A role for endocannabinoid signaling in neuronal morphogenesis as the brain develops has recently been suggested. Here we used the developing somatosensory circuit as a model system to examine the role of endocannabinoid signaling in neural circuit formation. We first show that a deficiency in cannabinoid receptor type 1 (CB(1)R), but not G-protein-coupled receptor 55 (GPR55), leads to aberrant fasciculation and pathfinding in both corticothalamic and thalamocortical axons despite normal target recognition. Next, we localized CB(1)R expression to developing corticothalamic projections and found little if any expression in thalamocortical axons, using a newly established reporter mouse expressing GFP in thalamocortical projections. A similar thalamocortical projection phenotype was observed following removal of CB(1)R from cortical principal neurons, clearly demonstrating that CB(1)R in corticothalamic axons was required to instruct their complimentary connections, thalamocortical axons. When reciprocal thalamic and cortical connections meet, CB(1)R-containing corticothalamic axons are intimately associated with elongating thalamocortical projections containing DGLβ, a 2-arachidonoyl glycerol (2-AG) synthesizing enzyme. Thus, 2-AG produced in thalamocortical axons and acting at CB(1)Rs on corticothalamic axons is likely to modulate axonal patterning. The presence of monoglyceride lipase, a 2-AG degrading enzyme, in both thalamocortical and corticothalamic tracts probably serves to restrict 2-AG availability. In summary, our study provides strong evidence that endocannabinoids are a modulator for the proposed 'handshake' interactions between corticothalamic and thalamocortical axons, especially for fasciculation. These findings are important in understanding the long-term consequences of alterations in CB(1)R activity during development, a potential etiology for the mental health disorders linked to prenatal cannabis use.

Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

Laserspray ionization (LSI) mass spectrometry (MS) allows, for the first time, the analysis of proteins directly from tissue using high performance atmospheric pressure ionization mass spectrometers. Several abundant and numerous lower abundant protein ions with molecular masses up to ∼20,000 Da were detected as highly charged ions from delipified mouse brain tissue mounted on a common microscope slide and coated with 2,5-dihydroxyacetophenone as matrix. The ability of LSI to produce multiply charged ions by laser ablation at atmospheric pressure allowed protein analysis at 100,000 mass resolution on an Orbitrap Exactive Fourier transform mass spectrometer. A single acquisition was sufficient to identify the myelin basic protein N-terminal fragment directly from tissue using electron transfer dissociation on a linear trap quadrupole (LTQ) Velos. The high mass resolution and mass accuracy, also obtained with a single acquisition, are useful in determining protein molecular weights and from the electron transfer dissociation data in confirming database-generated sequences. Furthermore, microscopy images of the ablated areas show matrix ablation of ∼15 μm-diameter spots in this study. The results suggest that LSI-MS at atmospheric pressure potentially combines speed of analysis and imaging capability common to matrix-assisted laser desorption/ionization and soft ionization, multiple charging, improved fragmentation, and cross-section analysis common to electrospray ionization.

Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.

G protein coupled receptors (GPCRs) are one of the most widely studied gene superfamilies. Thousands of GPCR research studies have utilized heterologous expression systems such as human embryonic kidney cells (HEK293). Though often treated as 'blank slates', these cell lines nevertheless endogenously express GPCRs and related signaling proteins. The outcome of a given GPCR study can be profoundly influenced by this largely unknown complement of receptors and/or signaling proteins. Little easily accessible information exists that describes the expression profiles of the GPCRs in cell lines. What is accessible is often limited in scope - of the hundreds of GPCRs and related proteins, one is unlikely to find information on expression of more than a dozen proteins in a given cell line. Microarray technology has allowed rapid analysis of mRNA levels of thousands of candidate genes, but though often publicly available, the results can be difficult to efficiently access or even to interpret.

Cannabis continues to be the most accessible and popular illicit recreational drug. Whereas current data link adolescence cannabinoid exposure to increased risk for dependence on other drugs, depression, anxiety disorders and psychosis, the mechanism(s) underlying these adverse effects remains controversial. Here we show in a mouse model of female adolescent cannabinoid exposure deficient endocannabinoid (eCB)-mediated signaling and presynaptic forms of long-term depression at adult central glutamatergic synapses in the prefrontal cortex. Increasing endocannabinoid levels by blockade of monoacylglycerol lipase, the primary enzyme responsible for degrading the endocannabinoid 2-arachidonoylglycerol (2-AG), with the specific inhibitor JZL 184 ameliorates eCB-LTD deficits. The observed deficit in cortical presynaptic signaling may represent a neural maladaptation underlying network instability and abnormal cognitive functioning. Our study suggests that adolescent cannabinoid exposure may permanently impair brain functions, including the brain's intrinsic ability to appropriately adapt to external influences.

Indole-based compounds, such as the alkyl-indole (AI) compound WIN55212-2, activate the cannabinoid receptors, CB1 and CB2, two well-characterized G protein-coupled receptors (GPCR). Reports indicate that several indole-based cannabinoid agonists, including WIN55212-2, lack selectivity and interact with at least two additional targets: AI-sensitive GPCRs and microtubules. Studying how indole-based compounds modulate the activity of these 4 targets has been difficult as selective chemical tools were not available. Here we report the pharmacological characterization of six newly-developed indole-based compounds (ST-11, ST-23, ST-25, ST-29, ST-47 and ST-48) that exhibit distinct binding affinities at AI-sensitive receptors, cannabinoid CB1 and CB2 receptors and the colchicine site of tubulin. Several compounds exhibit some level of selectivity for AI-sensitive receptors, including ST-11 that binds AI-sensitive receptors with a Kd of 52nM and appears to have a weaker affinity for the colchicine site of tubulin (Kd=3.2μM) and does not bind CB1/CB2 receptors. Leveraging these characteristics, we show that activation of AI-sensitive receptors with ST-11 inhibits both the basal and stimulated migration of the Delayed Brain Tumor (DBT) mouse glioma cell line. Our study describes a new series of indole-based compounds that enable the pharmacological and functional differentiation of alkylindole-sensitive receptors from cannabinoid receptors and microtubules.

Variations in the gene LDAH (C2ORF43), which encodes lipid droplet-associated hydrolase (LDAH), are among few loci associated with human prostate cancer. Homologs of LDAH have been identified as proteins of lipid droplets (LDs). LDs are cellular organelles that store neutral lipids, such as triacylglycerols and sterol esters, as precursors for membrane components and as reservoirs of metabolic energy. LDAH is reported to hydrolyze cholesterol esters and to be important in macrophage cholesterol ester metabolism. Here, we confirm that LDAH is localized to LDs in several model systems. We generated a murine model in which Ldah is disrupted but found no evidence for a major function of LDAH in cholesterol ester or triacylglycerol metabolism in vivo, nor a role in energy or glucose metabolism. Our data suggest that LDAH is not a major cholesterol ester hydrolase, and an alternative metabolic function may be responsible for its possible effect on development of prostate cancer.

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

Orexins are associated with drug relapse in rodents. Here, we show that acute restraint stress in mice activates lateral hypothalamic (LH) orexin neurons, increases levels of orexin A and 2-arachidonoylglycerol (2-AG) in the ventral tegmental area (VTA), and reinstates extinguished cocaine-conditioned place preference (CPP). This stress-induced reinstatement of cocaine CPP depends on type 1 orexin receptors (OX1Rs), type 1 cannabinoid receptors (CB1Rs) and diacylglycerol lipase (DAGL) in the VTA. In dopaminergic neurons of VTA slices, orexin A presynaptically inhibits GABAergic transmission. This effect is prevented by internal GDP-β-S or inhibiting OX1Rs, CB1Rs, phospholipase C or DAGL, and potentiated by inhibiting 2-AG degradation. These results suggest that restraint stress activates LH orexin neurons, releasing orexins into the VTA to activate postsynaptic OX1Rs of dopaminergic neurons and generate 2-AG through a Gq-protein-phospholipase C-DAGL cascade. 2-AG retrogradely inhibits GABA release through presynaptic CB1Rs, leading to VTA dopaminergic disinhibition and reinstatement of cocaine CPP.

More than 30 years ago, a calcium-dependent enzyme activity was described that generates N-acyl phosphatidylethanolamines (NAPEs), which are precursors for N-acyl ethanolamine (NAE) lipid transmitters, including the endocannabinoid anandamide. The identity of this calcium-dependent N-acyltransferase (Ca-NAT) has remained mysterious. Here, we use activity-based protein profiling to identify the poorly characterized serine hydrolase PLA2G4E as a mouse brain Ca-NAT and show that this enzyme generates NAPEs and NAEs in mammalian cells.

The endocannabinoid system may regulate glial cell functions and their responses to pathological stimuli, specifically, Alzheimer's disease. One experimental approach is the enhancement of endocannabinoid tone by blocking the activity of degradative enzymes, such as fatty acid amide hydrolase (FAAH).

The nucleus accumbens (Acb) shell and caudate-putamen nucleus (CPu) are respectively implicated in the motivational and motor effects of dopamine, which are mediated in part through dopamine D₂-like receptors (D₂Rs) and modulated by activation of the cannabinoid-1 receptor (CB₁R). The dopamine D(₂/D3) receptor agonist, quinpirole elicits internalization of D₂Rs in isolated cells; however, dendritic and axonal targeting of D₂Rs may be highly influenced by circuit-dependent changes in vivo and potentially influenced by endogenous CB₁R activation.

Selective activation of the peripheral cannabinoid receptor 1 (CB1R) has been shown to suppress neuropathic pain symptoms in rodents. However, relatively little is known about changes in CB1R and its endogenous ligands during development or maintenance of neuropathic pain. Using immunohistochemistry, Western blot, real-time reverse transcription polymerase chain reaction, as well as liquid chromatography/mass spectrometry, we studied the changes in CB1Rs and endocannabinoids N-arachidonoylethanolamine/anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat lumbar (L4 and L5) dorsal root ganglia (DRG) after neuropathic pain induction (L5 spinal nerve ligation: SNL). Immunohistochemistry revealed that in control rats, CB1R is expressed in the majority (76-83%) of nociceptive neurons as indicated by co-labeling with isolectin B4 (IB4) or antibodies recognizing transient receptor potential vanilloid (TRPV1), calcitonin gene related peptide (CGRP), and the NR2C/2D subunits of the N-methyl-D-aspartate receptor. After L5 SNL, CB1R mRNA and protein increases in the ipsilateral uninjured L4 DRG whereas the percentages of CB1R immunoreactive (CB1R-ir) neurons remain unchanged in L4 and L5 DRG. However, for these CB1R-ir neurons, we observe significant increases in percentage of TRPV1-ir cells in ipsilateral L4 DRG, and decreases in percentage of IB4- and CGRP-co-labeled cells in ipsilateral L5 DRG. Levels of both AEA and 2-AG increase significantly only in the ipsilateral L5 DRG. These results are consistent with the preserved analgesic effects of cannabinoids in neuropathic pain and provide a rational framework for the development of peripherally acting endocannabinoid-based therapeutic interventions for neuropathic pain.

N-acylethanolamines (NAEs) are membrane-derived lipids that are utilized as signaling molecules in the nervous system (e.g., the endocannabinoid anandamide). An N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) that catalyzes formation of NAEs was recently identified as a member of the zinc metallohydrolase family of enzymes. NAPE-PLD(-/-) mice have greatly reduced brain levels of long-chain saturated NAEs but wild-type levels of polyunsaturated NAEs (e.g., anandamide), suggesting an important role for NAPE-PLD in the biosynthesis of at least a subset of endogenous NAEs in the mammalian nervous system. To provide a neuroanatomical basis for investigation of NAPE-PLD function, here we have analyzed expression of NAPE-PLD in the mouse brain using mRNA in situ hybridization and immunocytochemistry. NAPE-PLD(-/-) mice were utilized to establish the specificity of probes/antibodies used. The most striking feature of NAPE-PLD expression in the brain was in the dentate gyrus, where a strong mRNA signal was detected in granule cells. Accordingly, immunocytochemical analysis revealed intense NAPE-PLD immunoreactivity in the axons of granule cells (mossy fibers). Intense NAPE-PLD immunoreactivity was also detected in axons of the vomeronasal nerve that project to the accessory olfactory bulb. NAPE-PLD expression was detected in other brain regions (e.g., hippocampus, cortex, thalamus, hypothalamus), but the intensity of immunostaining was weaker than in mossy fibers. Collectively, the data obtained indicate that NAPE-PLD is expressed by specific populations of neurons in the brain and targeted to axonal processes. We suggest that NAEs generated by NAPE-PLD in axons may act as anterograde synaptic signaling molecules that regulate the activity of postsynaptic neurons.

In the era of combined antiretroviral therapy, HIV-1 infected individuals are living longer lives; however, longevity is met with an increasing number of HIV-1 associated neurocognitive disorders (HAND) diagnoses. The transactivator of transcription (Tat) is known to mediate the neurotoxic effects in HAND by acting directly on neurons and also indirectly via its actions on glia. The Go/No-Go (GNG) task was used to examine HAND in the Tat transgenic mouse model. The GNG task involves subjects discriminating between two stimuli sets in order to determine whether or not to inhibit a previously trained response. Data reveal inhibitory control deficits in female Tat(+) mice (p = .048) and an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group (p < .05). A significant negative correlation was noted between inhibitory control and IL CB1R expression (r = -.543, p = .045), with CB1R expression predicting 30% of the variance of inhibitory control (R2 = .295, p = .045). Furthermore, there was a significant increase in spontaneous excitatory postsynaptic current (sEPSC) frequencies in Tat(+) compared to Tat(-) mice (p = .008, across sexes). The increase in sEPSC frequency was significantly attenuated by bath application of PF3845, a fatty acid amide hydrolase (FAAH) enzyme inhibitor (p < .001). Overall, the GNG task is a viable measure to assess inhibitory control deficits in Tat transgenic mice and results suggest a potential therapeutic treatment for the observed deficits with drugs which modulate endocannabinoid enzyme activity. Graphical Abstract Results of the Go/No-Go operant conditioning task reveal inhibitory control deficits in female transgenic Tat(+) mice without significantly affecting males. The demonstrated inhibitory control deficits appear to be associated with an upregulation of cannabinoid type 1 receptors (CB1R) in the infralimbic (IL) cortex in the same female Tat(+) group.

The transcription factor NRF2 is a master regulator of the cellular antioxidant response, and it is often genetically activated in non-small-cell lung cancers (NSCLCs) by, for instance, mutations in the negative regulator KEAP1. While direct pharmacological inhibition of NRF2 has proven challenging, its aberrant activation rewires biochemical networks in cancer cells that may create special vulnerabilities. Here, we use chemical proteomics to map druggable proteins that are selectively expressed in KEAP1-mutant NSCLC cells. Principal among these is NR0B1, an atypical orphan nuclear receptor that we show engages in a multimeric protein complex to regulate the transcriptional output of KEAP1-mutant NSCLC cells. We further identify small molecules that covalently target a conserved cysteine within the NR0B1 protein interaction domain, and we demonstrate that these compounds disrupt NR0B1 complexes and impair the anchorage-independent growth of KEAP1-mutant cancer cells. Our findings designate NR0B1 as a druggable transcriptional regulator that supports NRF2-dependent lung cancers.

Rpn13 is one of several ubiquitin receptors in the 26S proteasome. Cys88 of Rpn13 has been proposed to be the principal target of RA190, an electrophilic small molecule with interesting anti-cancer activities. Here, we examine the claim that RA190 mediates its cytotoxic effects through engagement with Rpn13. We find no evidence that this is the case. In vitro, RA190 is has no measurable effect on any of the known interactions of Rpn13. In cellulo, we see no physical engagement of Rpn13 by RA190, either on C88 or any other residue. However, chemical proteomics experiments in two different cell lines reveal that dozens of other proteins are heavily engaged by RA190. Finally, increasing or reducing the level of Rpn13 in HeLa and melanoma cells had no effect on the sensitivity of HeLa or melanoma cells to RA190. We conclude that Rpn13 is not the physiologically relevant target of RA190.

Introduction: The high prevalence of adolescent cannabis use, the association between this use and later psychiatric disease, and increased access to high-potency cannabis highlight the need for a better understanding of the long-term effects of adolescent cannabis use on cognitive and behavioral outcomes. Furthermore, increasing Δ9-tetrahydrocannabinol (THC) in high-potency cannabis is accompanied by a decrease in cannabidiol (CBD), thus an understanding of the interactions between CBD and THC in the neurodevelopmental effects of THC is also important. The current study examined the immediate and long-term behavioral consequences of THC, CBD, and their combination in a mouse model of adolescent cannabis use. Materials and Methods: Male CD1 mice received daily injections of THC (3 mg/kg), CBD (3 mg/kg), CBD+THC (3 mg/kg each), vehicle, or remained undisturbed in their home cage (no handling/injections), either during adolescence (postnatal day [PND] 28-48) or during early adulthood (PND 69-89). Animals were then evaluated with a battery of behavioral tests 1 day after drug treatment, and again after 42 drug-free days. The tests included the following: open field (day 1), novel object recognition (NOR; day 2), marble burying (day 3), elevated plus maze (EPM; day 4), and Nestlet shredding (day 5). Results: Chronic administration of THC during adolescence led to immediate and long-term impairments in object recognition/working memory, as measured by the NOR task. In contrast, adult administration of THC caused immediate, but not long term, impairment of object/working memory. Adolescent chronic exposure to THC increased repetitive and compulsive-like behaviors, as measured by the Nestlet shredding task. Chronic administration of THC, either during adolescence or during adulthood, led to a delayed increase in anxiety as measured by the EPM. All THC-induced behavioral abnormalities were prevented by the coadministration of CBD+THC, whereas CBD alone did not influence behavioral outcomes. Conclusion: These data suggest that chronic exposure to THC during adolescence leads to some of the behavioral abnormalities common in schizophrenia. Interestingly, CBD appeared to antagonize all THC-induced behavioral abnormalities. These findings support the hypothesis that adolescent THC use can impart long-term behavioral deficits; however, cotreatment with CBD prevents these deficits.

Cannabinoid type 1 receptor (CB1R) is expressed and participates in several aspects of cerebral cortex embryonic development as demonstrated with whole-transcriptome mRNA sequencing and other contemporary methods. However, the cellular location of CB1R, which helps to specify molecular mechanisms, remains to be documented. Using three-dimensional (3D) electron microscopic reconstruction, we examined CB1R immunolabeling in proliferating neural stem cells (NSCs) and migrating neurons in the embryonic mouse (Mus musculus) and rhesus macaque (Macaca mulatta) cerebral cortex. We found that the mitotic and postmitotic ventricular and subventricular zone (VZ and SVZ) cells are immunonegative in both species while radially migrating neurons in the intermediate zone (IZ) and cortical plate (CP) contain CB1R-positive intracellular vesicles. CB1R immunolabeling was more numerous and more extensive in monkeys compared to mice. In CB1R-knock out mice, projection neurons in the IZ show migration abnormalities such as an increased number of lateral processes. Thus, in radially migrating neurons CB1R provides a molecular substrate for the regulation of cell movement. Undetectable level of CB1R in VZ/SVZ cells indicates that previously suggested direct CB1R-transmitted regulation of cellular proliferation and fate determination demands rigorous re-examination. More abundant CB1R expression in monkey compared to mouse suggests that therapeutic or recreational cannabis use may be more distressing for immature primate neurons than inferred from experiments with rodents.