Cannabidiol negatively modulates adenosine A2A receptor functioning in living cells.

OBJECTIVES: Cannabidiol (CBD) is a phytocannabinoid with great potential in clinical applications. The mechanism(s) of action of CBD require further investigation. Previous studies suggested that adenosine A2A receptors (A2ARs) could play a role in CBD-induced effects. Here, we evaluated the ability of CBD to modify the function of A2AR. METHODS: We used HEK-293T cells transfected with the cDNA encoding the human A2AR and Gαs protein, both modified to perform bioluminescence-based assays. We first assessed the effect of CBD on A2AR ligand binding using an A2AR NanoLuciferase sensor. Next, we evaluated whether CBD modified A2AR coupling to mini-Gαs proteins using the NanoBiT™ assay. Finally, we further assessed CBD effects on A2AR intrinsic activity by recording agonist-induced cAMP accumulation. RESULTS: CBD did not bind orthosterically to A2AR but reduced the coupling of A2AR to Gαs protein and the subsequent generation of cAMP. CONCLUSION: CBD negatively modulates A2AR functioning.

Optical Control of Adenosine-Mediated Pain Modulation.

Adenosine receptors (ARs) play many important roles in physiology and have been recognized as potential targets for pain relief. Here, we introduce three photoswitchable adenosine derivatives that function as light-dependent agonists for ARs and confer optical control to these G protein-coupled receptors. One of our compounds, AzoAdenosine-3, was evaluated in the classical formalin model of pain. The molecule, active in the dark, was not metabolized by adenosine deaminase and effectively reduced pain perception in a light-dependent manner. These antinociceptive effects suggested a major role for A1R and A3R in peripheral-mediated pain sensitization, whereas an average adenosine-mediated antinociceptive effect will be facilitated by A2AR and A2BR. Our results demonstrate that a photoswitchable adenosine derivative can be used to map the contribution of ARs mediating analgesia in vivo.

Control of glutamate release by complexes of adenosine and cannabinoid receptors.

BACKGROUND: It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS: Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS: We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.

Design, Synthesis and Characterization of a New Series of Fluorescent Metabotropic Glutamate Receptor Type 5 Negative Allosteric Modulators.

In recent years, new drug discovery approaches based on novel pharmacological concepts have emerged. Allosteric modulators, for example, target receptors at sites other than the orthosteric binding sites and can modulate agonist-mediated activation. Interestingly, allosteric regulation may allow a fine-tuned regulation of unbalanced neurotransmitter' systems, thus providing safe and effective treatments for a number of central nervous system diseases. The metabotropic glutamate type 5 receptor (mGlu5R) has been shown to possess a druggable allosteric binding domain. Accordingly, novel allosteric ligands are being explored in order to finely regulate glutamate neurotransmission, especially in the brain. However, before testing the activity of these new ligands in the clinic or even in animal disease models, it is common to characterize their ability to bind mGlu5Rs in vitro. Here, we have developed a new series of fluorescent ligands that, when used in a new NanoBRET-based binding assay, will facilitate screening for novel mGlu5R allosteric modulators.

Chronic adenosine A2A receptor blockade induces locomotor sensitization and potentiates striatal LTD IN GPR37-deficient mice.

Adenosine A2A receptors (A2A R) play a key role in modulating dopamine-dependent locomotor activity, as heralded by the sensitization of locomotor activity upon chronic A2A R blockade, which is associated with elevated dopamine levels and altered corticostriatal synaptic plasticity. Since the orphan receptor GPR37 has been shown to modulate A2A R function in vivo, we aimed to test whether the A2A R-mediated sensitization of locomotor activity is GPR37-dependent and involves adaptations of synaptic plasticity. To this end, we administered a selective A2A R antagonist, SCH58261 (1 mg/kg, i.p.), daily for 14 days, and the locomotor sensitization, striatum-dependent cued learning, and corticostriatal synaptic plasticity (i.e., long-term depression) were compared in wild-type and GPR37-/- mice. Notably, GPR37 deletion promoted A2A R-associated locomotor sensitization but not striatum-dependent cued learning revealed upon chronic SCH58261 treatment of mice. Furthermore, chronic A2A R blockade potentiated striatal long-term depression in corticostriatal synapses of GPR37-/- but not of wild-type mice, thus correlating well with neurochemical alterations of the adenosinergic system. Overall, these results revealed the importance of GPR37 regulating A2A R-dependent locomotor sensitization and synaptic plasticity in the basal ganglia circuitry. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

Revealing Adenosine A2A-Dopamine D2 Receptor Heteromers in Parkinson's Disease Post-Mortem Brain through a New AlphaScreen-Based Assay.

Background: Several biophysical techniques have been successfully implemented to detect G protein-coupled receptors (GPCRs) heteromerization. Although these approaches have made it possible to ascertain the presence of GPCR heteromers in animal models of disease, no success has been accomplished in pathological human post-mortem brains. The AlphaScreen technology has been consistently used to quantify small analyte accumulation or depletion, bimolecular interactions, and post-translational modifications. The high signal-to-background, dynamic range and sensitivity exhibited by this technology support that it may be suitable to detect GPCR heteromers even under non-optimal conditions. Methods: Here, we describe the development of a new AlphaScreen assay to detect GPCR oligomers in human post-mortem brain. Results: Adenosine A2A-dopamine D2 receptor (A2AR/D2R) heteromer formation was monitored in caudate from healthy and Parkinson's disease (PD) subjects. The approach was first validated using striatal membranes from wild type and A2AR deficient mice. Secondly, we took advantage of the 6-hydroxydopamine hemiparkinsonian rat model to validate previous results. In addition, finally, A2AR/D2R heteromer formation was assessed in caudate membranes from human post-mortem brains. Importantly, our preliminary results revealed an increase in A2AR/D2R heteromer formation in PD brains. Conclusions: The new AlphaScreen assay allowed assessing GPCR heteromers in human post-mortem brains with high sensitivity.

Synthesis and Characterization of a New Bivalent Ligand Combining Caffeine and Docosahexaenoic Acid.

Caffeine is a promising drug for the management of neurodegenerative diseases such as Parkinson's disease (PD), demonstrating neuroprotective properties that have been attributed to its interaction with the basal ganglia adenosine A2A receptor (A2AR). However, the doses needed to exert these neuroprotective effects may be too high. Thus, it is important to design novel approaches that selectively deliver this natural compound to the desired target. Docosahexaenoic acid (DHA) is the major omega-3 fatty acid in the brain and can act as a specific carrier of caffeine. Furthermore, DHA displays properties that may lead to its use as a neuroprotective agent. In the present study, we constructed a novel bivalent ligand covalently linking caffeine and DHA and assessed its pharmacological activity and safety profile in a simple cellular model. Interestingly, the new bivalent ligand presented higher potency as an A2AR inverse agonist than caffeine alone. We also determined the range of concentrations inducing toxicity both in a heterologous system and in primary striatal cultures. The novel strategy presented here of attaching DHA to caffeine may enable increased effects of the drug at desired sites, which could be of interest for the treatment of PD.

Investigation of LRRC8-Mediated Volume-Regulated Anion Currents in Xenopus Oocytes.

Volume-regulated anion channels (VRACs) play an important role in controlling cell volume by opening upon cell swelling. Recent work has shown that heteromers of LRRC8A with other LRRC8 members (B, C, D, and E) form the VRAC. Here, we used Xenopus oocytes as a simple system to study LRRC8 proteins. We discovered that adding fluorescent proteins to the C-terminus resulted in constitutive anion channel activity. Using these constructs, we reproduced previous findings indicating that LRRC8 heteromers mediate anion and osmolyte flux with subunit-dependent kinetics and selectivity. Additionally, we found that LRRC8 heteromers mediate glutamate and ATP flux and that the inhibitor carbenoxolone acts from the extracellular side, binding to probably more than one site. Our results also suggest that the stoichiometry of LRRC8 heteromers is variable, with a number of subunits ≥6, and that the heteromer composition depends on the relative expression of different subunits. The system described here enables easy structure-function analysis of LRRC8 proteins.

Coassembly and coupling of SK2 channels and mGlu5 receptors.

Group I metabotropic glutamate (mGlu) receptors regulate hippocampal CA1 pyramidal neuron excitability via Ca(2+) wave-dependent activation of small-conductance Ca(2+)-activated K(+) (SK) channels. Here, we show that mGlu5 receptors and SK2 channels coassemble in heterologous coexpression systems and in rat brain. Further, in cotransfected cells or rat primary hippocampal neurons, mGlu5 receptor stimulation activated apamin-sensitive SK2-mediated K(+) currents. In addition, coexpression of mGlu5 receptors and SK2 channels promoted plasma membrane targeting of both proteins and correlated with increased mGlu5 receptor function that was unexpectedly blocked by apamin. These results demonstrate a reciprocal functional interaction between mGlu5 receptors and SK2 channels that reflects their molecular coassembly.

Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

The role of parkinson's disease-associated receptor GPR37 in the hippocampus: functional interplay with the adenosinergic system.

GPR37 is an orphan G protein-coupled receptor mostly enriched in brain areas such as the cerebellum, striatum, and hippocampus. Identified as a substrate of parkin, GPR37 has been suggested to play a role in Parkinson's disease. Distributed throughout the brain, the function of GPR37, however, remains unknown. We now provide the first mapping of GPR37 within the hippocampus, where GPR37 is widely expressed and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts, and axon terminals. GPR37 per se does not appear to play a role in learning and memory, since knocking out GPR37 (GPR37-KO) did not alter the performance in different hippocampal-related memory tasks. This is in agreement with slice electrophysiology experiments showing no differences both in short-term plasticity paired-pulse facilitation and long-term potentiation between WT and GPR37-KO mice. However, we report a potential functional interaction between GPR37 and adenosine A2A receptors (A2 A R) in the hippocampus, with A2 A R modulating the GPR37-associated phenotype. Thus, the absence of GPR37 appeared to sensitize mice to hippocampal A2 A R-mediated signaling, as observed by the effect of the A2 A R antagonist SCH58261 increasing synaptic depotentiation, reducing novel object recognition memory and reverting the anxiolytic effect of GPR37 deletion. Collectively, these findings afford insight into the localization and role of the orphan GPR37 within the hippocampus with potential involvement in A2 A R function (i.e., A2 A R sensitization). GPR37 is an orphan G protein-coupled receptor widely expressed in the hippocampus and localized at the level of the extrasynaptic plasma membrane of dendritic spines, dendritic shafts and axon terminals. This orphan receptor per se does not appear to directly control the learning and memory processes; however knocking-out GPR37 triggers anxiolytic-like effects and sensitizes mice to hippocampal A2A R-mediated signalling.

Photomodulation of G protein-coupled adenosine receptors by a novel light-switchable ligand.

The adenosinergic system operates through G protein-coupled adenosine receptors, which have become promising therapeutic targets for a wide range of pathological conditions. However, the ubiquity of adenosine receptors and the eventual lack of selectivity of adenosine-based drugs have frequently diminished their therapeutic potential. Accordingly, here we aimed to develop a new generation of light-switchable adenosine receptor ligands that change their intrinsic activity upon irradiation, thus allowing the spatiotemporal control of receptor functioning (i.e., receptor activation/inactivation dependent on location and timing). Therefore, we synthesized an orthosteric, photoisomerizable, and nonselective adenosine receptor agonist, nucleoside derivative MRS5543 containing an aryl diazo linkage on the N(6) substituent, which in the dark (relaxed isomer) behaved as a full adenosine A3 receptor (A3R) and partial adenosine A2A receptor (A2AR) agonist. Conversely, upon photoisomerization with blue light (460 nm), it remained a full A3R agonist but became an A2AR antagonist. Interestingly, molecular modeling suggested that structural differences encountered within the third extracellular loop of each receptor could modulate the intrinsic, receptor subtype-dependent, activity. Overall, the development of adenosine receptor ligands with photoswitchable activity expands the pharmacological toolbox in support of research and possibly opens new pharmacotherapeutic opportunities.

Striatal adenosine A2A receptor expression is controlled by S-adenosyl-L-methionine-mediated methylation.

Adenosine A2A receptor (A2AR) is a G protein-coupled receptor enriched in the striatum for which an increased expression has been demonstrated in certain neurological diseases. Interestingly, previous in vitro studies demonstrated that A2AR expression levels are reduced after treatment with S-adenosyl-L-methionine (SAM), a methyl donor molecule involved in the methylation of important biological structures such as DNA, proteins, and lipids. However, the in vivo effects of SAM treatment on A2AR expression are still obscure. Here, we demonstrated that 2 weeks of SAM treatment produced a significant reduction in the rat striatal A2AR messenger RNA (mRNA) and protein content as well as A2AR-mediated signaling. Furthermore, when the content of 5-methylcytosine levels in the 5'UTR region of ADORA2A was analyzed, this was significantly increased in the striatum of SAM-treated animals; thus, an unambiguous correlation between SAM-mediated methylation and striatal A2AR expression could be established. Overall, we concluded that striatal A2AR functionality can be controlled by SAM treatment, an issue that might be relevant for the management of these neurological conditions that course with increased A2AR expression.

The Parkinson's disease-associated GPR37 receptor-mediated cytotoxicity is controlled by its intracellular cysteine-rich domain.

GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor (GPCR) that aggregates intracellularly in a juvenile form of Parkinson's disease. However, little is known about the structure or function of this receptor. Here, in order to better understand the functioning of this receptor, we focused on the GPR37 C-terminal tail, in particular on a cystein-enriched region. Thus, we aimed to reveal the role of these residues on receptor plasma membrane expression and function, and also whether the presence of this cysteine-rich domain is linked to the previously described receptor-mediated cytotoxicity. Interestingly, while the deletion of six cysteine residues within this region did not affect receptor internalization it promoted GPR37 plasma membrane expression and signaling. Furthermore, the removal of the C-terminal cysteine-rich domain protected against GPR37-mediated apoptosis and cell death. Overall, we identified a GPR37 domain, namely the C-terminal tail cysteine-rich domain, which played a critical role in receptor cell surface expression, function and GPR37-mediated cytotoxicity. These results might contribute to better comprehend the pathophysiology (i.e. in Parkinson's disease) of this rather unknown member of the GPCR family.

Molecular mechanisms of MLC1 and GLIALCAM mutations in megalencephalic leukoencephalopathy with subcortical cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare leukodystrophy caused by mutations in MLC1 or GLIALCAM. The GLIALCAM gene product functions as an MLC1 beta-subunit. We aim to further clarify the molecular mechanisms of MLC caused by mutations in MLC1 or GLIALCAM. For this purpose, we analyzed a human post-mortem brain obtained from an MLC patient, who was homozygous for a missense mutation (S69L) in MLC1. We showed that this mutation affects the stability of MLC1 in vitro and reduces MLC1 protein levels in the brain to almost undetectable. However, the amount of GlialCAM and its localization were nearly unaffected, indicating that MLC1 is not necessary for GlialCAM expression or targeting. These findings were supported by experiments in primary astrocytes and in heterologous cells. In addition, we demonstrated that MLC1 and GlialCAM form homo- and hetero-complexes and that MLC-causing mutations in GLIALCAM mainly reduce the formation of GlialCAM homo-complexes, leading to a defect in the trafficking of GlialCAM alone to cell junctions. GLIALCAM mutations also affect the trafficking of its associated molecule MLC1, explaining why GLIALCAM and MLC1 mutations lead to the same disease: MLC.

Fluorescence resonance energy transfer-based technologies in the study of protein-protein interactions at the cell surface.

Understanding of the molecular mechanisms of protein-protein interactions (PPIs) at the cell surface of living cells is fundamental to comprehend the functional meaning of a large number of cellular processes. Here we discuss how new methodological strategies derived from non-invasive fluorescence-based approaches (i.e. fluorescence resonance energy transfer, FRET) have been successfully developed to characterize plasma membrane PPIs. Importantly, these technologies alone - or in concert with complementary methods (i.e. SNAP-tag/TR-FRET, TIRF/FRET) - can become extremely powerful approaches for visualizing cell surface PPIs, even between more than two proteins and also in native tissues. Interestingly, these methods would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Accordingly, herein we provide a thorough assessment on all biotechnological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied in the study of PPIs occurring at the cell surface of living cells.

Visualizing G Protein-Coupled Receptor-Receptor Interactions in Brain Using Proximity Ligation In Situ Assay.

G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors and the main drug targets in therapeutics. GPCRs can establish direct receptor-receptor interactions (oligomerization), which can also be considered as targets for drug development (GPCR oligomer-based drugs). However, prior to designing any novel GPCR oligomer-based drug development program, demonstrating the existence of a named GPCR oligomer in native tissues is needed as part of its target engagement definition. Here, we discuss the proximity ligation in situ assay (P-LISA), an experimental approach that reveals GPCR oligomerization in native tissues. We provide a detailed step-by-step protocol to perform P-LISA experiments and visualize GPCR oligomers in brain slices. We also provide instructions for slide observation, data acquisition, and quantification. Finally, we discuss the critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, this protocol may be used to straightforwardly visualize GPCR oligomers in the brain. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Visualization of GPCR oligomers by proximity ligation in situ assay (P-LISA) Support Protocol: Slide observation, image acquisition, and quantification.

Converging circuits between pain and depression: the ventral tegmental area as a therapeutic hub.

Chronic pain and depression are highly prevalent pathologies and cause a major socioeconomic burden to society. Chronic pain affects the emotional state of the individuals suffering from it, while depression worsens the prognosis of chronic pain patients and may diminish the effectiveness of pain treatments. There is a high comorbidity rate between both pathologies, which might share overlapping mechanisms. This review explores the evidence pinpointing a role for the ventral tegmental area (VTA) as a hub where both pain and emotional processing might converge. In addition, the feasibility of using the VTA as a possible therapeutic target is discussed. The role of the VTA, and the dopaminergic system in general, is highly studied in mood disorders, especially in deficits in reward-processing and motivation. Conversely, the VTA is less regarded where it concerns the study of central mechanisms of pain and its mood-associated consequences. Here, we first outline the brain circuits involving central processing of pain and mood disorders, focusing on the often-understudied role of the dopaminergic system and the VTA. Next, we highlight the state-of-the-art findings supporting the emergence of the VTA as a link where both pathways converge. Thus, we envision a promising part for the VTA as a putative target for innovative therapeutic approaches to treat chronic pain and its effects on mood. Finally, we emphasize the urge to develop and use animal models where both pain and depression-like symptoms are considered in conjunction.

Development of a Novel σ1 Receptor Biosensor Based on Its Heterodimerization with Binding Immunoglobulin Protein in Living Cells.

The σ1 receptor (S1R) is a ligand-regulated non-opioid intracellular receptor involved in several pathological conditions. The development of S1R-based drugs as therapeutic agents is a challenge due to the lack of simple functional assays to identify and classify S1R ligands. We have developed a novel nanoluciferase binary technology (NanoBiT) assay based on the ability of S1R to heteromerize with the binding immunoglobulin protein (BiP) in living cells. The S1R-BiP heterodimerization biosensor allows for rapid and accurate identification of S1R ligands by monitoring the dynamics of association-dissociation of S1R and BiP. Acute treatment of cells with the S1R agonist PRE-084 produced rapid and transient dissociation of the S1R-BiP heterodimer, which was blocked by haloperidol. The effect of PRE-084 was enhanced by calcium depletion, leading to a higher reduction in heterodimerization even in the presence of haloperidol. Prolonged incubation of cells with S1R antagonists (haloperidol, NE-100, BD-1047, and PD-144418) increased the formation of S1R-BiP heteromers, while agonists (PRE-084, 4-IBP, and pentazocine) did not alter heterodimerization under the same experimental conditions. The newly developed S1R-BiP biosensor is a simple and effective tool for exploring S1R pharmacology in an easy cellular setting. This biosensor is suitable for high-throughput applications and a valuable resource in the researcher's toolkit.