Adenosine receptors are the on-and-off switch of astrocytic cannabinoid type 1 (CB1) receptor effect upon synaptic plasticity in the medial prefrontal cortex.

The medial prefrontal cortex (mPFC) is involved in cognitive functions such as working memory. Astrocytic cannabinoid type 1 receptor (CB1R) induces cytosolic calcium (Ca2+) concentration changes with an impact on neuronal function. mPFC astrocytes also express adenosine A1 and A2A receptors (A1R, A2AR), being unknown the crosstalk between CB1R and adenosine receptors in these cells. We show here that a further level of regulation of astrocyte Ca2+ signaling occurs through CB1R-A2AR or CB1R-A1R heteromers that ultimately impact mPFC synaptic plasticity. CB1R-mediated Ca2+ transients increased and decreased when A1R and A2AR were activated, respectively, unveiling adenosine receptors as modulators of astrocytic CB1R. CB1R activation leads to an enhancement of long-term potentiation (LTP) in the mPFC, under the control of A1R but not of A2AR. Notably, in IP3R2KO mice, that do not show astrocytic Ca2+ level elevations, CB1R activation decreases LTP, which is not modified by A1R or A2AR. The present work suggests that CB1R has a homeostatic role on mPFC LTP, under the control of A1R, probably due to physical crosstalk between these receptors in astrocytes that ultimately alters CB1R Ca2+ signaling.

A metabolomics study in aqueous humor discloses altered arginine metabolism in Parkinson's disease.

BACKGROUND: The lack of accessible and informative biomarkers results in a delayed diagnosis of Parkinson's disease (PD), whose symptoms appear when a significant number of dopaminergic neurons have already disappeared. The retina, a historically overlooked part of the central nervous system (CNS), has gained recent attention. It has been discovered that the composition of cerebrospinal fluid influences the aqueous humor composition through microfluidic circulation. In addition, alterations found in the brain of patients with PD have a correlate in the retina. This new paradigm highlights the potential of the aqueous humor as a sample for identifying differentially concentrated metabolites that could, eventually, become biomarkers if also found altered in blood or CSF of patients. In this research we aim at analyzing the composition of the aqueous humor from healthy controls and PD patients. METHODS: A targeted metabolomics approach with concentration determination by mass spectrometry was used. Statistical methods including principal component analysis and linear discriminants were used to select differentially concentrated metabolites that allow distinguishing patients from controls. RESULTS: In this first metabolomics study in the aqueous humor of PD patients, elevated levels of 16 compounds were found; molecules differentially concentrated grouped into biogenic amines, amino acids, and acylcarnitines. A biogenic amine, putrescine, alone could be a metabolite capable of differentiating between PD and control samples. The altered levels of the metabolites were correlated, suggesting that the elevations stem from a common mechanism involving arginine metabolism. CONCLUSIONS: A combination of three metabolites, putrescine, tyrosine, and carnitine was able to correctly classify healthy participants from PD patients. Altered metabolite levels suggest altered arginine metabolism. The pattern of metabolomic disturbances was not due to the levodopa-based dopamine replacement medication because one of the patients was not yet taking levodopa but a dopamine receptor agonist.

Differential Gene Expression in Activated Microglia Treated with Adenosine A2A Receptor Antagonists Highlights Olfactory Receptor 56 and T-Cell Activation GTPase-Activating Protein 1 as Potential Biomarkers of the Polarization of Activated Microglia.

Microglial activation often accompanies the plastic changes occurring in the brain of patients with neurodegenerative diseases. A2A and A3 adenosine receptors have been proposed as therapeutic targets to combat neurodegeneration. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with SCH 58261, a selective A2A receptor antagonist, and with both SCH 58261 and 2-Cl-IB-MECA, a selective A3 receptor agonist. None of the treatments led to any clear microglial phenotype when gene expression for classical biomarkers of microglial polarization was assessed. However, many of the downregulated genes were directly or indirectly related to immune system-related events. Searching for genes whose expression was both significantly and synergistically affected when treated with the two adenosine receptor ligands, the AC122413.1 and Olfr56 were selected among those that were, respectively, upregulated and downregulated. We therefore propose that the products of these genes, olfactory receptor 56 and T-cell activation GTPase-activating protein 1, deserve attention as potential biomarkers of phenotypes that occur upon microglial activation.

The olfactory Olfr-78/51E2 receptor interacts with the adenosine A2A receptor. Effect of menthol and 1,8-cineole on A2A receptor-mediated signaling.

Heteromer formation is unknown for the olfactory family of G protein-coupled receptors (GPCRs). We here identified, in a heterologous system, heteromers formed by the adenosine A2A receptor (A2AR), which is a target for neuroprotection, and an olfactory receptor. A2AR interacts with the receptor family 51, subfamily E, member 2 (OR51E2), the human ortholog of the mouse Olfr-78, whose mRNA is differentially expressed in activated microglia treated with adenosine receptor ligands. Bioluminescence resonance energy transfer (BRET) assays were performed in HEK-293T cells expressing the human version of the receptors, OR51E2 and A2AR, fused, respectively, to Renilla luciferase (RLuc) and the yellow fluorescent protein (YFP). BRET data was consistent with a receptor-receptor interaction whose consequences at the functional level were measured by cAMP level determination in CHO cells. Results showed an olfactory receptor-mediated partial blockade of Gs coupling to the A2AR, i.e., the effect of the A2AR selective agonist on intracellular levels of cAMP was significantly reduced. Two odorants, menthol and 1,8-cineole, which failed to show Golf-mediated OR51E2 activation because they did not increase cytosolic cAMP levels, reduced the BRET readings in cells expressing A2AR-YFP and OR51E2-Rluc, most likely suggesting a conformational change of at least one receptor. These odorants led to an almost complete block of A2AR coupling to Gs.

The cannabinoid CB1 receptor interacts with the angiotensin AT2 receptor. Overexpression of AT2-CB1 receptor heteromers in the striatum of 6-hydroxydopamine hemilesioned rats.

It is of particular interest the potential of cannabinoid and angiotensin receptors as targets in the therapy of Parkinson's disease (PD). While endocannabinoids are neuromodulators that act through the CB1 and CB2 cannabinoid receptors, the renin angiotensin-system is relevant for regulation of the correct functioning of several brain circuits. Resonance energy transfer assays in a heterologous system showed that the CB1 receptor (CB1R) can directly interact with the angiotensin AT2 receptor (AT2R). Coactivation of the two receptors results in increased Gi-signaling. The AT2-CB1 receptor heteromer imprint consists of a blockade of AT2R-mediated signaling by rimonabant, a CB1R antagonist. Interestingly, the heteromer imprint, discovered in the heterologous system, was also found in primary striatal neurons thus demonstrating the expression of the heteromer in these cells. In situ proximity ligation assays confirmed the occurrence of AT2-CB1 receptor heteromers in striatal neurons. In addition, increased expression of the AT2-CB1 receptor heteromeric complexes was detected in the striatum of a rodent PD model consisting of rats hemilesioned using 6-hydroxydopamine. Expression of the heteromer was upregulated in the striatum of lesioned animals and, also, of lesioned animals that upon levodopa treatment became dyskinetic. In contrast, there was no upregulation in the striatum of lesioned rats that did not become dyskinetic upon chronic levodopa treatment. The results suggest that therapeutic developments focused on the CB1R should consider that this receptor can interact with the AT2R, which in the CNS is involved in mechanisms related to addictive behaviors and to neurodegenerative and neuroinflammatory diseases.

Gene regulation in activated microglia by adenosine A3 receptor agonists: a transcriptomics study.

Most neurodegenerative disorders, including the two most common, Alzheimer's disease (AD) and Parkinson's disease (AD), course with activation of microglia, the resident innate immune cells of the central nervous system. A3 adenosine receptor (A3R) agonists have been proposed to be neuroprotective by regulating the phenotype of activated microglia. RNAseq was performed using samples isolated from lipopolysaccharide/interferon-γ activated microglia treated with 2-Cl-IB-MECA, a selective A3R agonist. The results showed that the number of negatively regulated genes in the presence of 2-Cl-IB-MECA was greater than the number of positively regulated genes. Gene ontology enrichment analysis showed regulation of genes participating in several cell processes, including those involved in immune-related events. Analysis of known and predicted protein-protein interactions showed that Smad3 and Sp1 are transcription factors whose genes are regulated by A3R activation. Under the conditions of cell activation and agonist treatment regimen, 2-Cl-IB-MECA did not lead to any tendency to favor the expression of genes related to neuroprotective microglia (M2).

Biogenic Amine Levels Markedly Increase in the Aqueous Humor of Individuals with Controlled Type 2 Diabetes.

The composition of the aqueous humor of patients with type 2 diabetes is relevant to understanding the underlying causes of eye-related comorbidities. Information on the composition of aqueous humor in healthy subjects is limited due to the lack of adequate controls. To carry out a metabolomics study, 31 samples of aqueous humor from healthy subjects without ocular pathology, submitted to refractive surgery and seven samples from patients with type 2 diabetes without signs of ocular pathology related to diabetes were used. The level of 25 molecules was significantly (p < 0.001) altered in the aqueous humor of the patient group. The concentration of a single molecule, N-acetylornithine, makes it possible to discriminate between control and diabetes (sensitivity and specificity equal to 1). In addition, receptor operating characteristic curve and principal component analysis for the above-mentioned six molecules yielded significantly (p < 0.001) altered in the aqueous humor of the patient group. In addition, receptor operating characteristic curve and principal component analysis for six compounds yielded cut-off values and remarkable sensitivity, specificity, and segregation ability. The altered level of N-acetylornithine may be due to an increased amount of acetate in diabetes. It is of interest to further investigate whether this alteration is related to the pathogenesis of the disease. The increase in the amino form of pyruvate, alanine, in diabetes is also relevant because it could be a means of reducing the formation of lactate from pyruvate.

Antagonization of OX1 Receptor Potentiates CB2 Receptor Function in Microglia from APPSw/Ind Mice Model.

Microdialysis assays demonstrated a possible role of orexin in the regulation of amyloid beta peptide (Aß) levels in the hippocampal interstitial fluid in the APP transgenic model. CB2R is overexpressed in activated microglia, showing a neuroprotective effect. These two receptors may interact, forming CB2-OX1-Hets and becoming a new target to combat Alzheimer's disease. Aims: Demonstrate the potential role of CB2-OX1-Hets expression and function in microglia from animal models of Alzheimer's disease. Receptor heteromer expression was detected by immunocytochemistry, bioluminescence resonance energy transfer (BRET) and proximity ligation assay (PLA) in transfected HEK-293T cells and microglia primary cultures. Quantitation of signal transduction events in a heterologous system and in microglia cells was performed using the AlphaScreen® SureFire® kit, western blot, the GCaMP6 calcium sensor and the Lance Ultra cAMP kit (PerkinElmer). The formation of CB2-OX1 receptor complexes in transfected HEK-293T cells has been demonstrated. The tetrameric complex is constituted by one CB2R homodimer, one OX1R homodimer and two G proteins, a Gi and a Gq. The use of TAT interfering peptides showed that the CB2-OX1 receptor complex interface is TM4-TM5. At the functional level it has been observed that the OX1R antagonist, SB334867, potentiates the action induced by CB2R agonist JWH133. This effect is observed in transfected HEK-293T cells and microglia, and it is stronger in the Alzheimer's disease (AD) animal model APPSw/Ind where the expression of the complex assessed by the proximity ligation assay indicates an increase in the number of complexes compared to resting microglia. The CB2-OX1 receptor complex is overexpressed in microglia from AD animal models where OX1R antagonists potentiate the neuroprotective actions of CB2R activation. Taken together, these results point to OX1R antagonists as drugs with therapeutic potential to combat AD. Data access statement: Raw data will be provided by the corresponding author upon reasonable requirement.

The adenosine A2A receptor is a therapeutic target in neurological, heart and oncogenic diseases.

INTRODUCTION: Following approval of antagonists in Parkinson's disease therapy, the A2A adenosine receptor (A2AR) is gaining interest as a target to combat a variety of diseases. AREAS COVERED: This review focuses on the therapeutic potential of targeting A2AR inside but also outside the central nervous system, more precisely to combat cardiac arrhythmias and to boost immune-based cancer therapies. The mechanism of regulation of the immune system by adenosine (Ado) is complex since several actors are involved, namely the enzymes that produce and degrade the compound and the four Ado receptors. Antagonists of A2A or andadenosine A2B Ado receptors lead to reduce their activation intracellular cAMP levels thus boosting immune responses. The literature search methodology consisted of reviewing the authors own collection of papers plus searches in the PubMed and Google Scholar databases from March 2021 to August 2022. EXPERT OPINION: There is a pending issue, namely, how to demonstrate the neuroprotective potential of A2AR antagonists that would lead to delay neurodegenerative disease progression. In addition, A2AR antagonists and, eventually, dual A2A/A2B receptor antagonists have potential in the treatment of heart arrythmias and cancer. If adequate resources are allocated, it is a matter of time to confirm whether or not these possibilities become a reality.

Regulation of Expression of Cannabinoid CB2 and Serotonin 5HT1A Receptor Complexes by Cannabinoids in Animal Models of Hypoxia and in Oxygen/Glucose-Deprived Neurons.

Background: Cannabidiol (CBD) is a phytocannabinoid with potential in one of the most prevalent syndromes occurring at birth, the hypoxia of the neonate. CBD targets a variety of proteins, cannabinoid CB2 and serotonin 5HT1A receptors included. These two receptors may interact to form heteromers (CB2-5HT1A-Hets) that are also a target of CBD. Aims: We aimed to assess whether the expression and function of CB2-5HT1A-Hets is affected by CBD in animal models of hypoxia of the neonate and in glucose- and oxygen-deprived neurons. Methods: We developed a quantitation of signal transduction events in a heterologous system and in glucose/oxygen-deprived neurons. The expression of receptors was assessed by immuno-cyto and -histochemistry and, also, by using the only existing technique to visualize CB2-5HT1A-Hets fixed cultured cells and tissue sections (in situ proximity ligation PLA assay). Results: CBD and cannabigerol, which were used for comparative purposes, affected the structure of the heteromer, but in a qualitatively different way; CBD but not CBG increased the affinity of the CB2 and 5HT1A receptor-receptor interaction. Both cannabinoids regulated the effects of CB2 and 5HT1A receptor agonists. CBD was able to revert the upregulation of heteromers occurring when neurons were deprived of oxygen and glucose. CBD significantly reduced the increased expression of the CB2-5HT1A-Het in glucose/oxygen-deprived neurons. Importantly, in brain sections of a hypoxia/ischemia animal model, administration of CBD led to a significant reduction in the expression of CB2-5HT1A-Hets. Conclusions: Benefits of CBD in the hypoxia of the neonate are mediated by acting on CB2-5HT1A-Hets and by reducing the aberrant expression of the receptor-receptor complex in hypoxic-ischemic conditions. These results reinforce the potential of CBD for the therapy of the hypoxia of the neonate.

Targeted Metabolomics Shows That the Level of Glutamine, Kynurenine, Acyl-Carnitines and Lysophosphatidylcholines Is Significantly Increased in the Aqueous Humor of Glaucoma Patients.

The composition of the aqueous humor of patients with glaucoma is relevant to understand the underlying causes of the pathology. Information on the concentration of metabolites and small molecules in the aqueous humor of healthy subjects is limited. Among the causes of the limitations is the lack of healthy controls since, until recently, they were not surgically intervened; therefore, the aqueous humor of patients operated for cataract was used as a reference. Sixteen aqueous humor samples from healthy subjects undergoing refractive surgery and eight samples from glaucoma patients were used to assess the concentration of 188 compounds using chromatography and mass spectrometry. The concentration of 80 of the 188 was found to be reliable, allowing comparison of data from the two groups (glaucoma and control). The pattern found in the controls is similar to, but not the same as, that reported using samples from "controls" undergoing cataract surgery. Comparing data from glaucoma patients and healthy subjects, 57 of the 80 compounds were significantly (p < 0.05) altered in the aqueous humor. Kynurenine and glutamine, but not glutamate, were significantly increased in the glaucoma samples. Furthermore, 10 compounds were selected considering a statistical score of p < 0.0001 and the degree of change of more than double or less than half. The level of C10 (decanoyl)-carnitine decreased, while the concentration of spermidine and various acyl-carnitines and lysophosphatidylcholines increased in glaucoma. Principal component analysis showed complete segregation of controls and cases using the data for the 10 selected compounds. The receiver operating characteristic curve these 10 compounds and for glutamine allowed finding cut-off values and significant sensitivity and specificity scores. The concentration of small metabolites in the aqueous humor of glaucoma patients is altered even when they take medication and are well controlled. The imbalance affects membrane components, especially those of the mitochondria, suggesting that mitochondrial abnormalities are a cause or consequence of glaucoma. The increase in glutamine in glaucoma is also relevant because it could be a means of keeping the concentration of glutamate under control, thus avoiding its potential to induce the death of neurons and retinal cells. Equally notable was the increase in kynurenine, which is essential in the metabolism of nicotine adenine dinucleotides.

Expression of the Adenosine A2A-A3 Receptor Heteromer in Different Brain Regions and Marked Upregulation in the Microglia of the Transgenic APPSw,Ind Alzheimer's Disease Model.

Adenosine (Ado) receptors have been instrumental in the detection of heteromers and other higher-order receptor structures, mainly via interactions with other cell surface G-protein-coupled receptors. Apart from the first report of the A1 Ado receptor interacting with the A2A Ado receptor, there has been more recent data on the possibility that every Ado receptor type, A1, A2A, A2B, and A3, may interact with each other. The aim of this paper was to look for the expression and function of the A2A/A3 receptor heteromer (A2AA3Het) in neurons and microglia. In situ proximity ligation assays (PLA), performed in primary cells, showed that A2AA3Het expression was markedly higher in striatal than in cortical and hippocampal neurons, whereas it was similar in resting and activated microglia. Signaling assays demonstrated that the effect of the A2AR agonist, PSB 777, was reduced in the presence of the A3R agonist, 2-Cl-IB-MECA, whereas the effect of the A3R agonist was potentiated by the A2AR antagonist, SCH 58261. Interestingly, the expression of the heteromer was markedly enhanced in microglia from the APPSw,Ind model of Alzheimer's disease. The functionality of the heteromer in primary microglia from APPSw,Ind mice was more similar to that found in resting microglia from control mice.

Ghrelin and Cannabinoid Functional Interactions Mediated by Ghrelin/CB1 Receptor Heteromers That Are Upregulated in the Striatum From Offspring of Mice Under a High-Fat Diet.

There is evidence of ghrelinergic-cannabinoidergic interactions in the central nervous system (CNS) that may impact on the plasticity of reward circuits. The aim of this article was to look for molecular and/or functional interactions between cannabinoid CB1 and ghrelin GHS-R1a receptors. In a heterologous system and using the bioluminescence resonance energy transfer technique we show that human versions of cannabinoid CB1 and ghrelin GHS-R1a receptors may form macromolecular complexes. Such receptor heteromers have particular properties in terms of CB1/Gi-mediated signaling and in terms of GHS-R1a-Gq-mediated signaling. On the one hand, just co-expression of CB1R and GHS-R1a led to impairment of cannabinoid signaling. On the other hand, cannabinoids led to an increase in ghrelin-derived calcium mobilization that was stronger at low concentrations of the CB1 receptor agonist, arachidonyl-2'-chloroethylamide (ACEA). The expression of CB1-GHS-R1a receptor complexes in striatal neurons was confirmed by in situ proximity ligation imaging assays. Upregulation of CB1-GHS-R1a- receptor complexes was found in striatal neurons from siblings of pregnant female mice on a high-fat diet. Surprisingly, the expression was upregulated after treatment of neurons with ghrelin (200 nM) or with ACEA (100 nM). These results help to better understand the complexities underlying the functional interactions of neuromodulators in the reward areas of the brain.

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors.

We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.

N-Methyl-D-aspartate (NMDA) and cannabinoid CB2 receptors form functional complexes in cells of the central nervous system: insights into the therapeutic potential of neuronal and microglial NMDA receptors.

BACKGROUND: The cannabinoid CB2 receptor (CB2R), which is a target to afford neuroprotection, and N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors, which are key in mediating excitatory neurotransmission, are expressed in both neurons and glia. As NMDA receptors are the target of current medication in Alzheimer's disease patients and with the aim of finding neuromodulators of their actions that could provide benefits in dementia, we hypothesized that cannabinoids could modulate NMDA function. METHODS: Immunocytochemistry was used to analyze the colocalization between CB2 and NMDA receptors; bioluminescence resonance energy transfer was used to detect CB2-NMDA receptor complexes. Calcium and cAMP determination, mitogen-activated protein kinase (MAPK) pathway activation, and label-free assays were performed to characterize signaling in homologous and heterologous systems. Proximity ligation assays were used to quantify CB2-NMDA heteromer expression in mouse primary cultures and in the brain of APPSw/Ind transgenic mice, an Alzheimer's disease model expressing the Indiana and Swedish mutated version of the human amyloid precursor protein (APP). RESULTS: In a heterologous system, we identified CB2-NMDA complexes with a particular heteromer print consisting of impairment by cannabinoids of NMDA receptor function. The print was detected in activated primary microglia treated with lipopolysaccharide and interferon-γ. CB2R activation blunted NMDA receptor-mediated signaling in primary hippocampal neurons from APPSw/Ind mice. Furthermore, imaging studies showed that in brain slices and in primary cells (microglia or neurons) from APPSw/Ind mice, there was a marked overexpression of macromolecular CB2-NMDA receptor complexes thus becoming a tool to modulate excessive glutamate input by cannabinoids. CONCLUSIONS: The results indicate a negative cross-talk in CB2-NMDA complexes signaling. The expression of the CB2-NMDA receptor heteromers increases in both microglia and neurons from the APPSw/Ind transgenic mice, compared with levels in samples from age-matched control mice.

Identification of the Ghrelin and Cannabinoid CB2 Receptor Heteromer Functionality and Marked Upregulation in Striatal Neurons from Offspring of Mice under a High-Fat Diet.

Cannabinoids have been reported as orexigenic, i.e., as promoting food intake that, among others, is controlled by the so-called "hunger" hormone, ghrelin. The aim of this paper was to look for functional and/or molecular interactions between ghrelin GHSR1a and cannabinoid CB2 receptors at the central nervous system (CNS) level. In a heterologous system we identified CB2-GHSR1a receptor complexes with a particular heteromer print consisting of impairment of CB2 receptor/Gi-mediated signaling. The blockade was due to allosteric interactions within the heteromeric complex as it was reverted by antagonists of the GHSR1a receptor. Cannabinoids acting on the CB2 receptor did not affect cytosolic increases of calcium ions induced by ghrelin acting on the GHSR1a receptor. In situ proximity ligation imaging assays confirmed the expression of CB2-GHSR1a receptor complexes in both heterologous cells and primary striatal neurons. We tested heteromer expression in neurons from offspring of high-fat-diet mouse mothers as they have more risk to be obese. Interestingly, there was a marked upregulation of those complexes in striatal neurons from siblings of pregnant female mice under a high-fat diet.

Microglial Adenosine Receptors: From Preconditioning to Modulating the M1/M2 Balance in Activated Cells.

Neuronal survival depends on the glia, that is, on the astroglial and microglial support. Neurons die and microglia are activated not only in neurodegenerative diseases but also in physiological aging. Activated microglia, once considered harmful, express two main phenotypes: the pro-inflammatory or M1, and the neuroprotective or M2. When neuroinflammation, i.e., microglial activation occurs, it is important to achieve a good M1/M2 balance, i.e., at some point M1 microglia must be skewed into M2 cells to impede chronic inflammation and to afford neuronal survival. G protein-coupled receptors in general and adenosine receptors in particular are potential targets for increasing the number of M2 cells. This article describes the mechanisms underlying microglial activation and analyzes whether these cells exposed to a first damaging event may be ready to be preconditioned to better react to exposure to more damaging events. Adenosine receptors are relevant due to their participation in preconditioning. They can also be overexpressed in activated microglial cells. The potential of adenosine receptors and complexes formed by adenosine receptors and cannabinoids as therapeutic targets to provide microglia-mediated neuroprotection is here discussed.

Methamphetamine Blocks Adenosine A2A Receptor Activation via Sigma 1 and Cannabinoid CB1 Receptors.

Methamphetamine is, worldwide, one of the most consumed drugs of abuse. One important side effect is neurodegeneration leading to a decrease in life expectancy. The aim of this paper was to check whether the drug affects one of the receptors involved in neurodegeneration/neuroprotection events, namely the adenosine A2A receptor (A2AR). First, we noticed that methamphetamine does not affect A2A functionality if the receptor is expressed in a heterologous system. However, A2AR becomes sensitive to the drug upon complexes formation with the cannabinoid CB1 receptor (CB1R) and the sigma 1 receptor (σ1R). Signaling via both adenosine A2AR and cannabinoid CB1R was affected by methamphetamine in cells co-expressing the two receptors. In striatal primary cultures, the A2AR-CB1R heteromer complex was detected and methamphetamine not only altered its expression but completely blocked the A2AR- and the CB1R-mediated activation of the mitogen activated protein kinase (MAPK) pathway. In conclusion, methamphetamine, with the participation of σ1R, alters the expression and function of two interacting receptors, A2AR, which is a therapeutic target for neuroprotection, and CB1R, which is the most abundant G protein-coupled receptor (GPCR) in the brain.

Discovery of a macromolecular complex mediating the hunger suppressive actions of cocaine: Structural and functional properties.

Cocaine not only increases brain dopamine levels but also activates the sigma1 receptor (σ1 R) that in turn regulates orexigenic receptor function. Identification of interactions involving dopamine D1 (D1 R), ghrelin (GHS-R1a ), and σ1 receptors have been addressed by biophysical techniques and a complementation approach using interfering peptides. The effect of cocaine on receptor functionality was assayed by measuring second messenger, cAMP and Ca2+ , levels. The effect of acute or chronic cocaine administration on receptor complex expression was assayed by in situ proximity ligation assay. In silico procedures were used for molecular model building. σ1 R KO mice were used for confirming involvement of this receptor. Upon identification of protomer interaction and receptor functionality, a unique structural model for the macromolecular complex formed by σ1 R, D1 R, and GHS-R1a is proposed. The functionality of the complex, able to couple to both Gs and Gq proteins, is affected by cocaine binding to the σ1 R, as confirmed using samples from σ1 R-/- mice. The expression of the macromolecular complex was differentially affected upon acute and chronic cocaine administration to rats. The constructed 3D model is consistent with biochemical, biophysical, and available structural data. The σ1 R, D1 R, and GHS-R1a complex constitutes a functional unit that is altered upon cocaine binding to the σ1 R. Remarkably, the heteromer can simultaneously couple to two G proteins, thus allowing dopamine to signal via Ca2+ and ghrelin via cAMP. The anorexic action of cocaine is mediated by such complex whose expression is higher after acute than after chronic administration regimens.

Structure and function of adenosine receptor heteromers.

Adenosine is one of the most ancient signaling molecules and has receptors in both animals and plants. In mammals there are four specific receptors, A1, A2A, A2B, and A3, which belong to the superfamily of G-protein-coupled receptors (GPCRs). Evidence accumulated in the last 20 years indicates that GPCRs are often expressed as oligomeric complexes formed by a number of equal (homomers) or different (heteromers) receptors. This review presents the data showing the occurrence of heteromers formed by A1 and A2A, A2A and A2B, and A2A and A3 receptors highlighting (i) their tetrameric structural arrangements, and (ii) the functional diversity that those heteromers provide to adenosinergic signaling.