; multiple enzymes involved in the NAPE-PLD-independent multi-step pathways to generate NAE from NAPE, which came to light by the anal. of NAPE-PLD-deficient mice; and a lysosomal NAE-hydrolyzing acid amidase as a second NAE hydrolase. These newly recognized enzymes may become the targets for the development of new therapeutic drugs. Here, we focus on recent enzymol. findings in this area.
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Cravatt, B. F., Giang, D. K., Mayfield, S. P., Boger, D. L., Lerner, R. A., and Gilula, N. B. (1996) Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides Nature 384, 83– 87 DOI: 10.1038/384083a0
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Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides
Cravatt, Benjamin F.; Giang, Dan K.; Mayfield, Stephen P.; Boger, Dale L.; Lerner, Richard A.; Gilula, Norton B.
Nature (London) (1996), 384 (6604), 83-87CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
Endogenous neuromodulatory mols.. are commonly couples to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolyzed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-deproved cats. Cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling mols., the fatty-acid amides. Therefore we will hereafter refer to oelamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
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Belluomo, I., Matias, I., Pernegre, C., Marsicano, G., and Chaouloff, F. (2015) Opposite control of frontocortical 2-arachidonoylglycerol turnover rate by cannabinoid type-1 receptors located on glutamatergic neurons and on astrocytes J. Neurochem. 133, 26– 37 DOI: 10.1111/jnc.13044
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Opposite control of frontocortical 2-arachidonoylglycerol turnover rate by cannabinoid type-1 receptors located on glutamatergic neurons and on astrocytes
Belluomo, Ilaria; Matias, Isabelle; Pernegre, Camille; Marsicano, Giovanni; Chaouloff, Francis
Journal of Neurochemistry (2015), 133 (1), 26-37CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
This study examd. the resp. influences of cannabinoid type-1 (CB1) receptors expressed either in forebrain GABAergic neurons, in cortical glutamatergic neurons, or in astrocytes on the turnover rates of the endocannabinoids N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acylethanolamides, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), in mouse forebrain regions. To this end, conditional mutant mice lacking CB1 receptors from either of these cell types were pretreated systemically with JZL 195, a dual inhibitor of fatty acid amide hydrolase, the enzyme degrading AEA, PEA, and OEA, and of monoacylglycerol lipase, the main 2-AG-degrading enzyme. The analyses of frontocortical, hippocampal, and striatal AEA, 2-AG, PEA, and OEA concns. revealed that their resp. baseline concns. were not influenced by the mouse genotype. On the other hand, the accumulation of frontocortical and/or hippocampal 2-AG levels in JZL 195-pretreated mice was dependent on the mouse genotype. Thus, JZL 195-induced 2-AG accumulation rates were diminished in the frontal cortex of mice lacking CB1 receptors in glutamatergic neurons while their resp. values were increased in the frontal cortex and hippocampus of mice lacking these receptors in astrocytes. These genotypic differences occurred with parallel and proportionate changes in the fractional rate consts. for degrdn. of 2-AG, thus providing a mechanism whereby the baseline levels of 2-AG remained const. between genotypes. Besides suggesting a cell-type-specific control of frontocortical and/or hippocampal 2-AG synthesis and degrdn. rates by CB1 receptors, this study highlights the interest of assessing endocannabinoid turnover rates when questioning the status of the endocannabinoid system. In mice lacking CB1 receptors from glutamatergic neurons (Glu-CB1-/-) or from astrocytes (GFAP-CB1-/-), frontocortical and hippocampal 2-AG levels are not altered. Turnover studies indicate, however, that decreased 2-AG synthesis occurs with decreased degrdn. in Glu-CB1-/- mice, whereas the opposite is obsd. in GFAP-CB1-/- mice.
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Starke, K., Gothert, M., and Kilbinger, H. (1989) Modulation of neurotransmitter release by presynaptic autoreceptors Physiol. Rev. 69, 864– 989
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Langer, S. Z. (1997) 25 years since the discovery of presynaptic receptors: present knowledge and future perspectives Trends Pharmacol. Sci. 18, 95– 99 DOI: 10.1016/S0165-6147(96)01034-6
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There is no corresponding record for this reference.
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Di Marzo, V., Breivogel, C. S., Tao, Q., Bridgen, D. T., Razdan, R. K., Zimmer, A. M., Zimmer, A., and Martin, B. R. (2000) Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain J. Neurochem. 75, 2434– 2444 DOI: 10.1046/j.1471-4159.2000.0752434.x
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Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain
Di Marzo, Vincenzo; Breivogel, Chris S.; Tao, Qing; Bridgen, David T.; Razdan, Raj K.; Zimmer, Anne M.; Zimmer, Andreas; Martin, Billy R.
Journal of Neurochemistry (2000), 75 (6), 2434-2444CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB1), yet some of the neurobehavioral effects of this compd. in mice are unaffected by a selective CB1 antagonist. We studied the levels, pharmacol. actions, and degrdn. of AEA in transgenic mice lacking the CB1 gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-diln. liq. chromatog.-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB1 distribution only in part. In CB1 knockout homozygotes (CB1-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB1+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB1-/- with respect to CB1+/+ mice in all regions, AEA and Δ9-tetrahydrocannabinol (THC) were tested in CB1-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB1-/- mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB1-/- mice, and this stimulation was insensitive to CB1 and CB2 antagonists. We suggest that non-CB1, non-CB2 G protein-coupled receptors might mediate in mice some of the neurobehavioral actions of AEA.
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Di Marzo, V., Berrendero, F., Bisogno, T., Gonzalez, S., Cavaliere, P., Romero, J., Cebeira, M., Ramos, J. A., and Fernandez-Ruiz, J. J. (2000) Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of Delta(9)-tetrahydrocannabinol-tolerant rats J. Neurochem. 74, 1627– 1635 DOI: 10.1046/j.1471-4159.2000.0741627.x
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Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of Δ9-tetrahydrocannabinol-tolerant rats
Di Marzo, V.; Berrendero, F.; Bisogno, T.; Gonzalez, S.; Cavaliere, P.; Romero, J.; Cebeira, M.; Ramos, J. A.; Fernandez-Ruiz, J. J.
Journal of Neurochemistry (2000), 74 (4), 1627-1635CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
Recent studies have shown that the pharmacol. tolerance obsd. after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope diln. gas chromatog./mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with Δ9-tetrahydrocannabinol (Δ9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(γ-[35S]thio)-triphosphate ([35S]GTPγS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPγS binding decreased in most of the brain areas of Δ9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of Δ9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents obsd. in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from Δ9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiol. implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.
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Maccarrone, M., Attina, M., Bari, M., Cartoni, A., Ledent, C., and Finazzi-Agro, A. (2001) Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages J. Neurochem. 78, 339– 348 DOI: 10.1046/j.1471-4159.2001.00413.x
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Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages
Maccarrone, Mauro; Attina, Marina; Bari, Monica; Cartoni, Antonella; Ledent, Catherine; Finazzi-Agro, Alessandro
Journal of Neurochemistry (2001), 78 (2), 339-348CODEN: JONRA9; ISSN:0022-3042. (Blackwell Science Ltd.)
CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-mo-old knockout vs. wild-type mice, and even higher in 12-mo-old animals. The mol. mass (≈67 kDa) and isoelec. point (≈7.6) of mouse brain FAAH were detd. and the FAAH protein content was shown to parallel the enzyme activity. The kinetic consts. of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amts. of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compds. were twice as abundant in the hippocampus as in the cortex. Minor differences were obsd. in AEA or 2-arachidonoylglycerol content in knockout vs. wild-type mice, whereas the other compds. were lower in the hippocampus of knockout vs. wild-type animals.
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Maccarrone, M., Valverde, O., Barbaccia, M. L., Castane, A., Maldonado, R., Ledent, C., Parmentier, M., and Finazzi-Agro, A. (2002) Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour Eur. J. Neurosci. 15, 1178– 1186 DOI: 10.1046/j.1460-9568.2002.01957.x
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Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour
Maccarrone Mauro; Valverde Olga; Barbaccia Maria L; Castane Anna; Maldonado Rafael; Ledent Catherine; Parmentier Marc; Finazzi-Agro Alessandro
The European journal of neuroscience (2002), 15 (7), 1178-86 ISSN:0953-816X.
Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.
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Bequet, F., Uzabiaga, F., Desbazeille, M., Ludwiczak, P., Maftouh, M., Picard, C., Scatton, B., and Le Fur, G. (2007) CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus Eur. J. Neurosci. 26, 3458– 3464 DOI: 10.1111/j.1460-9568.2007.05900.x
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CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus
Bequet Frederic; Uzabiaga Francoise; Desbazeille Mathieu; Ludwiczak Pascal; Maftouh Mohamed; Picard Claudine; Scatton Bernard; Le Fur Gerard
The European journal of neuroscience (2007), 26 (12), 3458-64 ISSN:.
In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.
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Leishman, E., Cornett, B., Spork, K., Straiker, A., Mackie, K., and Bradshaw, H. B. (2016) Broad impact of deleting endogenous cannabinoid hydrolyzing enzymes and the CB1 cannabinoid receptor on the endogenous cannabinoid-related lipidome in eight regions of the mouse brain Pharmacol. Res. 110, 159– 172 DOI: 10.1016/j.phrs.2016.04.020
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Broad impact of deleting endogenous cannabinoid hydrolyzing enzymes and the CB1 cannabinoid receptor on the endogenous cannabinoid-related lipidome in eight regions of the mouse brain
Leishman, Emma; Cornett, Ben; Spork, Karl; Straiker, Alex; Mackie, Ken; Bradshaw, Heather B.
Pharmacological Research (2016), 110 (), 159-172CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
The enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) hydrolyze endogenous cannabinoids (eCBs), N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG), resp. These enzymes also metabolize eCB analogs such as lipoamines and 2-acyl glycerols, most of which are not ligands at CB1. To test the hypothesis that deleting eCB hydrolyzing enzymes and CB1 shifts lipid metab. more broadly and impacts more families of eCB structural analogs, targeted lipidomics analyses were performed on FAAH KO, MAGL KO, and CB1 KO mice and compared to WT controls in 8 brain regions. Methanolic exts. of discrete brain regions (brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, striatum and thalamus) were partially purified on C-18 solid-phase extn. columns. Over 70 lipids per sample were then analyzed with HPLC/MS/MS. AEA and 2-AG were unaffected throughout the brain in CB1 KO mice; however, there was an increase in the arachidonic acid (AA) metabolite, PGE2 in the majority of brain areas. By contrast, PGE2 and AA levels were significantly reduced throughout the brain in the MAGL KO corresponding to significant increases in 2-AG. No changes in AA or PGE2 were seen throughout in the FAAH KO brain, despite significant increases in AEA, suggesting AA liberated by FAAH does not contribute to steady state levels of AA or PGE2. Changes in the lipidome were not confined to the AA derivs. and showed regional variation in each of the eCB KO models. AEA and 2-AG hydrolyzing enzymes and the CB1 receptor link the eCB system to broader lipid signaling networks in contrasting ways, potentially altering neurotransmission and behavior independently of cannabinoid receptor signaling.
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Schlosburg, J. E., Blankman, J. L., Long, J. Z., Nomura, D. K., Pan, B., Kinsey, S. G., Nguyen, P. T., Ramesh, D., Booker, L., Burston, J. J., Thomas, E. A., Selley, D. E., Sim-Selley, L. J., Liu, Q. S., Lichtman, A. H., and Cravatt, B. F. (2010) Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system Nat. Neurosci. 13, 1113– 1119 DOI: 10.1038/nn.2616
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Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system
Schlosburg, Joel E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Nomura, Daniel K.; Pan, Bin; Kinsey, Steven G.; Nguyen, Peter T.; Ramesh, Divya; Booker, Lamont; Burston, James J.; Thomas, Elizabeth A.; Selley, Dana E.; Sim-Selley, Laura J.; Liu, Qing-song; Lichtman, Aron H.; Cravatt, Benjamin F.
Nature Neuroscience (2010), 13 (9), 1113-1119CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacol. tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused phys. dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and assocd. with agonism and functional antagonism of the brain cannabinoid system, resp.
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Kang, H. J., Kawasawa, Y. I., Cheng, F., Zhu, Y., Xu, X. M., Li, M. F., Sousa, A. M. M., Pletikos, M., Meyer, K. A., Sedmak, G., Guennel, T., Shin, Y., Johnson, M. B., Krsnik, Z., Mayer, S., Fertuzinhos, S., Umlauf, S., Lisgo, S. N., Vortmeyer, A., Weinberger, D. R., Mane, S., Hyde, T. M., Huttner, A., Reimers, M., Kleinman, J. E., and Sestan, N. (2011) Spatio-temporal transcriptome of the human brain Nature 478, 483– 489 DOI: 10.1038/nature10523
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Spatio-temporal transcriptome of the human brain
Kang, Hyo Jung; Kawasawa, Yuka Imamura; Cheng, Feng; Zhu, Ying; Xu, Xuming; Li, Mingfeng; Sousa, Andre M. M.; Pletikos, Mihovil; Meyer, Kyle A.; Sedmak, Goran; Guennel, Tobias; Shin, Yurae; Johnson, Matthew B.; Krsnik, Zeljka; Mayer, Simone; Fertuzinhos, Sofia; Umlauf, Sheila; Lisgo, Steven N.; Vortmeyer, Alexander; Weinberger, Daniel R.; Mane, Shrikant; Hyde, Thomas M.; Huttner, Anita; Reimers, Mark; Kleinman, Joel E.; Sestan, Nenad
Nature (London, United Kingdom) (2011), 478 (7370), 483-489CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and anal. of exon-level transcriptome and assocd. genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86% of the genes analyzed were expressed, and that 90% of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct coexpression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes assocd. with neurobiol. categories and diseases, and identified assocns. between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.
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Sharma, K., Schmitt, S., Bergner, C. G., Tyanova, S., Kannaiyan, N., Manrique-Hoyos, N., Kongi, K., Cantuti, L., Hanisch, U. K., Philips, M. A., Rossner, M. J., Mann, M., and Simons, M. (2015) Cell type- and brain region-resolved mouse brain proteome Nat. Neurosci. 18, 1819– 1831 DOI: 10.1038/nn.4160
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Cell type- and brain region-resolved mouse brain proteome
Sharma, Kirti; Schmitt, Sebastian; Bergner, Caroline G.; Tyanova, Stefka; Kannaiyan, Nirmal; Manrique-Hoyos, Natalia; Kongi, Karina; Cantuti, Ludovico; Hanisch, Uwe-Karsten; Philips, Mari-Anne; Rossner, Moritz J.; Mann, Matthias; Simons, Mikael
Nature Neuroscience (2015), 18 (12), 1819-1831CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
Brain transcriptome and connectome maps are being generated, but an equiv. effort on the proteome is currently lacking. We performed high-resoln. mass spectrometry-based proteomics for in-depth anal. of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than av. expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion mol. of the IgLON family, as a neg. regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.
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Lein, E. S., Hawrylycz, M. J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe, A. F., Boguski, M. S., Brockway, K. S., Byrnes, E. J., Chen, L., Chen, L., Chen, T. M., Chi Chin, M., Chong, J., Crook, B. E., Czaplinska, A., Dang, C. N., Datta, S., Dee, N. R., Desaki, A. L., Desta, T., Diep, E., Dolbeare, T. A., Donelan, M. J., Dong, H. W., Dougherty, J. G., Duncan, B. J., Ebbert, A. J., Eichele, G., Estin, L. K., Faber, C., Facer, B. A., Fields, R., Fischer, S. R., Fliss, T. P., Frensley, C., Gates, S. N., Glattfelder, K. J., Halverson, K. R., Hart, M. R., Hohmann, J. G., Howell, M. P., Jeung, D. P., Johnson, R. A., Karr, P. T., Kawal, R., Kidney, J. M., Knapik, R. H., Kuan, C. L., Lake, J. H., Laramee, A. R., Larsen, K. D., Lau, C., Lemon, T. A., Liang, A. J., Liu, Y., Luong, L. T., Michaels, J., Morgan, J. J., Morgan, R. J., Mortrud, M. T., Mosqueda, N. F., Ng, L. L., Ng, R., Orta, G. J., Overly, C. C., Pak, T. H., Parry, S. E., Pathak, S. D., Pearson, O. C., Puchalski, R. B., Riley, Z. L., Rockett, H. R., Rowland, S. A., Royall, J. J., Ruiz, M. J., Sarno, N. R., Schaffnit, K., Shapovalova, N. V., Sivisay, T., Slaughterbeck, C. R., Smith, S. C., Smith, K. A., Smith, B. I., Sodt, A. J., Stewart, N. N., Stumpf, K. R., Sunkin, S. M., Sutram, M., Tam, A., Teemer, C. D., Thaller, C., Thompson, C. L., Varnam, L. R., Visel, A., Whitlock, R. M., Wohnoutka, P. E., Wolkey, C. K., Wong, V. Y., Wood, M., Yaylaoglu, M. B., Young, R. C., Youngstrom, B. L., Feng Yuan, X., Zhang, B., Zwingman, T. A., and Jones, A. R. (2007) Genome-wide atlas of gene expression in the adult mouse brain Nature 445, 168– 176 DOI: 10.1038/nature05453
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Genome-wide atlas of gene expression in the adult mouse brain
Lein, Ed S.; Hawrylycz, Michael J.; Ao, Nancy; Ayres, Mikael; Bensinger, Amy; Bernard, Amy; Boe, Andrew F.; Boguski, Mark S.; Brockway, Kevin S.; Byrnes, Emi J.; Chen, Lin; Chen, Li; Chen, Tsuey-Ming; Chin, Mei Chi; Chong, Jimmy; Crook, Brian E.; Czaplinska, Aneta; Dang, Chinh N.; Datta, Suvro; Dee, Nick R.; Desaki, Aimee L.; Desta, Tsega; Diep, Ellen; Dolbeare, Tim A.; Donelan, Matthew J.; Dong, Hong-Wei; Dougherty, Jennifer G.; Duncan, Ben J.; Ebbert, Amanda J.; Eichele, Gregor; Estin, Lili K.; Faber, Casey; Facer, Benjamin A.; Fields, Rick; Fischer, Shanna R.; Fliss, Tim P.; Frensley, Cliff; Gates, Sabrina N.; Glattfelder, Katie J.; Halverson, Kevin R.; Hart, Matthew R.; Hohmann, John G.; Howell, Maureen P.; Jeung, Darren P.; Johnson, Rebecca A.; Karr, Patrick T.; Kawal, Reena; Kidney, Jolene M.; Knapik, Rachel H.; Kuan, Chihchau L.; Lake, James H.; Laramee, Annabel R.; Larsen, Kirk D.; Lau, Christopher; Lemon, Tracy A.; Liang, Agnes J.; Liu, Ying; Luong, Lon T.; Michaels, Jesse; Morgan, Judith J.; Morgan, Rebecca J.; Mortrud, Marty T.; Mosqueda, Nerick F.; Ng, Lydia L.; Ng, Randy; Orta, Geralyn J.; Overly, Caroline C.; Pak, Tu H.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Puchalski, Ralph B.; Riley, Zackery L.; Rockett, Hannah R.; Rowland, Stephen A.; Royall, Joshua J.; Ruiz, Marcos J.; Sarno, Nadia R.; Schaffnit, Katherine; Shapovalova, Nadiya V.; Sivisay, Taz; Slaughterbeck, Clifford R.; Smith, Simon C.; Smith, Kimberly A.; Smith, Bryan I.; Sodt, Andy J.; Stewart, Nick N.; Stumpf, Kenda-Ruth; Sunkin, Susan M.; Sutram, Madhavi; Tam, Angelene; Teemer, Carey D.; Thaller, Christina; Thompson, Carol L.; Varnam, Lee R.; Visel, Axel; Whitlock, Ray M.; Wohnoutka, Paul E.; Wolkey, Crissa K.; Wong, Victoria Y.; Wood, Matthew; Yaylaoglu, Murat B.; Young, Rob C.; Youngstrom, Brian L.; Yuan, Xu Feng; Zhang, Bin; Zwingman, Theresa A.; Jones, Allan R.
Nature (London, United Kingdom) (2007), 445 (7124), 168-176CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
Mol. approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behavior. The cellular diversity of the brain necessitates a cellular resoln. approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas contg. the expression patterns of ∼20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural anal. and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resoln. anal. has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.
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Vogel, C. and Marcotte, E. M. (2012) Insights into the regulation of protein abundance from proteomic and transcriptomic analyses Nat. Rev. Genet. 13, 227– 232 DOI: 10.1038/nrg3185
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Insights into the regulation of protein abundance from proteomic and transcriptomic analyses
Vogel, Christine; Marcotte, Edward M.
Nature Reviews Genetics (2012), 13 (4), 227-232CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)
A review. Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - i.e., post-transcriptional, translational and protein degrdn. regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.
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Kobe, B. and Kemp, B. E. (1999) Active site-directed protein regulation Nature 402, 373– 376 DOI: 10.1038/46478
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Active site-directed protein regulation
Kobe, Bostjan; Kemp, Bruce E.
Nature (London) (1999), 402 (6760), 373-376CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
A review, with 46 refs. Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effectors bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.
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Dotsey, E. Y., Jung, K. M., Basit, A., Wei, D., Daglian, J., Vacondio, F., Armirotti, A., Mor, M., and Piomelli, D. (2015) Peroxide-Dependent MGL Sulfenylation Regulates 2-AG-Mediated Endocannabinoid Signaling in Brain Neurons Chem. Biol. 22, 619– 628 DOI: 10.1016/j.chembiol.2015.04.013
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Peroxide-Dependent MGL Sulfenylation Regulates 2-AG-Mediated Endocannabinoid Signaling in Brain Neurons
Dotsey, Emmanuel Y.; Jung, Kwang-Mook; Basit, Abdul; Wei, Don; Daglian, Jennifer; Vacondio, Federica; Armirotti, Andrea; Mor, Marco; Piomelli, Daniele
Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (5), 619-628CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
The second messenger hydrogen peroxide transduces changes in the cellular redox state by reversibly oxidizing protein cysteine residues to sulfenic acid. This signaling event regulates many cellular processes but has never been shown to occur in the brain. Here, we report that hydrogen peroxide heightens endocannabinoid signaling in brain neurons through sulfenylation of cysteines C201 and C208 in monoacylglycerol lipase (MGL), a serine hydrolase that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in nerve terminals. The results suggest that MGL sulfenylation may provide a presynaptic control point for 2-AG-mediated endocannabinoid signaling.
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Shonesy, B. C., Wang, X., Rose, K. L., Ramikie, T. S., Cavener, V. S., Rentz, T., Baucum, A. J., 2nd, Jalan-Sakrikar, N., Mackie, K., Winder, D. G., Patel, S., and Colbran, R. J. (2013) CaMKII regulates diacylglycerol lipase-alpha and striatal endocannabinoid signaling Nat. Neurosci. 16, 456– 463 DOI: 10.1038/nn.3353
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CaMKII regulates diacylglycerol lipase-α and striatal endocannabinoid signaling
Shonesy, Brian C.; Wang, Xiaohan; Rose, Kristie L.; Ramikie, Teniel S.; Cavener, Victoria S.; Rentz, Tyler; Baucum, Anthony J., II; Jalan-Sakrikar, Nidhi; Mackie, Ken; Winder, Danny G.; Patel, Sachin; Colbran, Roger J.
Nature Neuroscience (2013), 16 (4), 456-463CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the mol. regulation of 2-AG synthesis is not well understood. Here we identify a functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in 2-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of 2-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of 2-AG breakdown using concns. of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the mol. regulation of striatal endocannabinoid signaling with implications for physiol. control of motor function.
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Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M. G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E. J., Gangadharan, U., Hobbs, C., Di Marzo, V., and Doherty, P. (2003) Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain J. Cell Biol. 163, 463– 468 DOI: 10.1083/jcb.200305129
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Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain
Bisogno, Tiziana; Howell, Fiona; Williams, Gareth; Minassi, Alberto; Grazia Cascio, Maria; Ligresti, Alessia; Matias, Isabel; Schiano-Moriello, Aniello; Paul, Praveen; Williams, Emma-Jane; Gangadharan, Uma; Hobbs, Carl; Di Marzo, Vincenzo; Doherty, Patrick
Journal of Cell Biology (2003), 163 (3), 463-468CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
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 enzymic 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.
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Cravatt, B. F., Wright, A. T., and Kozarich, J. W. (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry Annu. Rev. Biochem. 77, 383– 414 DOI: 10.1146/annurev.biochem.75.101304.124125
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Activity-based protein profiling: From enzyme chemistry to proteomic chemistry
Cravatt, Benjamin F.; Wright, Aaron T.; Kozarich, John W.
Annual Review of Biochemistry (2008), 77 (), 383-414CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
A review. Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chem. proteomic strategy to characterize enzyme function directly in native biol. systems on a global scale. Here, the authors review the basic technol. of ABPP, the enzyme classes addressable by this method, and the biol. discoveries attributable to its application.
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Niphakis, M. J. and Cravatt, B. F. (2014) Enzyme inhibitor discovery by activity-based protein profiling Annu. Rev. Biochem. 83, 341– 377 DOI: 10.1146/annurev-biochem-060713-035708
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Enzyme inhibitor discovery by activity-based protein profiling
Niphakis, Micah J.; Cravatt, Benjamin F.
Annual Review of Biochemistry (2014), 83 (), 341-377CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
Eukaryotic and prokaryotic organisms possess huge nos. of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biol. systems. Here, we discuss how the chem. proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochem. and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiol. and disease. These studies demonstrate the value of selective chem. probes as drivers of biol. inquiry.
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Baggelaar, M. P., Chameau, P. J., Kantae, V., Hummel, J., Hsu, K. L., Janssen, F., van der Wel, T., Soethoudt, M., Deng, H., den Dulk, H., Allara, M., Florea, B. I., Di Marzo, V., Wadman, W. J., Kruse, C. G., Overkleeft, H. S., Hankemeier, T., Werkman, T. R., Cravatt, B. F., and van der Stelt, M. (2015) Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons J. Am. Chem. Soc. 137, 8851– 8857 DOI: 10.1021/jacs.5b04883
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Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons
Baggelaar, Marc P.; Chameau, Pascal J. P.; Kantae, Vasudev; Hummel, Jessica; Hsu, Ku-Lung; Janssen, Freek; van der Wel, Tom; Soethoudt, Marjolein; Deng, Hui; den Dulk, Hans; Allara, Marco; Florea, Bogdan I.; Di Marzo, Vincenzo; Wadman, Wytse J.; Kruse, Chris G.; Overkleeft, Herman S.; Hankemeier, Thomas; Werkman, Taco R.; Cravatt, Benjamin F.; van der Stelt, Mario
Journal of the American Chemical Society (2015), 137 (27), 8851-8857CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
Diacylglycerol lipase (DAGL)-α and -β 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 β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metab. including abhydrolase domain-contg. protein 6, abhydrolase domain-contg. protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concn.-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 pharmacol. evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.
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Liu, Y., Patricelli, M. P., and Cravatt, B. F. (1999) Activity-based protein profiling: the serine hydrolases Proc. Natl. Acad. Sci. U. S. A. 96, 14694– 14699 DOI: 10.1073/pnas.96.26.14694
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Activity-based protein profiling: the serine hydrolases
Liu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.
Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
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Baggelaar, M. P., Janssen, F. J., van Esbroeck, A. C., den Dulk, H., Allara, M., Hoogendoorn, S., McGuire, R., Florea, B. I., Meeuwenoord, N., van den Elst, H., van der Marel, G. A., Brouwer, J., Di Marzo, V., Overkleeft, H. S., and van der Stelt, M. (2013) Development of an activity-based probe and in silico design reveal highly selective inhibitors for diacylglycerol lipase-alpha in brain Angew. Chem., Int. Ed. 52, 12081– 12085 DOI: 10.1002/anie.201306295
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Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain
Baggelaar, Marc P.; Janssen, Freek J.; van Esbroeck, Annelot C. M.; den Dulk, Hans; Allara, Marco; Hoogendoorn, Sascha; McGuire, Ross; Florea, Bogdan I.; Meeuwenoord, Nico; van den Elst, Hans; van der Marel, Gijsbert A.; Brouwer, Jaap; Di Marzo, Vincenzo; Overkleeft, Herman S.; van der Stelt, Mario
Angewandte Chemie, International Edition (2013), 52 (46), 12081-12085CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
A strategy that combines a knowledge-based in silico design approach and the development of novel activity-based probes for the detection of endogenous diacylglycerol lipase-α (DAGL-α) is presented. This approach resulted in the rapid identification of new DAGL-α inhibitors with high selectivity in the brain proteome.
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Cox, J. and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Nat. Biotechnol. 26, 1367– 1372 DOI: 10.1038/nbt.1511
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MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification
Cox, Juergen; Mann, Matthias
Nature Biotechnology (2008), 26 (12), 1367-1372CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
Efficient anal. of very large amts. of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resoln., quant. MS data. Using correlation anal. and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over std. techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome expt. and allows statistically robust identification and quantification of >4000 proteins in mammalian cell lysates.
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Kamat, S. S., Camara, K., Parsons, W. H., Chen, D. H., Dix, M. M., Bird, T. D., Howell, A. R., and Cravatt, B. F. (2015) Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay Nat. Chem. Biol. 11, 164– 171 DOI: 10.1038/nchembio.1721
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Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay
Kamat, Siddhesh S.; Camara, Kaddy; Parsons, William H.; Chen, Dong-Hui; Dix, Melissa M.; Bird, Thomas D.; Howell, Amy R.; Cravatt, Benjamin F.
Nature Chemical Biology (2015), 11 (2), 164-171CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunol. and neurol. processes. The metab. of lyso-PSs remains poorly understood in vivo. Recently, we detd. that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurol. disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacol. and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-mol. inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A resp. increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine prodn. Finally, Abhd16a-/- mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12-/- mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metab. in vivo, designating these enzymes as potential targets for treating neuroimmunol. disorders.
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Long, J. Z. and Cravatt, B. F. (2011) The metabolic serine hydrolases and their functions in mammalian physiology and disease Chem. Rev. 111, 6022– 6063 DOI: 10.1021/cr200075y
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The metabolic serine hydrolases and their functions in mammalian physiology and disease
Long, Jonathan Z.; Cravatt, Benjamin F.
Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 6022-6063CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
A review. Serine hydrolases (SHs) consist of >200 enzymes in humans which are characterized by the presence of an active site Ser residue that is used for the hydrolysis of substrates. The membership of this enzyme class is nearly equally split between serine proteases (trypsin/chymotrypsin/subtilisin enzymes) and "metabolic" SHs that cleave ester, amide, or thioester bonds in small mols., peptides, or proteins. The focus of this review is on the metabolic SHs. Here, the authors attempt to provide a comprehensive summary that captures the state of knowledge about metabolic SHs in their entirety, including those enzymes that remain mostly or completely uncharacterized. Particular emphasis is placed on relating the biochem. and enzymol. of individual SHs to the physiol. substrates and products they regulate in living systems, and how SHs, through the regulation of specific metabolic pathways impact health and disease.
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Herkenham, M., Lynn, A. B., Little, M. D., Johnson, M. R., Melvin, L. S., de Costa, B. R., and Rice, K. C. (1990) Cannabinoid receptor localization in brain Proc. Natl. Acad. Sci. U. S. A. 87, 1932– 1936 DOI: 10.1073/pnas.87.5.1932
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Cannabinoid receptor localization in brain
Herkenham, Miles; Lynn, Allison B.; Little, Mark D.; Johnson, M. Ross; Melvin, Lawrence S.; De Costa, Brian R.; Rice, Kenner C.
Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (5), 1932-6CODEN: PNASA6; ISSN:0027-8424.
[3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than Δ9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biol. assays, suggesting that the receptor characterized in this in vitro assay is the same receptor that mediates behavioral and pharmacol. effects of cannabinoids, including human subjective experience. Autoradiog. of cannabinoid receptors in brain sections from several mammalian species, including man, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia (the substantia nigra pars reticulata and globus pallidus) and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of Δ9-tetrahydrocannabinol are not lethal.
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Marsicano, G., Goodenough, S., Monory, K., Hermann, H., Eder, M., Cannich, A., Azad, S. C., Cascio, M. G., Gutierrez, S. O., van der Stelt, M., Lopez-Rodriguez, M. L., Casanova, E., Schutz, G., Zieglgansberger, W., Di Marzo, V., Behl, C., and Lutz, B. (2003) CB1 cannabinoid receptors and on-demand defense against excitotoxicity Science 302, 84– 88 DOI: 10.1126/science.1088208
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CB1 cannabinoid receptors and on-demand defense against excitotoxicity
Marsicano, Giovanni; Goodenough, Sharon; Monory, Krisztina; Hermann, Heike; Eder, Matthias; Cannich, Astrid; Azad, Shahnaz C.; Cascio, Maria Grazia; Gutierrez, Silvia Ortega; van der Stelt, Mario; Lopez-Rodriguez, Maria Luz; Casanova, Emilio; Schuetz, Guenther; Zieglgaensberger, Walter; Di Marzo, Vincenzo; Behl, Christian; Lutz, Beat
Science (Washington, DC, United States) (2003), 302 (5642), 84-88CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. The authors generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice, the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.
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van der Stelt, M., Trevisani, M., Vellani, V., De Petrocellis, L., Schiano Moriello, A., Campi, B., McNaughton, P., Geppetti, P., and Di Marzo, V. (2005) Anandamide acts as an intracellular messenger amplifying Ca2⁺ influx via TRPV1 channels EMBO J. 24, 3026– 3037 DOI: 10.1038/sj.emboj.7600784
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Maccarrone, M., Rossi, S., Bari, M., De Chiara, V., Fezza, F., Musella, A., Gasperi, V., Prosperetti, C., Bernardi, G., Finazzi-Agro, A., Cravatt, B. F., and Centonze, D. (2008) Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum Nat. Neurosci. 11, 152– 159 DOI: 10.1038/nn2042
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Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum
Maccarrone, Mauro; Rossi, Silvia; Bari, Monica; De Chiara, Valentina; Fezza, Filomena; Musella, Alessandra; Gasperi, Valeria; Prosperetti, Chiara; Bernardi, Giorgio; Finazzi-Agro, Alessandro; Cravatt, Benjamin F.; Centonze, Diego
Nature Neuroscience (2008), 11 (2), 152-159CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these mols. has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metab. and stimulation of presynaptic CB1 receptors. Elevation of AEA concns. by pharmacol. or genetic inhibition of AEA degrdn. reduced the levels, metab. and physiol. effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG prodn., confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metab. through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacol. and genetic inactivation of TRPV1 channels.
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Buczynski, M. W. and Parsons, L. H. (2010) Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls Br. J. Pharmacol. 160, 423– 442 DOI: 10.1111/j.1476-5381.2010.00787.x
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Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls
Buczynski, Matthew W.; Parsons, Loren H.
British Journal of Pharmacology (2010), 160 (3), 423-442CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
A review. Endocannabinoids play an important role in a diverse range of neurophysiol. processes including neural development, neuroimmune function, synaptic plasticity, pain, reward and affective state. This breadth of influence and evidence for altered endocannabinoid signaling in a variety of neuropathologies has fueled interest in the accurate quantification of these lipids in brain tissue. Established methods for endocannabinoid quantification primarily employ solvent-based lipid extn. with further sample purifn. by solid phase extn. In recent years in vivo microdialysis methods have also been developed for endocannabinoid sampling from the brain interstitial space. However, considerable variability in ests. of endocannabinoid content has led to debate regarding the physiol. range of concns. present in various brain regions. This paper provides a crit. review of factors that influence the quantification of brain endocannabinoid content as detd. by lipid extn. from bulk tissue and by in vivo microdialysis. A variety of methodol. issues are discussed including anal. approaches, endocannabinoid extn. and purifn., post-mortem changes in brain endocannabinoid content, cellular reactions to microdialysis probe implantation and caveats related to lipid sampling from the extracellular space. The application of these methods for estg. brain endocannabinoid content and the effects of endocannabinoid clearance inhibition are discussed. The benefits, limitations and pitfalls assocd. with each approach are emphasized, with an eye toward the appropriate interpretation of data gathered by each method.

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Abstract
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Figure 1
Figure 1. Biosynthetic and catabolic pathways of 2-AG and AEA.
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Figure 2
Figure 2. Gel-based mapping of brain region dependent hydrolase activity. (A) Activity-based protein profiling with MB064 in four mouse brain regions. (B) Quantification of endocannabinoid hydrolase activity as determined with MB064 (mean ± SD; n = 3). (C) Activity-based protein profiling with FP-TAMRA in four mouse brain regions. (D) Quantification of FAAH activity as measured with ABP FP-TAMRA (mean ± SD; n = 3). Statistical analysis was performed by means of two-way ANOVA with Tukey multiple comparisons test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; C = cerebellum, F = frontal cortex, H = hippocampus, S = striatum). Integrated band intensity is corrected for protein loading (coomassie).
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Figure 3
Figure 3. Brain region dependent chemoproteomic mapping of relative hydrolase activity. (A) Schematic representation of the chemoproteomic workflow. (B) Heat map of relative enzyme activity as measured by a fluorophosphonate (FP)-based activity-based probe conjugated to a biotin reporter tag (FP-biotin) and a β-lactone-based activity-based probe conjugated to a biotin reporter tag (MB108). Data are calculated from the mean ratios of the comparison between cerebellum and striatum, frontal cortex and striatum, and cerebellum and hippocampus. Each comparison was performed in three biological replicates. The relative enzyme activity in the brain region in which the serine hydrolase displayed highest activity was set to 100%. (C) Correlation graph for enzymes detected by both MB108 and FP-biotin. (D) Endocannabinoid regulating enzymes show significant difference between the studied brain regions (mean ± SEM; n = 3). Statistical analysis was performed by means of two-way ANOVA with Tukey multiple comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001) (C = cerebellum, F = frontal cortex, H = hippocampus, S = striatum).
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Figure 4
Figure 4. Gel based CB1^+/+ vs CB1^–/– comparison. Study of enzyme activity in CB1^+/+ and CB1^–/– mouse brain regions as measured by two activity based probes (MB064 and TAMRA-FP, n = 3). No difference in enzyme activity between CB1^–/– and CB1^+/+ was observed.
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Figure 5
Figure 5. Comparison of enzyme activity in CB1^+/+ and CB1^–/– mouse brain regions. Log₂ ratio of enzyme activity in CB1^–/– brain regions compared to CB1^+/+ (mean ± SD). Activity is measured by activity-based proteomics using FP-biotin (10 μM) and MB108 (10 μM). Combined data from both activity based probes (n = 4 for each brain region). Statistical analysis by means of one-way ANOVA, each CB1 WT/KO ratio was compared to a Log₂ ratio of 0; subsequently the p values were subjected to Benjamini Hochberg correction. Setting the false discovery rate at 10%.
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References
This article references 48 other publications.
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  Tsuboi, Kazuhito; Okamoto, Yoko; Rahman, Iffat Ara Sonia; Uyama, Toru; Inoue, Tomohito; Tokumura, Akira; Ueda, Natsuo
  Biochimica et Biophysica Acta, Molecular and Cell Biology of Lipids (2015), 1851 (5), 537-548CODEN: BBMLFG; ISSN:1388-1981. (Elsevier B. V.)
  Bioactive N-acylethanolamines include anti-inflammatory palmitoylethanolamide, anorexic oleoylethanolamide, and an endocannabinoid arachidonoylethanolamide (anandamide). In animal tissues, these mols. are biosynthesized from N-acylethanolamine phospholipids directly by phospholipase D-type enzyme or through multi-step routes via N-acylethanolamine lysophospholipids. We previously found that mouse brain has a lysophospholipase D (lysoPLD) activity hydrolyzing N-acylethanolamine lysophospholipids to N-acylethanolamines and that this activity could be partially attributed to glycerophosphodiesterase (GDE) 1. In the present study, we examd. catalytic properties of GDE4, another member of the GDE family. When overexpressed in HEK293 cells, murine GDE4 mostly resided in the membrane fraction. Purified GDE4 showed lysoPLD activity toward various lysophospholipids, including N-acylethanolamine lysophospholipids as well as lysophosphatidylethanolamine and lysophosphatidylcholine. When HEK293 cells were metabolically labeled with N-[14C]palmitoylethanolamine lysophospholipid, the transient expression of GDE4 increased the [14C]palmitoylethanolamide level, while the knockdown of endogenous GDE4 decreased this level. These results suggested that GDE4 functions as an N-acylethanolamine-generating lysoPLD in living cells. Moreover, the expression of GDE4 increased most species of lysophosphatidic acid (LPA), which can be produced from various lysophospholipids by the lysoPLD activity of GDE4. GDE4 mRNA was widely distributed among mouse tissues including brain, stomach, ileum, colon, and testis. In conclusion, GDE4 may act as a lysoPLD, which is involved in the generation of N-acylethanolamines and LPA.
15. 15
  Liu, J., Wang, L., Harvey-White, J., Huang, B. X., Kim, H. Y., Luquet, S., Palmiter, R. D., Krystal, G., Rai, R., Mahadevan, A., Razdan, R. K., and Kunos, G. (2008) Multiple pathways involved in the biosynthesis of anandamide Neuropharmacology 54, 1– 7 DOI: 10.1016/j.neuropharm.2007.05.020
  15
  Multiple pathways involved in the biosynthesis of anandamide
  Liu Jie; Wang Lei; Harvey-White Judith; Huang Bill X; Kim Hee-Yong; Luquet Serge; Palmiter Richard D; Krystal Gerald; Rai Ravi; Mahadevan Anu; Razdan Raj K; Kunos George
  Neuropharmacology (2008), 54 (1), 1-7 ISSN:0028-3908.
  Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by alpha,beta-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5' phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD(-/-) mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.
16. 16
  Rahman, I. A. S., Tsuboi, K., Uyama, T., and Ueda, N. (2014) New players in the fatty acyl ethanolamide metabolism Pharmacol. Res. 86, 1– 10 DOI: 10.1016/j.phrs.2014.04.001
  16
  New players in the fatty acyl ethanolamide metabolism
  Rahman, Iffat Ara Sonia; Tsuboi, Kazuhito; Uyama, Toru; Ueda, Natsuo
  Pharmacological Research (2014), 86 (), 1-10CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
  A review. Fatty acyl ethanolamides represent a class of endogenous bioactive lipid mols. and are generally referred to as N-acylethanolamines (NAEs). NAEs include palmitoylethanolamide (anti-inflammatory and analgesic substance), oleoylethanolamide (anorexic substance), and anandamide (endocannabinoid). The endogenous levels of NAEs are mainly regulated by enzymes responsible for their biosynthesis and degrdn. In mammalian tissues, the major biosynthetic pathway starts from glycerophospholipids and is composed of two enzyme reactions. The first step is N-acylation of ethanolamine phospholipids catalyzed by Ca2+-dependent N-acyltransferase and the second step is the release of NAEs from N-acylated ethanolamine phospholipids by N-acylphosphatidylethanolamine (NAPE)-hydrolyzing phospholipase D (NAPE-PLD). As for the degrdn. of NAEs, fatty acid amide hydrolase plays the central role. However, recent studies strongly suggest the involvement of other enzymes in the NAE metab. These enzymes include members of the HRAS-like suppressor family (also called phospholipase A/acyltransferase family), which were originally discovered as tumor suppressors but can function as Ca2+-independent NAPE-forming N-acyltransferases; multiple enzymes involved in the NAPE-PLD-independent multi-step pathways to generate NAE from NAPE, which came to light by the anal. of NAPE-PLD-deficient mice; and a lysosomal NAE-hydrolyzing acid amidase as a second NAE hydrolase. These newly recognized enzymes may become the targets for the development of new therapeutic drugs. Here, we focus on recent enzymol. findings in this area.
17. 17
  Cravatt, B. F., Giang, D. K., Mayfield, S. P., Boger, D. L., Lerner, R. A., and Gilula, N. B. (1996) Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides Nature 384, 83– 87 DOI: 10.1038/384083a0
  17
  Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid amides
  Cravatt, Benjamin F.; Giang, Dan K.; Mayfield, Stephen P.; Boger, Dale L.; Lerner, Richard A.; Gilula, Norton B.
  Nature (London) (1996), 384 (6604), 83-87CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
  Endogenous neuromodulatory mols.. are commonly couples to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolyzed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-deproved cats. Cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling mols., the fatty-acid amides. Therefore we will hereafter refer to oelamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
18. 18
  Belluomo, I., Matias, I., Pernegre, C., Marsicano, G., and Chaouloff, F. (2015) Opposite control of frontocortical 2-arachidonoylglycerol turnover rate by cannabinoid type-1 receptors located on glutamatergic neurons and on astrocytes J. Neurochem. 133, 26– 37 DOI: 10.1111/jnc.13044
  18
  Opposite control of frontocortical 2-arachidonoylglycerol turnover rate by cannabinoid type-1 receptors located on glutamatergic neurons and on astrocytes
  Belluomo, Ilaria; Matias, Isabelle; Pernegre, Camille; Marsicano, Giovanni; Chaouloff, Francis
  Journal of Neurochemistry (2015), 133 (1), 26-37CODEN: JONRA9; ISSN:0022-3042. (Wiley-Blackwell)
  This study examd. the resp. influences of cannabinoid type-1 (CB1) receptors expressed either in forebrain GABAergic neurons, in cortical glutamatergic neurons, or in astrocytes on the turnover rates of the endocannabinoids N-arachidonoylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), and the non-cannabinoid N-acylethanolamides, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA), in mouse forebrain regions. To this end, conditional mutant mice lacking CB1 receptors from either of these cell types were pretreated systemically with JZL 195, a dual inhibitor of fatty acid amide hydrolase, the enzyme degrading AEA, PEA, and OEA, and of monoacylglycerol lipase, the main 2-AG-degrading enzyme. The analyses of frontocortical, hippocampal, and striatal AEA, 2-AG, PEA, and OEA concns. revealed that their resp. baseline concns. were not influenced by the mouse genotype. On the other hand, the accumulation of frontocortical and/or hippocampal 2-AG levels in JZL 195-pretreated mice was dependent on the mouse genotype. Thus, JZL 195-induced 2-AG accumulation rates were diminished in the frontal cortex of mice lacking CB1 receptors in glutamatergic neurons while their resp. values were increased in the frontal cortex and hippocampus of mice lacking these receptors in astrocytes. These genotypic differences occurred with parallel and proportionate changes in the fractional rate consts. for degrdn. of 2-AG, thus providing a mechanism whereby the baseline levels of 2-AG remained const. between genotypes. Besides suggesting a cell-type-specific control of frontocortical and/or hippocampal 2-AG synthesis and degrdn. rates by CB1 receptors, this study highlights the interest of assessing endocannabinoid turnover rates when questioning the status of the endocannabinoid system. In mice lacking CB1 receptors from glutamatergic neurons (Glu-CB1-/-) or from astrocytes (GFAP-CB1-/-), frontocortical and hippocampal 2-AG levels are not altered. Turnover studies indicate, however, that decreased 2-AG synthesis occurs with decreased degrdn. in Glu-CB1-/- mice, whereas the opposite is obsd. in GFAP-CB1-/- mice.
19. 19
  Starke, K., Gothert, M., and Kilbinger, H. (1989) Modulation of neurotransmitter release by presynaptic autoreceptors Physiol. Rev. 69, 864– 989
  There is no corresponding record for this reference.
20. 20
  Langer, S. Z. (1997) 25 years since the discovery of presynaptic receptors: present knowledge and future perspectives Trends Pharmacol. Sci. 18, 95– 99 DOI: 10.1016/S0165-6147(96)01034-6
  There is no corresponding record for this reference.
21. 21
  Di Marzo, V., Breivogel, C. S., Tao, Q., Bridgen, D. T., Razdan, R. K., Zimmer, A. M., Zimmer, A., and Martin, B. R. (2000) Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: Evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain J. Neurochem. 75, 2434– 2444 DOI: 10.1046/j.1471-4159.2000.0752434.x
  21
  Levels, metabolism, and pharmacological activity of anandamide in CB1 cannabinoid receptor knockout mice: evidence for non-CB1, non-CB2 receptor-mediated actions of anandamide in mouse brain
  Di Marzo, Vincenzo; Breivogel, Chris S.; Tao, Qing; Bridgen, David T.; Razdan, Raj K.; Zimmer, Anne M.; Zimmer, Andreas; Martin, Billy R.
  Journal of Neurochemistry (2000), 75 (6), 2434-2444CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
  Anandamide [arachidonylethanolamide (AEA)] appears to be an endogenous agonist of brain cannabinoid receptors (CB1), yet some of the neurobehavioral effects of this compd. in mice are unaffected by a selective CB1 antagonist. We studied the levels, pharmacol. actions, and degrdn. of AEA in transgenic mice lacking the CB1 gene. We quantified AEA and the other endocannabinoid, 2-arachidonoyl glycerol, in six brain regions and the spinal cord by isotope-diln. liq. chromatog.-mass spectrometry. The distribution of endocannabinoids and their inactivating enzyme, fatty acid amide hydrolase, were found to overlap with CB1 distribution only in part. In CB1 knockout homozygotes (CB1-/-), the hippocampus and, to a lesser extent, the striatum exhibited lower AEA levels as compared with wild-type (CB1+/+) controls. These data suggest a ligand/receptor relationship between AEA and CB1 in these two brain regions, where tonic activation of the receptor may tightly regulate the biosynthesis of its endogenous ligand. 2-Arachidonoyl glycerol levels and fatty acid amide hydrolase activity were unchanged in CB1-/- with respect to CB1+/+ mice in all regions, AEA and Δ9-tetrahydrocannabinol (THC) were tested in CB1-/- mice for their capability of inducing analgesia and catalepsy and decreasing spontaneous activity. The effects of AEA, unlike THC, were not decreased in CB1-/- mice. AEA, but not THC, stimulated GTPγS binding in brain membranes from CB1-/- mice, and this stimulation was insensitive to CB1 and CB2 antagonists. We suggest that non-CB1, non-CB2 G protein-coupled receptors might mediate in mice some of the neurobehavioral actions of AEA.
22. 22
  Di Marzo, V., Berrendero, F., Bisogno, T., Gonzalez, S., Cavaliere, P., Romero, J., Cebeira, M., Ramos, J. A., and Fernandez-Ruiz, J. J. (2000) Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of Delta(9)-tetrahydrocannabinol-tolerant rats J. Neurochem. 74, 1627– 1635 DOI: 10.1046/j.1471-4159.2000.0741627.x
  22
  Enhancement of anandamide formation in the limbic forebrain and reduction of endocannabinoid contents in the striatum of Δ9-tetrahydrocannabinol-tolerant rats
  Di Marzo, V.; Berrendero, F.; Bisogno, T.; Gonzalez, S.; Cavaliere, P.; Romero, J.; Cebeira, M.; Ramos, J. A.; Fernandez-Ruiz, J. J.
  Journal of Neurochemistry (2000), 74 (4), 1627-1635CODEN: JONRA9; ISSN:0022-3042. (Lippincott Williams & Wilkins)
  Recent studies have shown that the pharmacol. tolerance obsd. after prolonged exposure to synthetic or plant-derived cannabinoids in adult rats is accompanied by down-regulation/desensitization of brain cannabinoid receptors. However, no evidence exists on possible changes in the contents of the endogenous ligands of cannabinoid receptors in the brain of cannabinoid-tolerant rats. The present study was designed to elucidate this possibility by measuring, by means of isotope diln. gas chromatog./mass spectrometry, the contents of both anandamide (arachidonoylethanolamide; AEA) and its biosynthetic precursor, N-arachidonoylphosphatidylethanolamine (NArPE), and 2-arachidonoylglycerol (2-AG) in several brain regions of adult male rats treated daily with Δ9-tetrahydrocannabinol (Δ9-THC) for a period of 8 days. The areas analyzed included cerebellum, striatum, limbic forebrain, hippocampus, cerebral cortex, and brainstem. The same regions were also analyzed for cannabinoid receptor binding and WIN-55,212-2-stimulated guanylyl-5'-O-(γ-[35S]thio)-triphosphate ([35S]GTPγS) binding to test the development of the well known down-regulation/desensitization phenomenon. Results were as follows: As expected, cannabinoid receptor binding and WIN-55,212-2-stimulated [35S]GTPγS binding decreased in most of the brain areas of Δ9-THC-tolerant rats. The only region exhibiting no changes in both parameters was the limbic forebrain. This same region exhibited a marked (almost fourfold) increase in the content of AEA after 8 days of Δ9-THC treatment. By contrast, the striatum exhibited a decrease in AEA contents, whereas no changes were found in the brainstem, hippocampus, cerebellum, or cerebral cortex. The increase in AEA contents obsd. in the limbic forebrain was accompanied by a tendency of NArPE levels to decrease, whereas in the striatum, no significant change in NArPE contents was found. The contents of 2-AG were unchanged in brain regions from Δ9-THC-tolerant rats, except for the striatum where they dropped significantly. In summary, the present results show that prolonged activation of cannabinoid receptors leads to decreased endocannabinoid contents and signaling in the striatum and to increased AEA formation in the limbic forebrain. The pathophysiol. implications of these findings are discussed in view of the proposed roles of endocannabinoids in the control of motor behavior and emotional states.
23. 23
  Maccarrone, M., Attina, M., Bari, M., Cartoni, A., Ledent, C., and Finazzi-Agro, A. (2001) Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages J. Neurochem. 78, 339– 348 DOI: 10.1046/j.1471-4159.2001.00413.x
  23
  Anandamide degradation and N-acylethanolamines level in wild-type and CB1 cannabinoid receptor knockout mice of different ages
  Maccarrone, Mauro; Attina, Marina; Bari, Monica; Cartoni, Antonella; Ledent, Catherine; Finazzi-Agro, Alessandro
  Journal of Neurochemistry (2001), 78 (2), 339-348CODEN: JONRA9; ISSN:0022-3042. (Blackwell Science Ltd.)
  CD1 mice lacking the CB1 receptors (knockout, KO) were compared with wild-type littermates for their ability to degrade N-arachidonoylethanolamine (anandamide, AEA) through a membrane transporter (AMT) and a fatty acid amide hydrolase (FAAH). The regional distribution and age-dependence of AMT and FAAH activity were investigated. Anandamide membrane transporter and FAAH increased with age in knockout mice, whereas they showed minor changes in wild-type animals. Remarkably, they were higher in all brain areas of 6-mo-old knockout vs. wild-type mice, and even higher in 12-mo-old animals. The mol. mass (≈67 kDa) and isoelec. point (≈7.6) of mouse brain FAAH were detd. and the FAAH protein content was shown to parallel the enzyme activity. The kinetic consts. of AMT and FAAH in the cortex of wild-type and knockout mice at different ages suggested that different amts. of the same proteins were expressed. The cortex and hippocampus of wild-type and knockout mice contained the following N-acylethanolamines: AEA (8% of total), 2-arachidonoylglycerol (5%), N-oleoylethanolamine (20%), N-palmitoylethanolamine (53%) and N-stearoylethanolamine (14%). These compds. were twice as abundant in the hippocampus as in the cortex. Minor differences were obsd. in AEA or 2-arachidonoylglycerol content in knockout vs. wild-type mice, whereas the other compds. were lower in the hippocampus of knockout vs. wild-type animals.
24. 24
  Maccarrone, M., Valverde, O., Barbaccia, M. L., Castane, A., Maldonado, R., Ledent, C., Parmentier, M., and Finazzi-Agro, A. (2002) Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour Eur. J. Neurosci. 15, 1178– 1186 DOI: 10.1046/j.1460-9568.2002.01957.x
  24
  Age-related changes of anandamide metabolism in CB1 cannabinoid receptor knockout mice: correlation with behaviour
  Maccarrone Mauro; Valverde Olga; Barbaccia Maria L; Castane Anna; Maldonado Rafael; Ledent Catherine; Parmentier Marc; Finazzi-Agro Alessandro
  The European journal of neuroscience (2002), 15 (7), 1178-86 ISSN:0953-816X.
  Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the most active endocannabinoids at brain (CB1) cannabinoid receptors. CD1 mice lacking the CB1 receptors ("knockout" [KO] mutants) were compared with wildtype (WT) littermates for their ability to degrade AEA through an AEA membrane transporter (AMT) and an AEA hydrolase (fatty acid amide hydrolase, FAAH). The age dependence of AMT and FAAH activity were investigated in 1- or 4-month-old WT and KO animals, and found to increase with age in KO, but not WT, mice and to be higher in the hippocampus than in the cortex of all animals. AEA and 2-AG were detected in nmol/mg protein (microm) concentrations in both regions, though the hippocampus showed approximately twice the amount found in the cortex. In the same regions, 2-AG failed to change across groups, while AEA was significantly decreased (approximately 30%) in hippocampus, but not in cortex, of old KO mice, when compared with young KO or age-matched WT animals. In the open-field test under bright light and in the lit-dark exploration model of anxiety, young KO mice, compared with old KO, exhibited a mild anxiety-related behaviour. In contrast, neither the increase in memory performance assessed by the object recognition test, nor the reduction of morphine withdrawal symptoms, showed age dependence in CB1 KO mice. These results suggest that invalidation of the CB1 receptor gene is associated with age-dependent adaptive changes of endocannabinoid metabolism which appear to correlate with the waning of the anxiety-like behaviour exhibited by young CB1 KO mice.
25. 25
  Bequet, F., Uzabiaga, F., Desbazeille, M., Ludwiczak, P., Maftouh, M., Picard, C., Scatton, B., and Le Fur, G. (2007) CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus Eur. J. Neurosci. 26, 3458– 3464 DOI: 10.1111/j.1460-9568.2007.05900.x
  25
  CB1 receptor-mediated control of the release of endocannabinoids (as assessed by microdialysis coupled with LC/MS) in the rat hypothalamus
  Bequet Frederic; Uzabiaga Francoise; Desbazeille Mathieu; Ludwiczak Pascal; Maftouh Mohamed; Picard Claudine; Scatton Bernard; Le Fur Gerard
  The European journal of neuroscience (2007), 26 (12), 3458-64 ISSN:.
  In the present study, we examined the occurrence and potential regulation of endocannabinoid release by cannabinoid CB1 receptors in the rat brain. To this end, we developed a highly sensitive (limit of sensitivity 30-300 amol) new analytical method, combining online brain microdialysis with solid-phase extraction-liquid chromatography-tandem mass spectrometry, which allowed the detection in real time of trace amounts of endocannabinoids in the extracellular fluid. In the hypothalamus, anandamide and 2-arachidonoyl-glycerol release was stimulated following depolarization via local administration of K(+), with or without addition of Ca(2+), or glutamate application. Inhibition of fatty acid amide hydrolase by systemic administration of intraperitoneal (i.p.) URB597 (0.5 mg/kg) induced an increase of anandamide, but not 2-arachidonoyl-glycerol, outflow. The CB1 receptor antagonist rimonabant (10 mg/kg i.p.) increased, whereas the CB1 agonist WIN55,212-2 (2.5 mg/kg i.p.) decreased, anandamide release. Interestingly, the same treatments induced opposite changes in 2-arachidonoyl-glycerol release. At a dose of 3 mg/kg i.p., which by itself did not affect endocannabinoid release, rimonabant fully antagonized the effect of WIN55,212-2 (2.5 mg/kg i.p.). Taken together, these results suggest that CB1 receptors are able to control the local release of endocannabinoids in the hypothalamus via a feedback mechanism and strengthen the view that anandamide and 2-arachidonoyl-glycerol have distinct physiological roles.
26. 26
  Leishman, E., Cornett, B., Spork, K., Straiker, A., Mackie, K., and Bradshaw, H. B. (2016) Broad impact of deleting endogenous cannabinoid hydrolyzing enzymes and the CB1 cannabinoid receptor on the endogenous cannabinoid-related lipidome in eight regions of the mouse brain Pharmacol. Res. 110, 159– 172 DOI: 10.1016/j.phrs.2016.04.020
  26
  Broad impact of deleting endogenous cannabinoid hydrolyzing enzymes and the CB1 cannabinoid receptor on the endogenous cannabinoid-related lipidome in eight regions of the mouse brain
  Leishman, Emma; Cornett, Ben; Spork, Karl; Straiker, Alex; Mackie, Ken; Bradshaw, Heather B.
  Pharmacological Research (2016), 110 (), 159-172CODEN: PHMREP; ISSN:1043-6618. (Elsevier Ltd.)
  The enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) hydrolyze endogenous cannabinoids (eCBs), N-arachidonoyl ethanolamine (AEA) and 2-arachidonoyl glycerol (2-AG), resp. These enzymes also metabolize eCB analogs such as lipoamines and 2-acyl glycerols, most of which are not ligands at CB1. To test the hypothesis that deleting eCB hydrolyzing enzymes and CB1 shifts lipid metab. more broadly and impacts more families of eCB structural analogs, targeted lipidomics analyses were performed on FAAH KO, MAGL KO, and CB1 KO mice and compared to WT controls in 8 brain regions. Methanolic exts. of discrete brain regions (brainstem, cerebellum, cortex, hippocampus, hypothalamus, midbrain, striatum and thalamus) were partially purified on C-18 solid-phase extn. columns. Over 70 lipids per sample were then analyzed with HPLC/MS/MS. AEA and 2-AG were unaffected throughout the brain in CB1 KO mice; however, there was an increase in the arachidonic acid (AA) metabolite, PGE2 in the majority of brain areas. By contrast, PGE2 and AA levels were significantly reduced throughout the brain in the MAGL KO corresponding to significant increases in 2-AG. No changes in AA or PGE2 were seen throughout in the FAAH KO brain, despite significant increases in AEA, suggesting AA liberated by FAAH does not contribute to steady state levels of AA or PGE2. Changes in the lipidome were not confined to the AA derivs. and showed regional variation in each of the eCB KO models. AEA and 2-AG hydrolyzing enzymes and the CB1 receptor link the eCB system to broader lipid signaling networks in contrasting ways, potentially altering neurotransmission and behavior independently of cannabinoid receptor signaling.
27. 27
  Schlosburg, J. E., Blankman, J. L., Long, J. Z., Nomura, D. K., Pan, B., Kinsey, S. G., Nguyen, P. T., Ramesh, D., Booker, L., Burston, J. J., Thomas, E. A., Selley, D. E., Sim-Selley, L. J., Liu, Q. S., Lichtman, A. H., and Cravatt, B. F. (2010) Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system Nat. Neurosci. 13, 1113– 1119 DOI: 10.1038/nn.2616
  27
  Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system
  Schlosburg, Joel E.; Blankman, Jacqueline L.; Long, Jonathan Z.; Nomura, Daniel K.; Pan, Bin; Kinsey, Steven G.; Nguyen, Peter T.; Ramesh, Divya; Booker, Lamont; Burston, James J.; Thomas, Elizabeth A.; Selley, Dana E.; Sim-Selley, Laura J.; Liu, Qing-song; Lichtman, Aron H.; Cravatt, Benjamin F.
  Nature Neuroscience (2010), 13 (9), 1113-1119CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
  Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacol. tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused phys. dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and assocd. with agonism and functional antagonism of the brain cannabinoid system, resp.
28. 28
  Kang, H. J., Kawasawa, Y. I., Cheng, F., Zhu, Y., Xu, X. M., Li, M. F., Sousa, A. M. M., Pletikos, M., Meyer, K. A., Sedmak, G., Guennel, T., Shin, Y., Johnson, M. B., Krsnik, Z., Mayer, S., Fertuzinhos, S., Umlauf, S., Lisgo, S. N., Vortmeyer, A., Weinberger, D. R., Mane, S., Hyde, T. M., Huttner, A., Reimers, M., Kleinman, J. E., and Sestan, N. (2011) Spatio-temporal transcriptome of the human brain Nature 478, 483– 489 DOI: 10.1038/nature10523
  28
  Spatio-temporal transcriptome of the human brain
  Kang, Hyo Jung; Kawasawa, Yuka Imamura; Cheng, Feng; Zhu, Ying; Xu, Xuming; Li, Mingfeng; Sousa, Andre M. M.; Pletikos, Mihovil; Meyer, Kyle A.; Sedmak, Goran; Guennel, Tobias; Shin, Yurae; Johnson, Matthew B.; Krsnik, Zeljka; Mayer, Simone; Fertuzinhos, Sofia; Umlauf, Sheila; Lisgo, Steven N.; Vortmeyer, Alexander; Weinberger, Daniel R.; Mane, Shrikant; Hyde, Thomas M.; Huttner, Anita; Reimers, Mark; Kleinman, Joel E.; Sestan, Nenad
  Nature (London, United Kingdom) (2011), 478 (7370), 483-489CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  Brain development and function depend on the precise regulation of gene expression. However, our understanding of the complexity and dynamics of the transcriptome of the human brain is incomplete. Here we report the generation and anal. of exon-level transcriptome and assocd. genotyping data, representing males and females of different ethnicities, from multiple brain regions and neocortical areas of developing and adult post-mortem human brains. We found that 86% of the genes analyzed were expressed, and that 90% of these were differentially regulated at the whole-transcript or exon level across brain regions and/or time. The majority of these spatio-temporal differences were detected before birth, with subsequent increases in the similarity among regional transcriptomes. The transcriptome is organized into distinct coexpression networks, and shows sex-biased gene expression and exon usage. We also profiled trajectories of genes assocd. with neurobiol. categories and diseases, and identified assocns. between single nucleotide polymorphisms and gene expression. This study provides a comprehensive data set on the human brain transcriptome and insights into the transcriptional foundations of human neurodevelopment.
29. 29
  Sharma, K., Schmitt, S., Bergner, C. G., Tyanova, S., Kannaiyan, N., Manrique-Hoyos, N., Kongi, K., Cantuti, L., Hanisch, U. K., Philips, M. A., Rossner, M. J., Mann, M., and Simons, M. (2015) Cell type- and brain region-resolved mouse brain proteome Nat. Neurosci. 18, 1819– 1831 DOI: 10.1038/nn.4160
  29
  Cell type- and brain region-resolved mouse brain proteome
  Sharma, Kirti; Schmitt, Sebastian; Bergner, Caroline G.; Tyanova, Stefka; Kannaiyan, Nirmal; Manrique-Hoyos, Natalia; Kongi, Karina; Cantuti, Ludovico; Hanisch, Uwe-Karsten; Philips, Mari-Anne; Rossner, Moritz J.; Mann, Matthias; Simons, Mikael
  Nature Neuroscience (2015), 18 (12), 1819-1831CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
  Brain transcriptome and connectome maps are being generated, but an equiv. effort on the proteome is currently lacking. We performed high-resoln. mass spectrometry-based proteomics for in-depth anal. of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type-specific proteins defined as tenfold more abundant than av. expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion mol. of the IgLON family, as a neg. regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.
30. 30
  Lein, E. S., Hawrylycz, M. J., Ao, N., Ayres, M., Bensinger, A., Bernard, A., Boe, A. F., Boguski, M. S., Brockway, K. S., Byrnes, E. J., Chen, L., Chen, L., Chen, T. M., Chi Chin, M., Chong, J., Crook, B. E., Czaplinska, A., Dang, C. N., Datta, S., Dee, N. R., Desaki, A. L., Desta, T., Diep, E., Dolbeare, T. A., Donelan, M. J., Dong, H. W., Dougherty, J. G., Duncan, B. J., Ebbert, A. J., Eichele, G., Estin, L. K., Faber, C., Facer, B. A., Fields, R., Fischer, S. R., Fliss, T. P., Frensley, C., Gates, S. N., Glattfelder, K. J., Halverson, K. R., Hart, M. R., Hohmann, J. G., Howell, M. P., Jeung, D. P., Johnson, R. A., Karr, P. T., Kawal, R., Kidney, J. M., Knapik, R. H., Kuan, C. L., Lake, J. H., Laramee, A. R., Larsen, K. D., Lau, C., Lemon, T. A., Liang, A. J., Liu, Y., Luong, L. T., Michaels, J., Morgan, J. J., Morgan, R. J., Mortrud, M. T., Mosqueda, N. F., Ng, L. L., Ng, R., Orta, G. J., Overly, C. C., Pak, T. H., Parry, S. E., Pathak, S. D., Pearson, O. C., Puchalski, R. B., Riley, Z. L., Rockett, H. R., Rowland, S. A., Royall, J. J., Ruiz, M. J., Sarno, N. R., Schaffnit, K., Shapovalova, N. V., Sivisay, T., Slaughterbeck, C. R., Smith, S. C., Smith, K. A., Smith, B. I., Sodt, A. J., Stewart, N. N., Stumpf, K. R., Sunkin, S. M., Sutram, M., Tam, A., Teemer, C. D., Thaller, C., Thompson, C. L., Varnam, L. R., Visel, A., Whitlock, R. M., Wohnoutka, P. E., Wolkey, C. K., Wong, V. Y., Wood, M., Yaylaoglu, M. B., Young, R. C., Youngstrom, B. L., Feng Yuan, X., Zhang, B., Zwingman, T. A., and Jones, A. R. (2007) Genome-wide atlas of gene expression in the adult mouse brain Nature 445, 168– 176 DOI: 10.1038/nature05453
  30
  Genome-wide atlas of gene expression in the adult mouse brain
  Lein, Ed S.; Hawrylycz, Michael J.; Ao, Nancy; Ayres, Mikael; Bensinger, Amy; Bernard, Amy; Boe, Andrew F.; Boguski, Mark S.; Brockway, Kevin S.; Byrnes, Emi J.; Chen, Lin; Chen, Li; Chen, Tsuey-Ming; Chin, Mei Chi; Chong, Jimmy; Crook, Brian E.; Czaplinska, Aneta; Dang, Chinh N.; Datta, Suvro; Dee, Nick R.; Desaki, Aimee L.; Desta, Tsega; Diep, Ellen; Dolbeare, Tim A.; Donelan, Matthew J.; Dong, Hong-Wei; Dougherty, Jennifer G.; Duncan, Ben J.; Ebbert, Amanda J.; Eichele, Gregor; Estin, Lili K.; Faber, Casey; Facer, Benjamin A.; Fields, Rick; Fischer, Shanna R.; Fliss, Tim P.; Frensley, Cliff; Gates, Sabrina N.; Glattfelder, Katie J.; Halverson, Kevin R.; Hart, Matthew R.; Hohmann, John G.; Howell, Maureen P.; Jeung, Darren P.; Johnson, Rebecca A.; Karr, Patrick T.; Kawal, Reena; Kidney, Jolene M.; Knapik, Rachel H.; Kuan, Chihchau L.; Lake, James H.; Laramee, Annabel R.; Larsen, Kirk D.; Lau, Christopher; Lemon, Tracy A.; Liang, Agnes J.; Liu, Ying; Luong, Lon T.; Michaels, Jesse; Morgan, Judith J.; Morgan, Rebecca J.; Mortrud, Marty T.; Mosqueda, Nerick F.; Ng, Lydia L.; Ng, Randy; Orta, Geralyn J.; Overly, Caroline C.; Pak, Tu H.; Parry, Sheana E.; Pathak, Sayan D.; Pearson, Owen C.; Puchalski, Ralph B.; Riley, Zackery L.; Rockett, Hannah R.; Rowland, Stephen A.; Royall, Joshua J.; Ruiz, Marcos J.; Sarno, Nadia R.; Schaffnit, Katherine; Shapovalova, Nadiya V.; Sivisay, Taz; Slaughterbeck, Clifford R.; Smith, Simon C.; Smith, Kimberly A.; Smith, Bryan I.; Sodt, Andy J.; Stewart, Nick N.; Stumpf, Kenda-Ruth; Sunkin, Susan M.; Sutram, Madhavi; Tam, Angelene; Teemer, Carey D.; Thaller, Christina; Thompson, Carol L.; Varnam, Lee R.; Visel, Axel; Whitlock, Ray M.; Wohnoutka, Paul E.; Wolkey, Crissa K.; Wong, Victoria Y.; Wood, Matthew; Yaylaoglu, Murat B.; Young, Rob C.; Youngstrom, Brian L.; Yuan, Xu Feng; Zhang, Bin; Zwingman, Theresa A.; Jones, Allan R.
  Nature (London, United Kingdom) (2007), 445 (7124), 168-176CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
  Mol. approaches to understanding the functional circuitry of the nervous system promise new insights into the relationship between genes, brain and behavior. The cellular diversity of the brain necessitates a cellular resoln. approach towards understanding the functional genomics of the nervous system. We describe here an anatomically comprehensive digital atlas contg. the expression patterns of ∼20,000 genes in the adult mouse brain. Data were generated using automated high-throughput procedures for in situ hybridization and data acquisition, and are publicly accessible online. Newly developed image-based informatics tools allow global genome-scale structural anal. and cross-correlation, as well as identification of regionally enriched genes. Unbiased fine-resoln. anal. has identified highly specific cellular markers as well as extensive evidence of cellular heterogeneity not evident in classical neuroanatomical atlases. This highly standardized atlas provides an open, primary data resource for a wide variety of further studies concerning brain organization and function.
31. 31
  Vogel, C. and Marcotte, E. M. (2012) Insights into the regulation of protein abundance from proteomic and transcriptomic analyses Nat. Rev. Genet. 13, 227– 232 DOI: 10.1038/nrg3185
  31
  Insights into the regulation of protein abundance from proteomic and transcriptomic analyses
  Vogel, Christine; Marcotte, Edward M.
  Nature Reviews Genetics (2012), 13 (4), 227-232CODEN: NRGAAM; ISSN:1471-0056. (Nature Publishing Group)
  A review. Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - i.e., post-transcriptional, translational and protein degrdn. regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.
32. 32
  Kobe, B. and Kemp, B. E. (1999) Active site-directed protein regulation Nature 402, 373– 376 DOI: 10.1038/46478
  32
  Active site-directed protein regulation
  Kobe, Bostjan; Kemp, Bruce E.
  Nature (London) (1999), 402 (6760), 373-376CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
  A review, with 46 refs. Regulation of protein function is vital for the control of cellular processes. Proteins are often regulated by allosteric mechanisms, in which effectors bind to regulatory sites distinct from the active sites and alter protein function. Intrasteric regulation, directed at the active site and thus the counterpart of allosteric control, is now emerging as an important regulatory mechanism.
33. 33
  Dotsey, E. Y., Jung, K. M., Basit, A., Wei, D., Daglian, J., Vacondio, F., Armirotti, A., Mor, M., and Piomelli, D. (2015) Peroxide-Dependent MGL Sulfenylation Regulates 2-AG-Mediated Endocannabinoid Signaling in Brain Neurons Chem. Biol. 22, 619– 628 DOI: 10.1016/j.chembiol.2015.04.013
  33
  Peroxide-Dependent MGL Sulfenylation Regulates 2-AG-Mediated Endocannabinoid Signaling in Brain Neurons
  Dotsey, Emmanuel Y.; Jung, Kwang-Mook; Basit, Abdul; Wei, Don; Daglian, Jennifer; Vacondio, Federica; Armirotti, Andrea; Mor, Marco; Piomelli, Daniele
  Chemistry & Biology (Oxford, United Kingdom) (2015), 22 (5), 619-628CODEN: CBOLE2; ISSN:1074-5521. (Elsevier Ltd.)
  The second messenger hydrogen peroxide transduces changes in the cellular redox state by reversibly oxidizing protein cysteine residues to sulfenic acid. This signaling event regulates many cellular processes but has never been shown to occur in the brain. Here, we report that hydrogen peroxide heightens endocannabinoid signaling in brain neurons through sulfenylation of cysteines C201 and C208 in monoacylglycerol lipase (MGL), a serine hydrolase that deactivates the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in nerve terminals. The results suggest that MGL sulfenylation may provide a presynaptic control point for 2-AG-mediated endocannabinoid signaling.
34. 34
  Shonesy, B. C., Wang, X., Rose, K. L., Ramikie, T. S., Cavener, V. S., Rentz, T., Baucum, A. J., 2nd, Jalan-Sakrikar, N., Mackie, K., Winder, D. G., Patel, S., and Colbran, R. J. (2013) CaMKII regulates diacylglycerol lipase-alpha and striatal endocannabinoid signaling Nat. Neurosci. 16, 456– 463 DOI: 10.1038/nn.3353
  34
  CaMKII regulates diacylglycerol lipase-α and striatal endocannabinoid signaling
  Shonesy, Brian C.; Wang, Xiaohan; Rose, Kristie L.; Ramikie, Teniel S.; Cavener, Victoria S.; Rentz, Tyler; Baucum, Anthony J., II; Jalan-Sakrikar, Nidhi; Mackie, Ken; Winder, Danny G.; Patel, Sachin; Colbran, Roger J.
  Nature Neuroscience (2013), 16 (4), 456-463CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
  The endocannabinoid 2-arachidonoylglycerol (2-AG) mediates activity-dependent depression of excitatory neurotransmission at central synapses, but the mol. regulation of 2-AG synthesis is not well understood. Here we identify a functional interaction between the 2-AG synthetic enzyme diacylglycerol lipase-α (DGLα) and calcium/calmodulin dependent protein kinase II (CaMKII). Activated CaMKII interacted with the C-terminal domain of DGLα, phosphorylated two serine residues and inhibited DGLα activity. Consistent with an inhibitory role for CaMKII in 2-AG synthesis, in vivo genetic inhibition of CaMKII increased striatal DGL activity and basal levels of 2-AG, and CaMKII inhibition augmented short-term retrograde endocannabinoid signaling at striatal glutamatergic synapses. Lastly, blockade of 2-AG breakdown using concns. of JZL-184 that have no effect in wild-type mice produced a hypolocomotor response in mice with reduced CaMKII activity. These findings provide mechanistic insights into the mol. regulation of striatal endocannabinoid signaling with implications for physiol. control of motor function.
35. 35
  Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M. G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E. J., Gangadharan, U., Hobbs, C., Di Marzo, V., and Doherty, P. (2003) Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain J. Cell Biol. 163, 463– 468 DOI: 10.1083/jcb.200305129
  35
  Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain
  Bisogno, Tiziana; Howell, Fiona; Williams, Gareth; Minassi, Alberto; Grazia Cascio, Maria; Ligresti, Alessia; Matias, Isabel; Schiano-Moriello, Aniello; Paul, Praveen; Williams, Emma-Jane; Gangadharan, Uma; Hobbs, Carl; Di Marzo, Vincenzo; Doherty, Patrick
  Journal of Cell Biology (2003), 163 (3), 463-468CODEN: JCLBA3; ISSN:0021-9525. (Rockefeller University Press)
  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 enzymic 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.
36. 36
  Cravatt, B. F., Wright, A. T., and Kozarich, J. W. (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry Annu. Rev. Biochem. 77, 383– 414 DOI: 10.1146/annurev.biochem.75.101304.124125
  36
  Activity-based protein profiling: From enzyme chemistry to proteomic chemistry
  Cravatt, Benjamin F.; Wright, Aaron T.; Kozarich, John W.
  Annual Review of Biochemistry (2008), 77 (), 383-414CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews Inc.)
  A review. Genome sequencing projects have provided researchers with a complete inventory of the predicted proteins produced by eukaryotic and prokaryotic organisms. Assignment of functions to these proteins represents one of the principal challenges for the field of proteomics. Activity-based protein profiling (ABPP) has emerged as a powerful chem. proteomic strategy to characterize enzyme function directly in native biol. systems on a global scale. Here, the authors review the basic technol. of ABPP, the enzyme classes addressable by this method, and the biol. discoveries attributable to its application.
37. 37
  Niphakis, M. J. and Cravatt, B. F. (2014) Enzyme inhibitor discovery by activity-based protein profiling Annu. Rev. Biochem. 83, 341– 377 DOI: 10.1146/annurev-biochem-060713-035708
  37
  Enzyme inhibitor discovery by activity-based protein profiling
  Niphakis, Micah J.; Cravatt, Benjamin F.
  Annual Review of Biochemistry (2014), 83 (), 341-377CODEN: ARBOAW; ISSN:0066-4154. (Annual Reviews)
  Eukaryotic and prokaryotic organisms possess huge nos. of uncharacterized enzymes. Selective inhibitors offer powerful probes for assigning functions to enzymes in native biol. systems. Here, we discuss how the chem. proteomic platform activity-based protein profiling (ABPP) can be implemented to discover selective and in vivo-active inhibitors for enzymes. We further describe how these inhibitors have been used to delineate the biochem. and cellular functions of enzymes, leading to the discovery of metabolic and signaling pathways that make important contributions to human physiol. and disease. These studies demonstrate the value of selective chem. probes as drivers of biol. inquiry.
38. 38
  Baggelaar, M. P., Chameau, P. J., Kantae, V., Hummel, J., Hsu, K. L., Janssen, F., van der Wel, T., Soethoudt, M., Deng, H., den Dulk, H., Allara, M., Florea, B. I., Di Marzo, V., Wadman, W. J., Kruse, C. G., Overkleeft, H. S., Hankemeier, T., Werkman, T. R., Cravatt, B. F., and van der Stelt, M. (2015) Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons J. Am. Chem. Soc. 137, 8851– 8857 DOI: 10.1021/jacs.5b04883
  38
  Highly Selective, Reversible Inhibitor Identified by Comparative Chemoproteomics Modulates Diacylglycerol Lipase Activity in Neurons
  Baggelaar, Marc P.; Chameau, Pascal J. P.; Kantae, Vasudev; Hummel, Jessica; Hsu, Ku-Lung; Janssen, Freek; van der Wel, Tom; Soethoudt, Marjolein; Deng, Hui; den Dulk, Hans; Allara, Marco; Florea, Bogdan I.; Di Marzo, Vincenzo; Wadman, Wytse J.; Kruse, Chris G.; Overkleeft, Herman S.; Hankemeier, Thomas; Werkman, Taco R.; Cravatt, Benjamin F.; van der Stelt, Mario
  Journal of the American Chemical Society (2015), 137 (27), 8851-8857CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  Diacylglycerol lipase (DAGL)-α and -β 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 β-lactone-based probes led to the discovery of α-ketoheterocycle LEI105 as a potent, highly selective, and reversible dual DAGL-α/DAGL-β inhibitor. LEI105 did not affect other enzymes involved in endocannabinoid metab. including abhydrolase domain-contg. protein 6, abhydrolase domain-contg. protein 12, monoacylglycerol lipase, and fatty acid amide hydrolase and did not display affinity for the cannabinoid CB1 receptor. Targeted lipidomics revealed that LEI105 concn.-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 pharmacol. evidence to support the hypothesis that "on demand biosynthesis" of 2-AG is responsible for retrograde signaling.
39. 39
  Liu, Y., Patricelli, M. P., and Cravatt, B. F. (1999) Activity-based protein profiling: the serine hydrolases Proc. Natl. Acad. Sci. U. S. A. 96, 14694– 14699 DOI: 10.1073/pnas.96.26.14694
  39
  Activity-based protein profiling: the serine hydrolases
  Liu, Yongsheng; Patricelli, Matthew P.; Cravatt, Benjamin F.
  Proceedings of the National Academy of Sciences of the United States of America (1999), 96 (26), 14694-14699CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
  With the postgenome era rapidly approaching, new strategies for the functional anal. of proteins are needed. To date, proteomics efforts have primarily been confined to recording variations in protein level rather than activity. The ability to profile classes of proteins on the basis of changes in their activity would greatly accelerate both the assignment of protein function and the identification of potential pharmaceutical targets. Here, we describe the chem. synthesis and utility of an active-site directed probe for visualizing dynamics in the expression and function of an entire enzyme family, the serine hydrolases. By reacting this probe, a biotinylated fluorophosphonate referred to as FP-biotin, with crude tissue exts., we quickly and with high sensitivity detect numerous serine hydrolases, many of which display tissue-restricted patterns of expression. Addnl., we show that FP-biotin labels these proteins in an activity-dependent manner that can be followed kinetically, offering a powerful means to monitor dynamics simultaneously in both protein function and expression.
40. 40
  Baggelaar, M. P., Janssen, F. J., van Esbroeck, A. C., den Dulk, H., Allara, M., Hoogendoorn, S., McGuire, R., Florea, B. I., Meeuwenoord, N., van den Elst, H., van der Marel, G. A., Brouwer, J., Di Marzo, V., Overkleeft, H. S., and van der Stelt, M. (2013) Development of an activity-based probe and in silico design reveal highly selective inhibitors for diacylglycerol lipase-alpha in brain Angew. Chem., Int. Ed. 52, 12081– 12085 DOI: 10.1002/anie.201306295
  40
  Development of an Activity-Based Probe and In Silico Design Reveal Highly Selective Inhibitors for Diacylglycerol Lipase-α in Brain
  Baggelaar, Marc P.; Janssen, Freek J.; van Esbroeck, Annelot C. M.; den Dulk, Hans; Allara, Marco; Hoogendoorn, Sascha; McGuire, Ross; Florea, Bogdan I.; Meeuwenoord, Nico; van den Elst, Hans; van der Marel, Gijsbert A.; Brouwer, Jaap; Di Marzo, Vincenzo; Overkleeft, Herman S.; van der Stelt, Mario
  Angewandte Chemie, International Edition (2013), 52 (46), 12081-12085CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)
  A strategy that combines a knowledge-based in silico design approach and the development of novel activity-based probes for the detection of endogenous diacylglycerol lipase-α (DAGL-α) is presented. This approach resulted in the rapid identification of new DAGL-α inhibitors with high selectivity in the brain proteome.
41. 41
  Cox, J. and Mann, M. (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Nat. Biotechnol. 26, 1367– 1372 DOI: 10.1038/nbt.1511
  41
  MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification
  Cox, Juergen; Mann, Matthias
  Nature Biotechnology (2008), 26 (12), 1367-1372CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)
  Efficient anal. of very large amts. of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe MaxQuant, an integrated suite of algorithms specifically developed for high-resoln., quant. MS data. Using correlation anal. and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over std. techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome expt. and allows statistically robust identification and quantification of >4000 proteins in mammalian cell lysates.
42. 42
  Kamat, S. S., Camara, K., Parsons, W. H., Chen, D. H., Dix, M. M., Bird, T. D., Howell, A. R., and Cravatt, B. F. (2015) Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay Nat. Chem. Biol. 11, 164– 171 DOI: 10.1038/nchembio.1721
  42
  Immunomodulatory lysophosphatidylserines are regulated by ABHD16A and ABHD12 interplay
  Kamat, Siddhesh S.; Camara, Kaddy; Parsons, William H.; Chen, Dong-Hui; Dix, Melissa M.; Bird, Thomas D.; Howell, Amy R.; Cravatt, Benjamin F.
  Nature Chemical Biology (2015), 11 (2), 164-171CODEN: NCBABT; ISSN:1552-4450. (Nature Publishing Group)
  Lysophosphatidylserines (lyso-PSs) are a class of signaling lipids that regulate immunol. and neurol. processes. The metab. of lyso-PSs remains poorly understood in vivo. Recently, we detd. that ABHD12 is a major brain lyso-PS lipase, implicating lyso-PSs in the neurol. disease polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataract (PHARC), which is caused by null mutations in the ABHD12 gene. Here, we couple activity-based profiling with pharmacol. and genetic methods to annotate the poorly characterized enzyme ABHD16A as a phosphatidylserine (PS) lipase that generates lyso-PS in mammalian systems. We describe a small-mol. inhibitor of ABHD16A that depletes lyso-PSs from cells, including lymphoblasts derived from subjects with PHARC. In mouse macrophages, disruption of ABHD12 and ABHD16A resp. increases and decreases both lyso-PSs and lipopolysaccharide-induced cytokine prodn. Finally, Abhd16a-/- mice have decreased brain lyso-PSs, which runs counter to the elevation in lyso-PS in Abhd12-/- mice. Our findings illuminate an ABHD16A-ABHD12 axis that dynamically regulates lyso-PS metab. in vivo, designating these enzymes as potential targets for treating neuroimmunol. disorders.
43. 43
  Long, J. Z. and Cravatt, B. F. (2011) The metabolic serine hydrolases and their functions in mammalian physiology and disease Chem. Rev. 111, 6022– 6063 DOI: 10.1021/cr200075y
  43
  The metabolic serine hydrolases and their functions in mammalian physiology and disease
  Long, Jonathan Z.; Cravatt, Benjamin F.
  Chemical Reviews (Washington, DC, United States) (2011), 111 (10), 6022-6063CODEN: CHREAY; ISSN:0009-2665. (American Chemical Society)
  A review. Serine hydrolases (SHs) consist of >200 enzymes in humans which are characterized by the presence of an active site Ser residue that is used for the hydrolysis of substrates. The membership of this enzyme class is nearly equally split between serine proteases (trypsin/chymotrypsin/subtilisin enzymes) and "metabolic" SHs that cleave ester, amide, or thioester bonds in small mols., peptides, or proteins. The focus of this review is on the metabolic SHs. Here, the authors attempt to provide a comprehensive summary that captures the state of knowledge about metabolic SHs in their entirety, including those enzymes that remain mostly or completely uncharacterized. Particular emphasis is placed on relating the biochem. and enzymol. of individual SHs to the physiol. substrates and products they regulate in living systems, and how SHs, through the regulation of specific metabolic pathways impact health and disease.
44. 44
  Herkenham, M., Lynn, A. B., Little, M. D., Johnson, M. R., Melvin, L. S., de Costa, B. R., and Rice, K. C. (1990) Cannabinoid receptor localization in brain Proc. Natl. Acad. Sci. U. S. A. 87, 1932– 1936 DOI: 10.1073/pnas.87.5.1932
  44
  Cannabinoid receptor localization in brain
  Herkenham, Miles; Lynn, Allison B.; Little, Mark D.; Johnson, M. Ross; Melvin, Lawrence S.; De Costa, Brian R.; Rice, Kenner C.
  Proceedings of the National Academy of Sciences of the United States of America (1990), 87 (5), 1932-6CODEN: PNASA6; ISSN:0027-8424.
  [3H]CP 55,940, a radiolabeled synthetic cannabinoid, which is 10-100 times more potent in vivo than Δ9-tetrahydrocannabinol, was used to characterize and localize a specific cannabinoid receptor in brain sections. The potencies of a series of natural and synthetic cannabinoids as competitors of [3H]CP 55,940 binding correlated closely with their relative potencies in several biol. assays, suggesting that the receptor characterized in this in vitro assay is the same receptor that mediates behavioral and pharmacol. effects of cannabinoids, including human subjective experience. Autoradiog. of cannabinoid receptors in brain sections from several mammalian species, including man, reveals a unique and conserved distribution; binding is most dense in outflow nuclei of the basal ganglia (the substantia nigra pars reticulata and globus pallidus) and in the hippocampus and cerebellum. Generally high densities in forebrain and cerebellum implicate roles for cannabinoids in cognition and movement. Sparse densities in lower brainstem areas controlling cardiovascular and respiratory functions may explain why high doses of Δ9-tetrahydrocannabinol are not lethal.
45. 45
  Marsicano, G., Goodenough, S., Monory, K., Hermann, H., Eder, M., Cannich, A., Azad, S. C., Cascio, M. G., Gutierrez, S. O., van der Stelt, M., Lopez-Rodriguez, M. L., Casanova, E., Schutz, G., Zieglgansberger, W., Di Marzo, V., Behl, C., and Lutz, B. (2003) CB1 cannabinoid receptors and on-demand defense against excitotoxicity Science 302, 84– 88 DOI: 10.1126/science.1088208
  45
  CB1 cannabinoid receptors and on-demand defense against excitotoxicity
  Marsicano, Giovanni; Goodenough, Sharon; Monory, Krisztina; Hermann, Heike; Eder, Matthias; Cannich, Astrid; Azad, Shahnaz C.; Cascio, Maria Grazia; Gutierrez, Silvia Ortega; van der Stelt, Mario; Lopez-Rodriguez, Maria Luz; Casanova, Emilio; Schuetz, Guenther; Zieglgaensberger, Walter; Di Marzo, Vincenzo; Behl, Christian; Lutz, Beat
  Science (Washington, DC, United States) (2003), 302 (5642), 84-88CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Abnormally high spiking activity can damage neurons. Signaling systems to protect neurons from the consequences of abnormal discharge activity have been postulated. The authors generated conditional mutant mice that lack expression of the cannabinoid receptor type 1 in principal forebrain neurons but not in adjacent inhibitory interneurons. In mutant mice, the excitotoxin kainic acid (KA) induced excessive seizures in vivo. The threshold to KA-induced neuronal excitation in vitro was severely reduced in hippocampal pyramidal neurons of mutants. KA administration rapidly raised hippocampal levels of anandamide and induced protective mechanisms in wild-type principal hippocampal neurons. These protective mechanisms could not be triggered in mutant mice. The endogenous cannabinoid system thus provides on-demand protection against acute excitotoxicity in central nervous system neurons.
46. 46
  van der Stelt, M., Trevisani, M., Vellani, V., De Petrocellis, L., Schiano Moriello, A., Campi, B., McNaughton, P., Geppetti, P., and Di Marzo, V. (2005) Anandamide acts as an intracellular messenger amplifying Ca2⁺ influx via TRPV1 channels EMBO J. 24, 3026– 3037 DOI: 10.1038/sj.emboj.7600784
  There is no corresponding record for this reference.
47. 47
  Maccarrone, M., Rossi, S., Bari, M., De Chiara, V., Fezza, F., Musella, A., Gasperi, V., Prosperetti, C., Bernardi, G., Finazzi-Agro, A., Cravatt, B. F., and Centonze, D. (2008) Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum Nat. Neurosci. 11, 152– 159 DOI: 10.1038/nn2042
  47
  Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum
  Maccarrone, Mauro; Rossi, Silvia; Bari, Monica; De Chiara, Valentina; Fezza, Filomena; Musella, Alessandra; Gasperi, Valeria; Prosperetti, Chiara; Bernardi, Giorgio; Finazzi-Agro, Alessandro; Cravatt, Benjamin F.; Centonze, Diego
  Nature Neuroscience (2008), 11 (2), 152-159CODEN: NANEFN; ISSN:1097-6256. (Nature Publishing Group)
  Of the endocannabinoids (eCBs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG) have received the most study. A functional interaction between these mols. has never been described. Using mouse brain slices, we found that stimulation of metabotropic glutamate 5 receptors by 3,5-dihydroxyphenylglycine (DHPG) depressed inhibitory transmission in the striatum through selective involvement of 2-AG metab. and stimulation of presynaptic CB1 receptors. Elevation of AEA concns. by pharmacol. or genetic inhibition of AEA degrdn. reduced the levels, metab. and physiol. effects of 2-AG. Exogenous AEA and the stable AEA analog methanandamide inhibited basal and DHPG-stimulated 2-AG prodn., confirming that AEA is responsible for the downregulation of the other eCB. AEA is an endovanilloid substance, and the stimulation of transient receptor potential vanilloid 1 (TRPV1) channels mimicked the effects of endogenous AEA on 2-AG metab. through a previously unknown glutathione-dependent pathway. Consistently, the interaction between AEA and 2-AG was lost after pharmacol. and genetic inactivation of TRPV1 channels.
48. 48
  Buczynski, M. W. and Parsons, L. H. (2010) Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls Br. J. Pharmacol. 160, 423– 442 DOI: 10.1111/j.1476-5381.2010.00787.x
  48
  Quantification of brain endocannabinoid levels: methods, interpretations and pitfalls
  Buczynski, Matthew W.; Parsons, Loren H.
  British Journal of Pharmacology (2010), 160 (3), 423-442CODEN: BJPCBM; ISSN:1476-5381. (Wiley-Blackwell)
  A review. Endocannabinoids play an important role in a diverse range of neurophysiol. processes including neural development, neuroimmune function, synaptic plasticity, pain, reward and affective state. This breadth of influence and evidence for altered endocannabinoid signaling in a variety of neuropathologies has fueled interest in the accurate quantification of these lipids in brain tissue. Established methods for endocannabinoid quantification primarily employ solvent-based lipid extn. with further sample purifn. by solid phase extn. In recent years in vivo microdialysis methods have also been developed for endocannabinoid sampling from the brain interstitial space. However, considerable variability in ests. of endocannabinoid content has led to debate regarding the physiol. range of concns. present in various brain regions. This paper provides a crit. review of factors that influence the quantification of brain endocannabinoid content as detd. by lipid extn. from bulk tissue and by in vivo microdialysis. A variety of methodol. issues are discussed including anal. approaches, endocannabinoid extn. and purifn., post-mortem changes in brain endocannabinoid content, cellular reactions to microdialysis probe implantation and caveats related to lipid sampling from the extracellular space. The application of these methods for estg. brain endocannabinoid content and the effects of endocannabinoid clearance inhibition are discussed. The benefits, limitations and pitfalls assocd. with each approach are emphasized, with an eye toward the appropriate interpretation of data gathered by each method.
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Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid HydrolasesClick to copy article linkArticle link copied!

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Abstract

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Results and Discussion

Gel-based Brain Region Comparison

Figure 2

Chemoproteomic Brain Region Comparison

Figure 3

Comparison of CB1+/+ and CB1–/– Brain Regions

Figure 4

Figure 5

Discussion

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Preparation of Mouse Tissue Proteome

Activity-based Protein Profiling

Proteomics

Statistical Analysis

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Chemical Proteomics Maps Brain Region Specific Activity of Endocannabinoid Hydrolases
Click to copy article linkArticle link copied!

Comparison of CB₁^+/+ and CB₁^–/– Brain Regions