Up-regulation Of Adenosine A2A Receptors Research Articles

Neuronal A2A receptor exacerbates synapse loss and memory deficits in APP/PS1 mice.

Early pathological upregulation of adenosine A2A receptors (A2ARs), one of the caffeine targets, by neurons is thought to be involved in the development of synaptic and memory deficits in Alzheimer's disease (AD) but mechanisms remain ill-defined. To tackle this question, we promoted a neuronal upregulation of A2AR in the hippocampus of APP/PS1 mice developing AD-like amyloidogenesis. Our findings revealed that the early upregulation of A2AR in the presence of an ongoing amyloid pathology exacerbates memory impairments of APP/PS1 mice. These behavioural changes were not linked to major change in the development of amyloid pathology but rather associated with increased phosphorylated tau at neuritic plaques. Moreover, proteomic and transcriptomic analyses coupled with quantitative immunofluorescence studies indicated that neuronal upregulation of the receptor promoted both neuronal and non-neuronal autonomous alterations, i.e. enhanced neuroinflammatory response but also loss of excitatory synapses and impaired neuronal mitochondrial function, presumably accounting for the detrimental effect on memory. Overall, our results provide compelling evidence that neuronal A2AR dysfunction, as seen in the brain of patients, contributes to amyloid-related pathogenesis and underscores the potential of A2AR as a relevant therapeutic target for mitigating cognitive impairments in this neurodegenerative disorder.

Choroid plexus-selective inactivation of adenosine A2A receptors protects against T cell infiltration and experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is one of the most common autoimmune disorders characterized by the infiltration of immune cells into the brain and demyelination. The unwanted immunosuppressive side effect of therapeutically successful natalizumab led us to focus on the choroid plexus (CP), a key site for the first wave of immune cell infiltration in experimental autoimmune encephalomyelitis (EAE), for the control of immune cells trafficking. Adenosine A2A receptor (A2AR) is emerging as a potential pharmacological target to control EAE pathogenesis. However, the cellular basis for the A2AR-mediated protection remains undetermined.MethodsIn the EAE model, we assessed A2AR expression and leukocyte trafficking determinants in the CP by immunohistochemistry and qPCR analyses. We determined the effect of the A2AR antagonist KW6002 treatment at days 8–12 or 8–14 post-immunization on T cell infiltration across the CP and EAE pathology. We determined the critical role of the CP-A2AR on T cell infiltration and EAE pathology by focal knock-down of CP-A2AR via intracerebroventricular injection of CRE-TAT recombinase into the A2ARflox/flox mice. In the cultured CP epithelium, we also evaluated the effect of overexpression of A2ARs or the A2AR agonist CGS21680 treatment on the CP permeability and lymphocytes migration.ResultsWe found the specific upregulation of A2AR in the CP associated with enhanced CP gateway activity peaked at day 12 post-immunization in EAE mice. Furthermore, the KW6002 treatment at days 8–12 or 8–14 post-immunization reduced T cell trafficking across the CP and attenuated EAE pathology. Importantly, focal CP-A2AR knock-down attenuated the pathogenic infiltration of Th17+ cells across the CP via inhibiting the CCR6–CCL20 axis through NFκB/STAT3 pathway and protected against EAE pathology. Lastly, activation of A2AR in the cultured epithelium by A2AR overexpression or CGS21680 treatment increased the permeability of the CP epithelium and facilitated lymphocytes migration.ConclusionThese findings define the CP niche as one of the primary sites of A2AR action, whereby A2AR antagonists confer protection against EAE pathology. Thus, pharmacological targeting of the CP-A2AR represents a novel therapeutic strategy for MS by controlling immune cell trafficking across CP.

Association Between Adenosine A2A Receptors and Connexin 43 Regulates Hemichannels Activity and ATP Release in Astrocytes Exposed to Amyloid-β Peptides.

Increasing evidence implicates astrocytes and the associated purinergic modulation in Alzheimer's disease (AD), characterized by cognitive deficits involving the extracellular amyloid-β peptides (Aβ) accumulation. Aβ can affect astrocytic gliotransmitters release, namely ATP, which is rapidly metabolized into adenosine by ecto-5'-nucleotidase, CD73, resulting in adenosine A2A receptors (A2AR) activation that bolsters neurodegeneration. AD's brains exhibit an upregulation of A2AR and of connexin 43 (Cx43), which in astrocytes forms hemichannels that can mediate ATP release. However, a coupling between astrocytic A2AR and Cx43 remains to be established. This was now investigated using astrocytic primary cultures exposed to Aβ1-42 peptides. Aβ triggered ATP release through Cx43 hemichannels, a process blocked by A2AR antagonists and mimicked by selective A2AR activation. A2AR directly regulated hemichannels activity and prevented Cx43 upregulation and phosphorylation observed in Aβ1-42-exposed astrocytes. Moreover, a proximity ligand assay revealed a physical association between astrocytic A2AR and Cx43. Finally, the blockade of CD73-mediated extracellular formation of ATP-derived adenosine prevented the Aβ-induced increase of Cx43 hemichannel activity and of ATP release. Overall, the data identify a feed-forward loop involving astrocytic A2AR and Cx43 hemichannels, whereby A2AR increase Cx43 hemichannel activity leading to increased ATP release, which is converted into adenosine by CD73, sustaining the increased astrocytic A2AR activity in AD-like conditions.

Adenosine A2a Receptors Improve Hypoxic Pulmonary Arterial Hypertension Via Mitochondrial ATP-sensitive Potassium Channels

IntroductionThis study is aimed to explore the effects of Adenosine A2a receptors (A2aR) on hypoxia-induced pulmonary hypertension (HPH) via mitochondrial ATP-sensitive potassium channels (MitoKATP) in vivo and in vitro.Material and methodsUsing wild-type (WT) and A2aR-deficient (A2aR-/-) mice; hypoxic pulmonary artery smooth muscle cells (PASMCs) were induced by a 24-hours hypoxia exposure. Mice and PASMCs were treated with the A2aR agonist CGS21680, MitoKATP blocker 5-hydroxydecanoic acid sodium salt (5HD), or MitoKATP agonist diazoxide. Mitochondrial morphology was observed by electron microscopy. The mitochondrial membrane potential (Δψm); invasive hemodynamic parameters; right ventricular (RV) hypertrophy index; pulmonary arterial remodeling index; proliferative and apoptotic indexes; protein expression levels of A2aR, Bax, Bcl-2, and Caspase-9; and release of cytochrome C from the mitochondria to the cytoplasm were measured.ResultsIn vitro, hypoxia induced the opening of MitoKATP. The up-regulation of A2aR reduced the opening of MitoKATP, and the blocking of MitoKATP or activating A2aR promoted mitochondria-dependent apoptosis of PASMCs. In vivo, compared with WT mice, A2aR-/- mice displayed increased RV systolic pressure, RV hypertrophy index, and pulmonary arterial remodeling index. The expression levels of Bax, cytochrome C, and Caspase-9 were higher and Bcl-2 expression was lower in A2aR-/- mice than in WT mice. CGS21680 could reverse hypoxia-induced hemodynamic changes, RV hypertrophy, and pulmonary arterial remodeling as well as abnormal proliferation and apoptosis resistance in WT mice with pulmonary hypertension (PH).ConclusionsA2aR induced the mitochondrial-dependent apoptosis pathway and inhibited PASMC proliferation by blocking MitoKATP, thereby inhibiting pulmonary vascular structural remodeling and reducing PH.

Upregulation of adenosine A2A receptor and downregulation of GLT1 is associated with neuronal cell death in Rasmussen's encephalitis.

Rasmussen encephalitis (RE) is a severe pediatric inflammatory brain disease characterized by unilateral inflammation and atrophy of the cerebral cortex, drug-resistant focal epilepsy and progressive neurological and cognitive deterioration. The etiology and pathogenesis of RE remain unclear. Our previous results demonstrated that the adenosine A1 receptor (A1R) and the major adenosine-removing enzyme adenosine kinase play an important role in the etiology of RE. Because the downstream pathways of inhibitory A1R signaling are modulated by stimulatory A2AR signaling, which by itself controls neuro-inflammation, glial activation and glial glutamate homeostasis through interaction with glutamate transporter GLT-1, we hypothesized that maladaptive changes in adenosine A2A receptor (A2AR) expression are associated with RE. We used immunohistochemistry and Western blot analysis to examine the expression of A2ARs, glutamate transporter-I (GLT-1) and the apoptotic marker Bcl-2 in surgically resected cortical specimens from RE patients (n=18) in comparison with control cortical tissue. In lesions of the RE specimen we found upregulation of A2ARs, downregulation of GLT-1 and increased apoptosis of both neurons and astroglia. Double staining revealed colocalization of A2ARs and Bcl-2 in RE lesions. These results suggest that maladaptive changes in A2AR expression are associated with a decrease in GLT-I expression as a possible precipitator for apoptotic cell loss in RE. Because A2AR antagonists are already under clinical evaluation for Parkinson's disease, the A2AR might likewise be a tractable target for the treatment of RE.

Spironolactone inhibits endothelial-mesenchymal transition via the adenosine A2A receptor to reduce cardiorenal fibrosis in rats

AimsThe mechanisms underlying cardiorenal syndromes are complex and not fully understood; Fibrosis seems to be a primary driver of the diseases' pathophysiology. Spironolactone can reduce cardiac or renal fibrosis by inhibiting endothelial-mesenchymal transition (EndMT). Spironolactone protection may rely on activation of adenosine receptors, but the role of the adenosine A2A receptor (A2AR) is unclear. We hypothesize that spironolactone may modulate A2AR to suppress EndMT and reduce cardiorenal remodeling. Main methodsA model of renal injury followed by heart failure was established by subcutaneous administration of isoprenaline (Iso) to rats. Assessment of cardiac and renal function, fibrosis, EndMT markers, adenosine and A2AR expression was performed. TGF-β was used to induce EndMT in primary human umbilical vein endothelial cells (HUVECs). Rats or cells were divided into four groups: those that treated with spironolactone alone or in combination with A2AR antagonist ZM241385 or neither, and compared to normal controls. Key findingsIsoprenaline-treated rats exhibited cardiac and renal fibrosis, impaired cardiac and renal function, enhanced EndMT, and lower A2AR expression. Spironolactone significantly up-regulated A2AR expression and inhibited EndMT in vivo and in vitro. Moreover, spironolactone improved cardiorenal remodeling and reduced dysfunction. These changes were exacerbated by administration of ZM241385. Together, these findings show that spironolactone up-regulated A2AR to reduce EndMT and ameliorate cardiorenal fibrosis. SignificanceThe anti-fibrotic effects of spironolactone may partly depend on the up-regulation of A2AR, and that A2AR might be a potential therapeutic target for the treatment of cardiorenal syndrome.

Adenosine A2A receptor involves in neuroinflammation-mediated cognitive decline through activating microglia under acute hypobaric hypoxia

Hypobaric hypoxia (HH) at high altitudes leads to a wide range of cognitive impairments which can handicap human normal activities and performances. However, the underlying mechanism is still unclear. Adenosine A2A receptors (A2ARs) of the brain are pivotal to synaptic plasticity and cognition. Besides, insult-induced up-regulation of A2AR regulates neuroinflammation and therefore induces brain damages in various neuropathological processes. The present study was designed to determine whether A2AR-mediate neuroinflammation involves in cognitive impairments under acute HH. A2AR knock-out and wild-type male mice were exposed to a simulated altitude of 8000 m for 7 consecutive days in a hypobaric chamber and simultaneously received behavioral tests including Morris water maze test and open filed test. A2AR expression, the activation of microglia and the production of TNF-α were evaluated in the hippocampus by immunohistochemistry and ELISA, respectively. Behavioral tests showed that acute HH exposure caused the dysfunction of spatial memory and mood, while genetic inactivation of A2AR attenuated the impairment of spatial memory but not that of mood. Double-labeled immunofluorescence showed that A2ARs were mainly expressed on microglia and up-regulated in the hippocampus of acute HH model mice. Acute HH also induced the accumulation of microglia and increased production of TNF-α in the hippocampus, which could be markedly inhibited by A2AR inactivation. These findings indicate that microglia-mediated neuroinflammation triggered by A2AR activation involves in acute HH-induced spatial memory impairment and that A2AR could be a new target for the pharmacotherapy of cognitive dysfunction at high altitudes.

Adenosine receptors as markers of brain iron deficiency: Implications for Restless Legs Syndrome

Deficits of sensorimotor integration with periodic limb movements during sleep (PLMS) and hyperarousal and sleep disturbances in Restless Legs Syndrome (RLS) constitute two pathophysiologically distinct but interrelated clinical phenomena, which seem to depend mostly on alterations in dopaminergic and glutamatergic neurotransmission, respectively. Brain iron deficiency is considered as a main pathogenetic mechanism in RLS. Rodents with brain iron deficiency represent a valuable pathophysiological model of RLS, although they do not display motor disturbances. Nevertheless, they develop the main neurochemical dopaminergic changes found in RLS, such as decrease in striatal dopamine D2 receptor density. On the other hand, brain iron deficient mice exhibit the characteristic pattern of hyperarousal in RLS, providing a tool to find the link between brain iron deficiency and sleep disturbances in RLS. The present study provides evidence for a role of the endogenous sleep-promoting factor adenosine. Three different experimental preparations, long-term (22 weeks) severe or moderate iron-deficient (ID) diets (3- or 7-ppm iron diet) in mice and short-term (3 weeks) severe ID diet (3-ppm iron diet) in rats, demonstrated a significant downregulation (Western blotting in mouse and radioligand binding saturation experiments in rat brain tissue) of adenosine A1 receptors (A1R) in the cortex and striatum, concomitant to striatal D2R downregulation. On the other hand, the previously reported upregulation of adenosine A2A receptors (A2AR) was only observed with severe ID in both mice and rats. The results suggest a key role for A1R downregulation in the PLMS and hyperarousal in RLS.

Dual roles of the adenosine A2a receptor in autoimmune neuroinflammation.

BackgroundConditions of inflammatory tissue distress are associated with high extracellular levels of adenosine, due to increased adenosine triphosphate (ATP) degradation upon cellular stress or the release of extracellular ATP upon cell death, which can be degraded to adenosine by membrane-bound ecto-enzymes like CD39 and CD73. Adenosine is recognised to mediate anti-inflammatory effects via the adenosine A2a receptor (A2aR), as shown in experimental models of arthritis. Here, using pharmacological interventions and genetic inactivation, we investigated the roles of A2aR in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).MethodsWe used two independent mouse EAE variants, i.e. active immunization in C57BL/6 with myelin oligodendrocyte glycoprotein (MOG)35-55 or transfer-EAE by proteolipid protein (PLP)139-155-stimulated T lymphocytes and EAE in mice treated with A2aR-agonist CGS21680 at different stages of disease course and in mice lacking A2aR (A2aR−/−) compared to direct wild-type littermates. In EAE, we analysed myelin-specific proliferation and cytokine synthesis ex vivo, as well as inflammation and demyelination by immunohistochemistry. In vitro, we investigated the effect of A2aR on migration of CD4+ T cells, macrophages and microglia, as well as the impact of A2aR on phagocytosis of macrophages and microglia. Statistical tests were Mann-Whitney U and Student’s t test.ResultsWe found an upregulation of A2aR in the central nervous system (CNS) in EAE, predominantly detected on T cells and macrophages/microglia within the inflamed tissue. Preventive EAE treatment with A2aR-specific agonist inhibited myelin-specific T cell proliferation ex vivo and ameliorated disease, while application of the same agonist after disease onset exacerbated non-remitting EAE progression and resulted in more severe tissue destruction. Accordingly, A2aR-deficient mice showed accelerated and exacerbated disease manifestation with increased frequencies of IFN-γ-, IL-17- and GM-CSF-producing CD4+ T helper cells and higher numbers of inflammatory lesions in the early stage. However, EAE quickly ameliorated and myelin debris accumulation was lower in A2aR−/− mice. In vitro, activation of A2aR inhibited phagocytosis of myelin by macrophages and primary microglia as well as migration of CD4+ T cells, macrophages and primary microglia.ConclusionsA2aR activation exerts a complex pattern in chronic autoimmune neurodegeneration: while providing anti-inflammatory effects on T cells and thus protection at early stages, A2aR seems to play a detrimental role during later stages of disease and may thus contribute to sustained tissue damage within the inflamed CNS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0512-z) contains supplementary material, which is available to authorized users.

Upregulation of adenosine A2A receptors induced by atypical antipsychotics and its correlation with sensory gating in schizophrenia patients

Sensory gating deficits have been found in patients with schizophrenia and their unaffected relatives. However, the underlying neurobiological mechanism of this deficit remains unclear. Pre-clinical studies have implicated adenosine in sensory gating deficits in schizophrenia. Therefore, the current study investigated a possible relationship between peripheral adenosine A2A receptor (ADORA2A) and sensory gating indices (P50 measures) in medication-free schizophrenia (n=31) and healthy (n=21) groups. The effects of six-week antipsychotic treatment were examined. At baseline, schizophrenia patients showed impaired sensory gating compared to healthy controls. However, there was no significant difference in ADORA2A gene expression among groups. In addition, ADORA2A expression was not correlated with sensory gating at any time point. Following treatment, we found a significant upregulation of ADORA2A expression. Intriguingly, we observed a significant positive association between ADORA2A upregulation and baseline P50 amplitudes in the schizophrenia group. A main finding of the current pilot study is the upregulation of ADORA2A expression following treatment with antipsychotics. In addition, this upregulation was predicted by baseline P50 amplitude, an observation that awaits replication in an expanded sample.

Chronic hypoxia upregulates adenosine 2a receptor expression in chromaffin cells via hypoxia inducible factor-2α: Role in modulating secretion

Catecholamine (CAT) release from chromaffin tissue plays an essential role in the fetus which develops in a low O 2 environment (hypoxia). To address molecular mechanisms regulating CAT secretion in low O 2, we exposed a fetal chromaffin-derived cell line (MAH cells) to chronic hypoxia (CHox; 2% O 2, 24 h) and assessed gene expression using microarrays, quantitative RT-PCR, and western blot. CHox caused a dramatic ∼12× upregulation of adenosine A2a receptor (A2aR) mRNA, an effect critically dependent upon hypoxia-inducible factor (HIF)-2α which bound the promoter of the A2aR gene. In amperometric studies, acute hypoxia and high K + (30 mM) evoked quantal CAT secretion that was enhanced after CHox, and further potentiated during simultaneous A2aR activation by adenosine. A2aR activation also enhanced stimulus-induced rise in intracellular Ca 2+ in control, but not HIF-2α-deficient, MAH cells. Thus, A2aR, adenosine, and HIF-2α are key contributors to the potentiation of CAT secretion in developing chromaffin cells during chronic hypoxia.

Abnormal calcium handling in atrial fibrillation is linked to up-regulation of adenosine A2A receptors

Atrial fibrillation (AF) is associated with abnormal sarcoplasmic reticulum (SR) calcium release, which is promoted by adenosine A(2A) receptor (A(2A)R) activation. Here, we tested the hypothesis that abnormal calcium release in AF is linked to A(2A)R remodelling. Western blotting and quantitative real-time PCR were used to determine A(2A)R mRNA and protein levels in right atrial samples from patients with and without AF. Effects of A(2A)R activation on calcium handling were assessed with patch-clamp technique and confocal calcium imaging. A(2A)R mRNA levels and functional A(2A)Rs were moderately up-regulated in patients with atrial dilation and markedly up-regulated in those with AF. Accordingly, A(2A)R stimulation significantly increased ryanodine receptor phosphorylation in AF patients, and spontaneous calcium waves increased moderately in myocytes from patients with atrial dilation and strongly in patients with AF (2.2 ± 2.1 to 14.3 ± 8.8 min(-1), n = 6, P = 0.01). Moreover, the high baseline level of calcium waves in AF was reduced by A(2A)R antagonists (3.5 ± 2.0 to 1.3 ± 1.3 min(-1), n = 6, P = 0.007) or adenosine deaminase (1.7 ± 1.5 to 0.5 ± 0.6 min(-1), n = 10, P = 0.02) suggesting that A(2A)Rs are activated by endogenous adenosine. Indeed, intracellular perfusion with adenosine significantly increased the calcium wave frequency (1.1 ± 0.8 to 8.2 ± 3.3 min(-1), n = 8), whereas adenosine removal from the cytosol decreased it (2.1 ± 0.9 to 0.3 ± 0.3 min(-1), n = 8, P = 0.04). Atrial fibrillation patients show increased A(2A)R expression that may account for the high baseline level of spontaneous SR calcium release seen in myocytes from these patients, and the ability of A(2A)R antagonists to reduce this abnormal calcium release points to the A(2A)R as a novel molecular target in AF.

Increased expression of adenosine A2A receptors in patients with spontaneous and head-up-tilt-induced syncope

Adenosine may play a role in the triggering of neurocardiogenic syncope, but no information on adenosine receptors is available at the present time. The purpose of this study was to investigate whether adenosine A2A receptors expression is altered in patients with neurocardiogenic syncope. Adenosine plasma levels (APLs), the expression of A2A receptors, were measured (mean +/- standard error of the mean) during tilt testing. Expression of receptors was assessed on mononuclear cells using a selective receptor ligand. At baseline, the APLs of 16 patients with a positive test were higher than those of 17 patients with a negative test and of those of a control group (2.10 +/- 0.30 vs. 0.40 +/- 0.05 and 0.41 +/- 0.06 muM, respectively; P <.0001). The number of receptors was higher in patients tested positive than in patients tested negative or in the control group (122 +/- 10 vs. 38 +/- 4 and 44 +/- 4 fmol/g of proteins, respectively; P <.0001). No difference was found in the affinity or synthesis among the three groups. This study showed an increased number and an up-regulation of adenosine A2A receptors in patients with spontaneous syncope and a positive head-up tilt, which in the context of high APLs may play a role in the recurrence of syncopal episodes.