A2A Receptor Research Articles

Expression and Impact of Adenosine A3 Receptors on Calcium Homeostasis in Human Right Atrium.

Increased adenosine A2A receptor (A2AR) expression and activation underlies a higher incidence of spontaneous calcium release in atrial fibrillation (AF). Adenosine A3 receptors (A3R) could counteract excessive A2AR activation, but their functional role in the atrium remains elusive, and we therefore aimed to address the impact of A3Rs on intracellular calcium homeostasis. For this purpose, we analyzed right atrial samples or myocytes from 53 patients without AF, using quantitative PCR, patch-clamp technique, immunofluorescent labeling or confocal calcium imaging. A3R mRNA accounted for 9% and A2AR mRNA for 32%. At baseline, A3R inhibition increased the transient inward current (ITI) frequency from 0.28 to 0.81 events/min (p < 0.05). Simultaneous stimulation of A2ARs and A3Rs increased the calcium spark frequency seven-fold (p < 0.001) and the ITI frequency from 0.14 to 0.64 events/min (p < 0.05). Subsequent A3R inhibition caused a strong additional increase in the ITI frequency (to 2.04 events/min; p < 0.01) and increased phosphorylation at s2808 1.7-fold (p < 0.001). These pharmacological treatments had no significant effects on L-type calcium current density or sarcoplasmic reticulum calcium load. In conclusion, A3Rs are expressed and blunt spontaneous calcium release at baseline and upon A2AR-stimulation in human atrial myocytes, pointing to A3R activation as a means to attenuate physiological and pathological elevations of spontaneous calcium release events.

Ejaculatory Duct Obstruction Affects Seminal Vesicle Contractile Efficacy and Smooth Muscle Ultrastructure in a Rat Model

The purpose of this study was to investigate the effects of ejaculatory duct obstruction (EDO) on contractile efficacy, smooth muscle ultrastructure, and α1A and M3 receptors of rat seminal vesicles (SVs). A total of 48 male rats, aged 14-15 weeks, were randomly divided into three groups, namely, the control, complete EDO, and partial EDO. SV tissues were collected at 4 and 8 weeks postoperatively for further experiments. Results revealed a marked reduction in SV contractile efficacy over time following obstruction in the complete EDO group. The contractile force and frequency decreased and increased in the partial EDO group at week 4, respectively, whereas contractile efficacy significantly reduced at week 8. Moreover, obstruction resulted in significant downregulation in expression of α1A and M3 proteins and mRNAs in rats from the complete EDO group over time. Rats in the partial EDO group initially exhibited an increase followed by a decrease. Analysis of the ultrastructure of SV smooth muscles confirmed the above changes. In conclusion, complete EDO can lead to a progressive decrease in contractile efficiency of SVs. On the other hand, partial EDO can first compensate for the contraction of SVs and gradually decompensate afterwards.

A Competition between Relative Stability and Binding Energy in Caffeine Phenyl-Glucose Aggregates: Implications in Biological Mechanisms.

Hydrogen bonds and stacking interactions are pivotal in biological mechanisms, although their proper characterisation within a molecular complex remains a difficult task. We used quantum mechanical calculations to characterise the complex between caffeine and phenyl-β-D-glucopyranoside, in which several functional groups of the sugar derivative compete with each other to attract caffeine. Calculations at different levels of theory (M06-2X/6-311++G(d,p) and B3LYP-ED=GD3BJ/def2TZVP) agree to predict several structures similar in stability (relative energy) but with different affinity (binding energy). These computational results were experimentally verified by laser infrared spectroscopy, through which the caffeine·phenyl-β-D-glucopyranoside complex was identified in an isolated environment, produced under supersonic expansion conditions. The experimental observations correlate with the computational results. Caffeine shows intermolecular interaction preferences that combine both hydrogen bonding and stacking interactions. This dual behaviour had already been observed with phenol, and now with phenyl-β-D-glucopyranoside, it is confirmed and maximised. In fact, the size of the complex's counterparts affects the maximisation of the intermolecular bond strength because of the conformational adaptability given by the stacking interaction. Comparison with the binding of caffeine within the orthosteric site of the A2A adenosine receptor shows that the more strongly bound caffeine·phenyl-β-D-glucopyranoside conformer mimics the interactions occurring within the receptor.

DrugEx v3: scaffold-constrained drug design with graph transformer-based reinforcement learning

Rational drug design often starts from specific scaffolds to which side chains/substituents are added or modified due to the large drug-like chemical space available to search for novel drug-like molecules. With the rapid growth of deep learning in drug discovery, a variety of effective approaches have been developed for de novo drug design. In previous work we proposed a method named DrugEx, which can be applied in polypharmacology based on multi-objective deep reinforcement learning. However, the previous version is trained under fixed objectives and does not allow users to input any prior information (i.e. a desired scaffold). In order to improve the general applicability, we updated DrugEx to design drug molecules based on scaffolds which consist of multiple fragments provided by users. Here, a Transformer model was employed to generate molecular structures. The Transformer is a multi-head self-attention deep learning model containing an encoder to receive scaffolds as input and a decoder to generate molecules as output. In order to deal with the graph representation of molecules a novel positional encoding for each atom and bond based on an adjacency matrix was proposed, extending the architecture of the Transformer. The graph Transformer model contains growing and connecting procedures for molecule generation starting from a given scaffold based on fragments. Moreover, the generator was trained under a reinforcement learning framework to increase the number of desired ligands. As a proof of concept, the method was applied to design ligands for the adenosine A2A receptor (A2AAR) and compared with SMILES-based methods. The results show that 100% of the generated molecules are valid and most of them had a high predicted affinity value towards A2AAR with given scaffolds.

Chemoimmunotherapy with doxorubicin and caffeine combination enhanced ICD induction and T-cell infiltration in B16F10 melanoma tumors.

Majority of chemotherapeutic agents can elicit antitumor immunity and modulate the composition, density, function, and distribution of tumor infiltrating lymphocytes (TILs), to influence differential therapeutic responses and prognosis in cancer patients. The clinical success of these agents, particularly anthracyclines like doxorubicin, not only depends on their cytotoxic activity but also by the enhancement of pre-existing immunity primarily through induction of immunogenic cell death(ICD). However, resistance for the induction of ICD either intrinsic or acquired is a major hurdle for most of these drugs. To enhance ICD by these agents, it has become clear that blockade of adenosine production or its signaling need to be specifically targeted as they represent highly resistant mechanisms. Given the prominent role of adenosine mediated immunosuppression and resistance to ICD induction in tumor microenvironment, combination strategies that involve ICD induction and adenosine signaling blockade are further warranted. In the present study, we investigated the antitumor effect of caffeine and doxorubicin combination therapy against 3-MCA-induced and cell-line induced tumors in mice. Our results demonstrated significant tumor growth inhibition by the combination therapy of doxorubicin and caffeine against both carcinogen-induced and cell-line induced tumor models. In addition, significant T-cell infiltration and enhanced ICD induction evidenced by increased intratumoral calreticulin and HMGB1 levels, was observed in B16F10 melanoma mice. The possible mechanism behind the observed antitumor activity might be due to the enhanced ICD induction and subsequent T-cell infiltration by the combination therapy. To prevent the development of resistance and to enhance the antitumor activity of ICD inducing drugs like doxorubicin, combination with adenosine-A2A receptor pathway inhibitors like caffeine might be a potential strategy.

USP20 is a predictor of poor prognosis in colorectal cancer and associated with lymph node metastasis, immune infiltration and chemotherapy resistance.

Colorectal cancer (CRC) is a highly prevalent malignancy with a poor prognosis. USP20 can support progression of variety of tumors. USP20 was shown to promote breast tumor metastasis, and proliferation of oral squamous carcinoma cells. However, the role of USP20 in CRC remains unclear. We used bioinformatics to analyze the expression and prognosis of USP20 in pan-cancer and explore the relationship between USP20 expression and immune infiltration, immune checkpoints, and chemotherapy resistance in CRC. The differential expression and prognostic role of USP20 in CRC was validated by qRT-PCR and immunohistochemistry. Cox univariate and multivariate analyses were performed to assess risk factors for poor prognosis of CRC, and new prognostic prediction models were constructed and evaluated by decision curve analysis (ROC) and receiver operating characteristic (DCA). USP20 was overexpressed in CRC cell lines to explore the effect of USP20 on the functionalities of CRC cells. Enrichment analyses were used to explore the possible mechanism of USP20 in CRC. The expression of USP20 was lower in CRC tissues than adjacent normal tissues. Compared with low USP20 expression patients, CRC patients with high USP20 expression level had shorter OS. Correlation analysis showed that USP20 expression was associated with lymph node metastasis. Cox regression analysis revealed USP20 as an independent risk factor for poor prognosis in CRC patients. ROC and DCA analyses showed that the performance of the newly constructed prediction model was better than the traditional TNM model. Immune infiltration analysis shown that USP20 expression is closely associated with T cell infiltration in CRC. A co-expression analysis showed that USP20 expression was positively correlated with several immune checkpoint genes including ADORA2A, CD160, CD27 and TNFRSF25 genes and positively associated with multiple multi-drug resistance genes such as MRP1, MRP3, and MRP5 genes. USP20 expression positively correlated with the sensitivity of cells to multiple anticancer drugs. Overexpression of USP20 enhanced the migration and invasive ability of CRC cells. Enrichment pathway analyses showed the USP20 may play a role via the Notch pathway, Hedgehog pathway and beta-catenin pathway. USP20 is downregulated in CRC and associated with prognosis in CRC. USP20 enhances CRC cells metastasis and is associated with immune infiltration, immune checkpoints, and chemotherapy resistance.

Anionic phospholipids control mechanisms of GPCR-G protein recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we explore the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids prime the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeds through a less favorable induced fit mechanism. In computational models, anionic lipids mimic interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly alters the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

Adenosine Receptors Expression in Human Retina and Choroid with Age-related Macular Degeneration.

Adenosine signaling modulates ocular inflammatory processes, and its antagonism mitigates neovascularization in both newborns and preclinical models of ocular neovascularization including age-related macular degeneration (AMD). The adenosine receptor expression patterns have not been well characterized in the human retina and choroid. Here we examined the expression of adenosine receptor subtypes within the retina and choroid of human donor eyes with and without AMD. Antibodies specifically targeting adenosine receptor subtypes A1, A2A, A2B, and A3 were used to assess their expression patterns. Quantitative real-time PCR analysis was used to confirm gene expression of these receptors within the normal human retina and choroid. We found that all four receptor subtypes were expressed in several layers of the retina, and within the retinal pigment epithelium and choroid. The expression of A1 receptors was more prominent in the inner and outer plexiform layers, where microglia normally reside, and supported by RNA expression in the retina. A2A and A2B showed similar expression patterns with prominent expression in the vasculature and retinal pigment epithelium. No dramatic differences in expression of these receptors were observed in eyes from patients with dry or wet AMD compared to control, with the exception A3 receptors. Eyes with dry AMD lost expression of A3 in the photoreceptor outer segments compared with eyes from control or wet AMD. The ocular presence of adenosine receptors is consistent with their proposed role in modulation of inflammation in both the retina and choroid, and their potential targeting for AMD treatment.

Increased Density of Endogenous Adenosine A2A Receptors in Atrial Fibrillation: From Cellular and Porcine Models to Human Patients.

Adenosine, an endogenous nucleoside, plays a critical role in maintaining homeostasis during stressful situations, such as energy deprivation or cellular damage. Therefore, extracellular adenosine is generated locally in tissues under conditions such as hypoxia, ischemia, or inflammation. In fact, plasma levels of adenosine in patients with atrial fibrillation (AF) are elevated, which also correlates with an increased density of adenosine A2A receptors (A2ARs) both in the right atrium and in peripheral blood mononuclear cells (PBMCs). The complexity of adenosine-mediated effects in health and disease requires simple and reproducible experimental models of AF. Here, we generate two AF models, namely the cardiomyocyte cell line HL-1 submitted to Anemonia toxin II (ATX-II) and a large animal model of AF, the right atrium tachypaced pig (A-TP). We evaluated the density of endogenous A2AR in those AF models. Treatment of HL-1 cells with ATX-II reduced cell viability, while the density of A2AR increased significantly, as previously observed in cardiomyocytes with AF. Next, we generated the animal model of AF based on tachypacing pigs. In particular, the density of the key calcium regulatory protein calsequestrin-2 was reduced in A-TP animals, which is consistent with the atrial remodelling shown in humans suffering from AF. Likewise, the density of A2AR in the atrium of the AF pig model increased significantly, as also shown in the biopsies of the right atrium of subjects with AF. Overall, our findings revealed that these two experimental models of AF mimicked the alterations in A2AR density observed in patients with AF, making them attractive models for studying the adenosinergic system in AF.

Biophysical Dissection of Isolated GPCRs: The Adenosine A2A Receptor under the Bistouries

In an effort to provide an overview of the biophysical approaches used to study G-protein-coupled receptors, we chose to consider the adenosine A2A receptor as a model, as it is widely reported in the literature to explore the way GPCRs are studied nowadays. After a brief introduction of the receptor, we gathered descriptions of the various tools used to investigate the pharmacology and structure of the A2A receptor. We began by describing the key developments which have led to successful studies of GPCRs including the cloning, expression and purification of A2A, and the subsequent characterizations including quality control, binding and functional studies that have been necessary for the further understanding of the receptor. Then, we reviewed the reconstitution of A2A into nanodiscs as well as the use of this biological material in structural mass spectrometry, NMR, calorimetry and various other approaches to gain not only information about the structure and function of A2A, but also the dynamics of the receptor and the tools necessary to pursue such investigations. The body of techniques presented herein are applicable to all GPCRs amenable to purification.

The Effect of Adenosine Signaling on Memory Impairment Induced by Pentylenetetrazole in Zebrafish.

Epilepsy is characterized by the manifestation of spontaneous and recurrent seizures. The high prevalence of comorbidities associated with epilepsy, such as cognitive dysfunction, affects the patients quality oflife. Adenosine signaling modulation might be an effective alternative to control seizures and epilepsy-associated comorbidities. This study aimed to verify the role of adenosine modulation on the seizure development and cognitive impairment induced by pentylenetetrazole (PTZ) in zebrafish. At first, animals were submitted to a training session in the inhibitory avoidance test and, after 10min, they received an intraperitoneal injection of valproate, adenosine A1 receptor agonist cyclopentyladenosine (CPA), adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), adenosine A2A receptor antagonist ZM 241385, adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nony1)-adenine hydrochloride (EHNA) or the nucleoside transporter inhibitor dipyridamole. Thirty min after the intraperitoneal injection, the animals were exposed to 7.5mM PTZ for 10min, where they were evaluated for latency to reach the seizure stages (I, II, and III). Finally, 24h after the training session, the animals were submitted to the inhibitory avoidance test to verify their cognitive performance during the test session. Valproate, CPA, and EHNA showed antiseizure effects and prevented the memory impairment induced by PTZ exposure. DPCPX, ZM 241385, and dipyridamole pretreatments caused no changes in seizure development; however, these drugs prevented memory impairment without altering locomotion. Our results reinforce the antiseizure effects of adenosine signaling and support the idea that the involvement of adenosine in memory processes may be a target for preventive strategies against cognitive impairment associated with epilepsy.

Activation of adenosine A2A receptors induces SUMOylation of HIF-1α.

Hypoxia is an established driver of numerous deleterious cell processes, including immunosuppression and tumor growth. Hypoxia-inducible factor 1α (HIF-1α) accumulates during periods of sustained hypoxia due to the action of multiple post-translational modifications, including the Small Ubiquitin-like modifier (SUMO) pathway. Although enhanced activity of the SUMO-pathway is upregulated during hypoxia, the mechanistic basis for this effect remains unclear. It has been proposed that the Gαs protein-coupled adenosine A2A receptor (A2AR), which is also activated during sustained hypoxia, mediates SUMOylation at a number of target proteins. We sought to determine if activation of A2AR by hypoxia induces SUMOylation of HIF-1α and which of three potential SUMO-sites on HIF-1α are modified by this mechanism. HEK293T cells expressing A2AR and treated with an A2AR agonist (CGS) showed significantly increased HIF-1α accumulation compared to untreated controls after exposure to hypoxia. Similarly, CGS and hypoxia enhanced SUMOylation of HIF-1α in human Jurkat T cells. Using site-directed mutagenesis and Förster resonance energy transfer (FRET), we determined that A2AR-mediated SUMOylation of HIF-1α occurs at lysine 532. Finally, we show that (1) this mechanism of regulation increases the mRNA levels of multiple HIF-1α transcriptional targets; and (2) this mechanism can be precluded by treating cells with a small molecule inhibitor of the SUMO pathway. Together, these data highlight a central role for A2AR-mediated SUMOylation of HIF-1α in mediating the effects of chronic hypoxia in Jurkat T cells.

Unique effect of clozapine on adenosine A2A-dopamine D2 receptor heteromerization

The striatal dopamine D2 receptor (D2R) is generally accepted to be involved in positive symptoms of schizophrenia and is a main target for clinically used antipsychotics. D2R are highly expressed in the striatum, where they form heteromers with the adenosine A2A receptor (A2AR). Changes in the density of A2AR-D2R heteromers have been reported in postmortem tissue from patients with schizophrenia, but the degree to which A2R are involved in schizophrenia and the effect of antipsychotic drugs is unknown. Here, we examine the effect of exposure to three prototypical antipsychotic drugs on A2AR-D2R heteromerization in mammalian cells using a NanoBiT assay. After 16 h of exposure, a significant increase in the density of A2AR-D2R heteromers was found with haloperidol and aripiprazole, but not with clozapine. On the other hand, clozapine, but not haloperidol or aripiprazole, was associated with a significant decrease in A2AR-D2R heteromerization after 2 h of treatment. Computational binding models of these compounds revealed distinctive molecular signatures that explain their different influence on heteromerization. The bulky tricyclic moiety of clozapine displaces TM 5 of D2R, inducing a clash with A2AR, while the extended binding mode of haloperidol and aripiprazole stabilizes a specific conformation of the second extracellular loop of D2R that enhances the interaction with A2AR. It is proposed that an increase in A2AR-D2R heteromerization is involved in the extrapyramidal side effects (EPS) of antipsychotics and that the specific clozapine-mediated destabilization of A2AR-D2R heteromerization can explain its low EPS liability.

Measuring the rupture force between adenosine A2A G-protein coupled receptors and G-proteins using axial optical tweezers.

G-protein-coupled receptors (GPCRs) are the largest group of membrane receptors. Here we used axial optical tweezers to measure the rupture force between the adenosine A2A receptor in a nanodisc and its associated G-protein. We also measured the interaction between the receptor and an engineered G-protein, mini-Gs, which is a truncated Gs alpha-subunit that can increase agonist affinity and stabilize the active state of GPCRs. These measurements required us to extend the force range accessible with axial optical tweezers to approximately 50 pN. This involved strengthening the DNA handles to which we attach the G-protein or mini-Gs and improving the force calibration of the axial optical tweezers by employing a twistable worm-like chain (tWLC) model. These advances enabled us to measure the rupture force between the adenosine A2A receptor and the G-proteins under various physiological conditions.

Protein engineering approaches to determine adenosine receptor-G protein interactions.

The adenosine receptor subfamily of G protein-coupled receptors is an important family of membrane receptors that modulate blood pressure and more recently have been implicated in cancer, neurodegenerative diseases, and diabetes, making them a significant fraction of drug discovery efforts. Adenosine A2A receptor (A2AR), one of four subfamily members, has a longer C-terminus than the other adenosine receptor subtypes, which may contribute to its exceptional trafficking to the plasma membrane, but its flexibility has been attributed to protein-protein interactions. Chimeric A1 and A3 receptors with A2AR C-termini showed improved localization to the plasma membrane and are functionally active, with greater than three-fold higher yields than previously reported from other heterologous expression systems. Interestingly, these chimeras are able to bind to their native G proteins, suggesting the specificity of interaction does not rely on the C-terminus. To investigate this further using purified A2AR and Gαs, we determined the affinity and binding rates via surface plasmon resonance (SPR). Together with in vivo signaling data, our results suggest that loss of downstream signaling for the truncation may result from a reduced GPCR-G protein complex interaction, which may be important for interpretation of structure-based drug discovery screening. Current studies are focused on the effect of lipid nanodiscs on A2AR-G protein interactions.

Loss of C3a and C5a receptors promotes adipocyte browning and attenuates diet-induced obesity via activating inosine/A2aR pathway.

Complement activation is thought to underline the pathologic progression of obesity-related metabolic disorders; however, its role in adaptive thermogenesis has scarcely been explored. Here, we identify complement C3a receptor (C3aR) and C5a receptor (C5aR) as critical switches to control adipocyte browning and energy balance in male mice. Loss of C3aR and C5aR in combination, more than individually, increases cold-induced adipocyte browning and attenuates diet-induced obesity in male mice. Mechanistically, loss of C3aR and C5aR increases regulatory Tcell (Treg) accumulation in the subcutaneous white adipose tissue during cold exposure or high-fat diet. Activated Tregs produce adenosine, which is converted to inosine by adipocyte-derived adenosine deaminases. Inosine promotes adipocyte browning in a manner dependent on activating adenosine A2a receptor. These data reveal a regulatory mechanism of complement in controlling adaptive thermogenesis and suggest that targeting the C3aR/C5aR pathways may represent a therapeutic strategy in treating obesity-related metabolic diseases.

Targets of Immune Escape Mechanisms in Cancer: Basis for Development and Evolution of Cancer Immune Checkpoint Inhibitors.

Immune checkpoint blockade (ICB) has emerged as a novel therapeutic tool for cancer therapy in the last decade. Unfortunately, a small number of patients benefit from approved immune checkpoint inhibitors (ICIs). Therefore, multiple studies are being conducted to find new ICIs and combination strategies to improve the current ICIs. In this review, we discuss some approved immune checkpoints, such as PD-L1, PD-1, and CTLA-4, and also highlight newer emerging ICIs. For instance, HLA-E, overexpressed by tumor cells, represents an immune-suppressive feature by binding CD94/NKG2A, on NK and T cells. NKG2A blockade recruits CD8+ T cells and activates NK cells to decrease the tumor burden. NKG2D acts as an NK cell activating receptor that can also be a potential ICI. The adenosine A2A and A2B receptors, CD47-SIRPα, TIM-3, LAG-3, TIGIT, and VISTA are targets that also contribute to cancer immunoresistance and have been considered for clinical trials. Their antitumor immunosuppressive functions can be used to develop blocking antibodies. PARPs, mARTs, and B7-H3 are also other potential targets for immunosuppression. Additionally, miRNA, mRNA, and CRISPR-Cas9-mediated immunotherapeutic approaches are being investigated with great interest. Pre-clinical and clinical studies project these targets as potential immunotherapeutic candidates in different cancer types for their robust antitumor modulation.

The ectonucleotidases CD39 and CD73 on T cells: The new pillar of hematological malignancy.

Hematological malignancy develops and applies various mechanisms to induce immune escape, in part through an immunosuppressive microenvironment. Adenosine is an immunosuppressive metabolite produced at high levels within the tumor microenvironment (TME). Adenosine signaling through the A2A receptor expressed on immune cells, such as T cells, potently dampens immune responses. Extracellular adenosine generated by ectonucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5'-nucleotidase (CD73) molecules is a newly recognized 'immune checkpoint mediator' and leads to the identification of immunosuppressive adenosine as an essential regulator in hematological malignancies. In this Review, we provide an overview of the detailed distribution and function of CD39 and CD73 ectoenzymes in the TME and the effects of CD39 and CD73 inhibition on preclinical hematological malignancy data, which provides insights into the potential clinical applications for immunotherapy.

Phenotypic Assessment of Pathogenic Variants in GNAO1 and Response to Caffeine in C. elegans Models of the Disease.

De novo mutations affecting the G protein α o subunit (Gαo)-encoding gene (GNAO1) cause childhood-onset developmental delay, hyperkinetic movement disorders, and epilepsy. Recently, we established Caenorhabditis elegans as an informative experimental model for deciphering pathogenic mechanisms associated with GNAO1 defects and identifying new therapies. In this study, we generated two additional gene-edited strains that harbor pathogenic variants which affect residues Glu246 and Arg209-two mutational hotspots in Gαo. In line with previous findings, biallelic changes displayed a variable hypomorphic effect on Gαo-mediated signaling that led to the excessive release of neurotransmitters by different classes of neurons, which, in turn, caused hyperactive egg laying and locomotion. Of note, heterozygous variants showed a cell-specific dominant-negative behavior, which was strictly dependent on the affected residue. As with previously generated mutants (S47G and A221D), caffeine was effective in attenuating the hyperkinetic behavior of R209H and E246K animals, indicating that its efficacy is mutation-independent. Conversely, istradefylline, a selective adenosine A2A receptor antagonist, was effective in R209H animals but not in E246K worms, suggesting that caffeine acts through both adenosine receptor-dependent and receptor-independent mechanisms. Overall, our findings provide new insights into disease mechanisms and further support the potential efficacy of caffeine in controlling dyskinesia associated with pathogenic GNAO1 mutations.

Enhancement of adenosine A2A signaling improves dopamine D2 receptor antagonist-induced dyskinesia via β-arrestin signaling.

Repeated administration of dopamine D2 receptor (D2R) antagonists, which is the treatment for psychosis, often causes tardive dyskinesia (TD). Despite notable clinical demand, effective treatment for TD has not been established yet. The neural mechanism involving the hyperinhibition of indirect pathway medium spiny neurons (iMSNs) in the striatum is considered one of the main causes of TD. In this study, we focused on adenosine A2A receptors (A2ARs) expressed in iMSNs and investigated whether pharmacological activation of A2ARs improves dyskinetic symptoms in a TD mouse model. A 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) and decreased the number of c-Fos+/ppENK+ iMSNs in the dorsal striatum. Haloperidol-induced VCMs were reduced by acute intraperitoneal administration of an A2AR agonist, CGS 21680A. Consistently, haloperidol-induced VCMs and decrease in the number of c-Fos+/ppENK+ iMSNs were also mitigated by intrastriatal injection of CGS 21680A. The effects of intrastriatal CGS 21680A were not observed when it was concomitantly administered with a β-arrestin inhibitor, barbadin. Finally, intrastriatal injection of an arrestin-biased D2R agonist, UNC9994, also inhibited haloperidol-induced VCMs. These results suggest that A2AR agonists mitigate TD symptoms by activating striatal iMSNs via β-arrestin signaling.