Adenosine A2A Receptor Knockout Mice Research Articles

Cardiovascular and respiratory evaluation in adenosine A2A receptor knockout mice submitted to short-term sustained hypoxia.

Sustained hypoxia (SH) in mice induces changes in the respiratory pattern and increase in the parasympathetic tone to the heart. Among adenosine G-protein-coupled receptors (GPCRs), the A2A receptors are especially important in mediating adenosine actions during hypoxia due to their expression in neurons involved with the generation and modulation of the autonomic and respiratory functions. Herein, we performed an in vivo evaluation of the baseline cardiovascular and respiratory parameters and their changes in response to SH in knockout mice for A2A receptors (A2A KO). SH produced similar and significant reductions in mean arterial pressure and heart rate in both wild-type (WT) and A2A KO mice when compared to their respective normoxic controls. Mice from WT and A2A KO groups submitted to normoxia or SH presented similar cardiovascular responses to peripheral chemoreflex activation (KCN). Under normoxic conditions A2A KO mice presented a respiratory frequency (fR ) significantly higher in relation to the WT group, which was reduced in response to SH. These data show that the lack of adenosine A2A receptors in mice does not affect the cardiovascular parameters and the autonomic responses to chemoreflex activation in control (normoxia) and SH mice. We conclude that the A2A receptors play a major role in the control of respiratory frequency and in the tachypnoeic response to SH in mice. NEW FINDINGS: What is the central question of this study? Are cardiovascular and respiratory parameters and their changes in response to sustained hypoxia (SH) altered in adenosine A2A receptor knockout mice? What is the main finding and its importance? Cardiovascular parameters and their changes in response to SH were not altered in A2A KO mice. The respiratory frequency in A2A KO was higher than in WT mice. In response to SH the respiratory frequency increased in WT, while it was reduced in A2A KO mice. A2A receptors play a major role in the modulation of respiratory frequency and in the tachypnoeic response to SH in mice.

A2AR-mediated lymphangiogenesis via VEGFR2 signaling prevents salt-sensitive hypertension.

Excess dietary sodium intake and retention lead to hypertension. Impaired dermal lymphangiogenesis and lymphatic dysfunction-mediated sodium and fluid imbalance are pathological mechanisms. The adenosine A2A receptor (A2AR) is expressed in lymphatic endothelial cells (LECs), while the roles and mechanisms of LEC-A2AR in skin lymphangiogenesis during salt-induced hypertension are not clear. The expression of LEC-A2AR correlated with lymphatic vessel density in both high-salt diet (HSD)-induced hypertensive mice and hypertensive patients. Lymphatic endothelial cell-specific A2AR knockout mice fed HSD exhibited 17 ± 2% increase in blood pressure and 17 ± 3% increase in Na+ content associated with decreased lymphatic density (-19 ± 2%) compared with HSD-WT mice. A2AR activation by agonist CGS21680 increased lymphatic capillary density and decreased blood pressure in HSD-WT mice. Furthermore, this A2AR agonist activated MSK1 directly to promote VEGFR2 activation and endocytosis independently of VEGF as assessed by phosphoprotein profiling and immunoprecipitation assays in LECs. VEGFR2 kinase activity inhibitor fruquintinib or VEGFR2 knockout in LECs but not VEGF-neutralizing antibody bevacizumab suppressed A2AR activation-mediated decrease in blood pressure. Immunostaining revealed phosphorylated VEGFR2 and MSK1 expression in the LECs were positively correlated with skin lymphatic vessel density and A2AR level in hypertensive patients. The study highlights a novel A2AR-mediated VEGF-independent activation of VEGFR2 signaling in dermal lymphangiogenesis and sodium balance, which might be a potential therapeutic target in salt-sensitive hypertension.

Adenosine A2A receptor activation reduces chondrocyte senescence.

Osteoarthritis (OA) pathogenesis is associated with reduced chondrocyte homeostasis and increased levels of cartilage cellular senescence. Chondrosenescence is the development of cartilage senescence that increases with aging joints and disrupts chondrocyte homeostasis and is associated with OA. Adenosine A2A receptor (A2AR) activation in cartilage via intra-articular injection of liposomal A2AR agonist, liposomal-CGS21680, leads to cartilage regeneration in vivo and chondrocyte homeostasis. A2AR knockout mice develop early OA isolated chondrocytes demonstrate upregulated expression of cellular senescence and aging-associated genes. Based on these observations, we hypothesized that A2AR activation would ameliorate cartilage senescence. We found that A2AR stimulation of chondrocytes reduced beta-galactosidase staining and regulated levels and cell localization of common senescence mediators p21 and p16 in vitro in the human TC28a2 chondrocyte cell line. In vivo analysis similarly showed A2AR activation reduced nuclear p21 and p16 in obesity-induced OA mice injected with liposomal-CGS21680 and increased nuclear p21 and p16 in A2AR knockout mouse chondrocytes compared to wild-type mice. A2AR agonism also increased activity of the chondrocyte Sirt1/AMPK energy-sensing pathway by enhancing nuclear Sirt1 localization and upregulating T172-phosphorylated (active) AMPK protein levels. Lastly, A2AR activation in TC28a2 and primary human chondrocytes reduced wild-type p53 and concomitantly increased p53 alternative splicing leading to increase in an anti-senescent p53 variant, Δ133p53α. The results reported here indicate that A2AR signaling promotes chondrocyte homeostasis in vitro and reduces OA cartilage development in vivo by reducing chondrocyte senescence.

The mGlu5 Receptor Protomer-Mediated Dopamine D2 Receptor Trans-Inhibition Is Dependent on the Adenosine A2A Receptor Protomer: Implications for Parkinson’s Disease

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.

Defibrotide Inhibits Antiphospholipid Antibody-Mediated Neutrophil Extracellular Trap Formation and Venous Thrombosis.

Defibrotide is a heterogenous mixture of polyanionic oligonucleotides currently approved for treatment of transplant-associated venoocclusive disease. While defibrotide has a known role in limiting endothelial cell activation, some studies have also demonstrated anti-leukocyte properties. In a recent study, we found that neutrophil extracellular traps (NETs) play a role in the thrombotic complications of antiphospholipid syndrome (APS). In the present study, we investigated the hypothesis that defibrotide might act to mitigate APS-relevant NET formation in vitro and in mouse models. We used in vitro assays and a mouse model to determine the mechanisms by which defibrotide inhibits NET formation and venous thrombosis in APS. At doses ranging from 1 to 10 μg/ml, defibrotide significantly suppressed NET formation from control neutrophils stimulated with IgG isolated from patients with APS. Defibrotide increased levels of intracellular cyclic AMP in neutrophils, and its suppressive effects on NET formation were mitigated by blocking adenosine A2A receptor or by inhibiting the cyclic AMP-dependent kinase protein kinase A. Defibrotide at doses ranging from 15 to 150 mg/kg/day inhibited NET formation and venous thrombosis in a model of antiphospholipid antibody-accelerated thrombosis-an effect that was reduced in adenosine A2A receptor-knockout mice. This study is the first to demonstrate mechanisms by which defibrotide counteracts neutrophil-mediated thrombotic inflammation inherent to APS.

Caffeine Functions by Inhibiting Dorsal and Ventral Hippocampal Adenosine 2A Receptors to Modulate Memory and Anxiety, Respectively.

As a nonspecific antagonist of the adenosine A2A receptor (A2AR), caffeine enhances learning and improves memory impairment. Simultaneously, the consumption of caffeine correlates with a feeling of anxiety. The hippocampus is functionally differentiated along its dorsal/ventral axis and plays a crucial role both in memory and anxiety. Whether caffeine exerts its regulation by inhibiting A2ARs in different subregions of the hippocampus is still unknown. In the present study, we found that after chronic intake of drinking water containing caffeine (1 g/L, 3 weeks), mice exhibited aggravated anxiety-like behavior and enhanced memory function. Tissue-specific, functional disruption of dorsal hippocampal A2ARs by the CRE-LoxP system prevented the memory-enhancing effect of caffeine, while selective disruption of ventral hippocampal A2ARs blocked the impact of caffeine on anxiety. These results, together with the enhanced memory of dorsal hippocampus A2AR knockout mice and greater anxiety-like behavior of ventral hippocampus A2AR knockout mice without caffeine, indicates a dissociation between the roles of ventral and dorsal hippocampal A2A receptors in caffeine’s effects on anxiety-like and memory-related behavioral measures, respectively. Furthermore, optogenetic activation of dorsal or ventral hippocampal A2ARs reversed the behavioral alterations caused by drinking caffeine, leading to impaired memory or decreased anxiety-like behaviors, respectively. Taken together, our findings suggest that the memory- and anxiety-enhancing effects of caffeine are related to the differential effects of inhibiting A2ARs in the dorsal and ventral hippocampus, respectively.

Adenosine A2A Receptor in Bone Marrow-Derived Cells Mediated Macrophages M2 Polarization via PPARγ-P65 Pathway in Chronic Hypoperfusion Situation.

Background: The role of adenosine A2A receptor (A2AR) in the ischemic white matter damage induced by chronic cerebral hypoperfusion remains obscure. Here we investigated the role of A2AR in the process of macrophage polarizations in the white matter damage induced by chronic cerebral hypoperfusion and explored the involved signaling pathways.Methods: We combined mouse model and macrophage cell line for our study. White matter lesions were induced in A2AR knockout mice, wild-type mice, and chimeric mice generated by bone marrow cells transplantation through bilateral common carotid artery stenosis. Microglial/macrophage polarization in the corpus callosum was detected by immunofluorescence. For the cell line experiments, RAW264.7 macrophages were treated with the A2AR agonist CHS21680 or A2AR antagonist SCH58261 for 30 min and cultured under low-glucose and hypoxic conditions. Macrophage polarization was examined by immunofluorescence. The expression of peroxisome proliferator activated receptor gamma (PPARγ) and transcription factor P65 was examined by western blotting and real-time polymerase chain reaction (RT-PCR). Inflammatory cytokine factors were assessed by enzyme-linked immunosorbent assay (ELISA) and RT-PCR.Results: Both global A2AR knockout and inactivation of A2AR in bone marrow-derived cells enhanced M1 marker expression in chronic ischemic white matter lesions. Under low-glucose and hypoxic conditions, CGS21680 treatment promoted macrophage M2 polarization, increased the expression of PPARγ, P65, and interleukin-10 (IL-10) and suppressed the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). The CGS21680-induced upregulation of P65 and IL-10 was abolished in macrophages upon PPARγ knockdown. The downregulation of TNF-α and IL-1β by CGS21680 was less affected by PPARγ knockdown.Conclusions: In the cerebral hypoperfusion induced white matter damage, A2AR signaling in bone marrow-derived cells induces macrophage M2 polarization and increases the expression of the anti-inflammatory factor IL-10 via the PPARγ-P65 pathway, both of which might explain its neuroprotective effect.

A Novel Role of A2AR in the Maintenance of Intestinal Barrier Function of Enteric Glia from Hypoxia-Induced Injury by Combining with mGluR5.

During acute intestinal ischemia reperfusion (IR) injury, the intestinal epithelial barrier (IEB) function is often disrupted. Enteric glial cells (EGCs) play an important role in maintaining the integrity of IEB functions. However, how EGCs regulate IEB function under IR stimulation is unknown. The present study reveals that the adenosine A2A receptor (A2AR) is important for mediating the barrier-modulating roles of EGCs. A2AR knockout (KO) experiments revealed more serious intestinal injury in A2AR KO mice than in WT mice after IR stimulation. Moreover, A2AR expression was significantly increased in WT mice when challenged by IR. To further investigate the role of A2AR in IEB, we established an in vitro EGC-Caco-2 co-culture system. Hypoxia stimulation was used to mimic the process of in vivo IR. Treating EGCs with the CGS21680 A2AR agonist attenuated hypoxia-induced intestinal epithelium damage through up-regulating ZO-1 and occludin expression in cocultured Caco-2 monolayers. Furthermore, we showed that A2AR and metabotropic glutamate receptor 5 (mGluR5) combine to activate the PKCα-dependent pathway in conditions of hypoxia. This study shows, for the first time, that hypoxia induces A2AR-mGluR5 interaction in EGCs to protect IEB function via the PKCα pathway.

Extracellular adenosine and slow-wave sleep are increased after ablation of nucleus accumbens core astrocytes and neurons in mice

Sleep and wakefulness are controlled by a wide range of neuronal populations in the mammalian brain. Activation of adenosine A2A receptor (A2AR)-expressing neurons in the nucleus accumbens (NAc) core promotes slow-wave sleep (SWS). The neuronal mechanism by which activation of NAc A2AR neurons induces SWS, however, is unknown. We hypothesized that the ability of NAc activation to induce sleep is mediated by the classic somnogen adenosine, which can be formed by various processes in all types of cells. Here, to investigate whether astrocytes are involved in the ability of the NAc to regulate SWS, we ablated glial fibrillary acidic protein (GFAP)-positive cells in the NAc core of mice by virus-mediated expression of diphtheria toxin (DT) receptors and intraperitoneal administration of DT. Analysis of electroencephalogram and electromyogram recordings of DT-treated wild-type mice revealed that SWS was remarkably increased at 1 week after DT treatment, whereas sleep-wake behavior was unchanged in DT-treated A2AR knockout mice. Cell ablation was associated with an increased number of GFAP-positive cells and activation of microglia in the NAc. In-vivo microdialysis revealed significantly increased levels of extracellular adenosine in the NAc at 1 week after DT treatment. Our findings suggest that elevated adenosine levels in the NAc core promote SWS by acting on A2ARs and provide the first evidence that adenosine is an endogenous candidate for activating NAc A2AR neurons that have the ability to induce SWS.

Adenosine A2A receptor mediates hypnotic effects of ethanol in mice

Ethanol has extensive effects on sleep and daytime alertness, causing premature disability and death. Adenosine, as a potent sleep-promoting substance, is involved in many cellular and behavioral responses to ethanol. However, the mechanisms of hypnotic effects of ethanol remain unclear. In this study, we investigated the role of adenosine in ethanol-induced sleep using C57BL/6Slac mice, adenosine A2A receptor (A2AR) knockout mice, and their wild-type littermates. The results showed that intraperitoneal injection of ethanol (3.0 g/kg) at 21:00 decreased the latency to non-rapid eye movement (NREM) sleep and increased the duration of NREM sleep for 5 h. Ethanol dose-dependently increased NREM sleep, which was consistent with decreases in wakefulness in C57BL/6Slac mice compared with their own control. Caffeine (5, 10, or 15 mg/kg), a nonspecific adenosine receptor antagonist, dose-dependently and at high doses completely blocked ethanol-induced NREM sleep when administered 30 min prior to (but not after) ethanol injection. Moreover, ethanol-induced NREM sleep was completely abolished in A2AR knockout mice compared with wild-type mice. These findings strongly indicate that A2AR is a key receptor for the hypnotic effects of ethanol, and pretreatment of caffeine might be a strategy to counter the hypnotic effects of ethanol.

Perivascular AQP4 dysregulation in the hippocampal CA1 area after traumatic brain injury is alleviated by adenosine A2A receptor inactivation

Traumatic brain injury (TBI) can induce cognitive dysfunction due to the regional accumulation of hyperphosphorylated tau protein (p-tau). However, the factors that cause p-tau to concentrate in specific brain regions remain unclear. Here, we show that AQP4 polarization in the perivascular astrocytic end feet was impaired after TBI, which was most prominent in the ipsilateral brain tissue surrounding the directly impacted region and the contralateral hippocampal CA1 area and was accompanied by increased local p-tau, changes in dendritic spine density and morphology, and upregulation of the adenosine A2A receptor (A2AR). The critical role of the A2AR signaling in these pathological changes was confirmed by alleviation of the impairment of AQP4 polarity and accumulation of p-tau in the contralateral CA1 area in A2AR knockout mice. Given that p-tau can be released to the extracellular space and that the astroglial water transport via AQP4 is involved in tau clearance from the brain interstitium, our results suggest that regional disruption of AQP4 polarity following TBI may reduce the clearance of the toxic interstitial solutes such as p-tau and lead to changes in dendritic spine density and morphology. This may explain why TBI patients are more vulnerable to cognitive dysfunction.

Adenosine A2A receptor deficiency attenuates the somnogenic effect of prostaglandin D2 in mice.

Prostaglandin D2 (PGD2) is one of the most potent endogenous sleep promoting substances. PGD2 activates the PGD2 receptor (DPR) and increases the extracellular level of adenosine in wild-type (WT) mice but not DPR knockout (KO) mice, suggesting that PGD2-induced sleep is DPR-dependent, and adenosine may be the signaling molecule that mediates the somnogenic effect of PGD2. The aim of this study was to determine the involvement of the adenosine A2A receptor (A2AR) in PGD2-induced sleep. We infused PGD2 into the lateral ventricle of WT and A2AR KO mice between 20:00 and 2:00 for 6 h, and electroencephalograms and electromyograms were simultaneously recorded. In WT mice, PGD2 infusion dose-dependently increased non-rapid eye movement (non-REM, NREM) sleep, which was 139.1%, 145.0% and 202.7% as large as that of vehicle-treated mice at doses of 10, 20 and 50 pmol/min, respectively. PGD2 infusion at doses of 20 and 50 pmol/min also increased REM sleep during the 6-h PGD2 infusion and 4-h post-dosing periods in WT mice to 148.9% and 166.7%, respectively. In A2AR KO mice, however, PGD2 infusion at 10 pmol/min did not change the sleep profile, whereas higher doses at 20 and 50 pmol/min increased the NREM sleep during the 6-h PGD2 infusion to 117.5% and 155.6%, respectively, but did not change the sleep in the post-dosing period. Moreover, PGD2 infusion at 50 pmol/min significantly increased the episode number in both genotypes but only enhanced the episode duration in WT mice. The results demonstrate that PGD2-induced sleep in mice is mediated by both adenosine A2AR-dependent and -independent systems.

Absence of the Adenosine A2A Receptor Confers Pulmonary Arterial Hypertension Through RhoA/ROCK Signaling Pathway in Mice.

Numerous evidence suggests that RhoA/Rho kinase (ROCK) signaling pathway plays an important role in the pathogenesis of pulmonary arterial hypertension (PAH), but little is known about its effects on the development of PAH in mice with absence of the adenosine A2A receptor (A2AR). Eight A2AR knockout (KO) and 8 wild-type mice were used. Morphometric analysis of pulmonary arterioles included right ventricle/left ventricle plus ventricular septum (Fulton index), vessel wall thickness/total vascular diameter (WT%), and vessel wall area/total vascular area (WA%). The expression of RhoA and ROCK1 mRNA was determined by real-time polymerase chain reaction. The expression of RhoA, ROCK1, and phosphorylation of myosin phosphatase target subunit 1 proteins in pulmonary tissue was tested by Western blot. The position of ROCK1 protein was evaluated by immunohistochemistry. Compared with wild-type mice, A2AR KO mice displayed (1) increased Fulton index, WT%, and WA% (P < 0.01); (2) increased mRNA expression of RhoA and ROCK1 (each P < 0.05); (3) increased protein expression of RhoA, ROCK1, and phosphorylation of myosin phosphatase target subunit 1 (each P < 0.01); (4) increased location of ROCK1 protein in endothelial and smooth muscle cells of pulmonary artery, bronchial, and alveolar epithelial cells. Activation of RhoA/ROCK signaling pathway may cause pulmonary vascular constriction, pulmonary artery remodeling, and PAH in adenosine A2A receptor KO mice.

Choosing voluntary exercise over sucrose consumption depends upon dopamine transmission: effects of haloperidol in wild type and adenosine A₂AKO mice.

Mesolimbic dopamine (DA) regulates behavioral activation and effort-related decision-making in motivated behaviors. Mesolimbic DA D2 receptors are co-localized with adenosine A2A receptors, and they interact in an antagonistic manner. A T-maze task was developed to assess dopaminergic involvement in preference between a reinforcer that involves vigorous voluntary activity (running wheel) and a reinforcer that requires minimal behavioral activation (sucrose pellets). Haloperidol (D2 antagonist) was administered to adenosine A2A receptor knockout (A2AKO) and wild-type (WT) littermate controls to assess the involvement of these two receptors in the selection of running wheel activity versus sucrose consumption. Under control conditions, mice spent more time running and less time eating. In WT mice, haloperidol reduced time running but actually increased time-consuming sucrose. However, A2AKO mice did not show the haloperidol-induced shift from running wheel activity to sucrose intake. Prefeeding reduced sucrose consumption in the T-maze in both strains, indicating that this paradigm is sensitive to motivational devaluation. Haloperidol increased c-Fos immunoreactivity in anterior cingulate cortex (ACg) and nucleus accumbens (Acb) core of WT but not KO mice. These results indicate that after DA antagonism, the preference for vigorous physical activity is reduced, while palatable food selection increases. Adenosine A2A receptor deletion provides resistance to these effects of D2 receptor antagonism. These two receptors in Acb core and ACg seem to be involved in the regulation of the intrinsic reinforcing characteristics of voluntary exercise but not in the regulation of the primary reinforcing characteristics of palatable sedentary reinforcers.

Bone regeneration in critical bone defects using three-dimensionally printed β-tricalcium phosphate/hydroxyapatite scaffolds is enhanced by coating scaffolds with either dipyridamole or BMP-2.

Bone defects resulting from trauma or infection need timely and effective treatments to restore damaged bone. Using specialized three-dimensional (3D) printing technology we have created custom 3D scaffolds of hydroxyapatite (HA)/beta-tri-calcium phosphate (β-TCP) to promote bone repair. To further enhance bone regeneration we have coated the scaffolds with dipyridamole, an agent that increases local adenosine levels by blocking cellular uptake of adenosine. Nearly 15% HA:85% β-TCP scaffolds were designed using Robocad software, fabricated using a 3D Robocasting system, and sintered at 1100°C for 4 h. Scaffolds were coated with BMP-2 (200 ng mL-1 ), dypiridamole 100 µM or saline and implanted in C57B6 and adenosine A2A receptor knockout (A2AKO) mice with 3 mm cranial critical bone defects for 2-8 weeks. Dipyridamole release from scaffold was assayed spectrophotometrically. MicroCT and histological analysis were performed. Micro-computed tomography (microCT) showed significant bone formation and remodeling in HA/β-TCP-dipyridamole and HA/β-TCP-BMP-2 scaffolds when compared to scaffolds immersed in vehicle at 2, 4, and 8 weeks (n = 5 per group; p ≤ 0.05, p ≤ 0.05, and p ≤ 0.01, respectively). Histological analysis showed increased bone formation and a trend toward increased remodeling in HA/β-TCP- dipyridamole and HA/β-TCP-BMP-2 scaffolds. Coating scaffolds with dipyridamole did not enhance bone regeneration in A2AKO mice. In conclusion, scaffolds printed with HA/β-TCP promote bone regeneration in critical bone defects and coating these scaffolds with agents that stimulate A2A receptors and growth factors can further enhance bone regeneration. These coated scaffolds may be very useful for treating critical bone defects due to trauma, infection or other causes. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 366-375, 2017.

Adenosine A2A Receptor Deficiency Alleviates Blast-Induced Cognitive Dysfunction

Traumatic brain injury (TBI), particularly explosive blast-induced TBI (bTBI), has become the most prevalent injury among military personnel. The disruption of cognitive function is one of the most serious consequences of bTBI because its long-lasting effects prevent survivors fulfilling their active duty and resuming normal civilian life. However, the mechanisms are poorly understood and there is no treatment available. This study investigated the effects of adenosine A2A receptor (A2AR) on bTBI-induced cognitive deficit, and explored the underlying mechanisms. After being subjected to moderate whole-body blast injury, mice lacking the A2AR (A2AR knockout (KO)) showed less severity and shorter duration of impaired spatial reference memory and working memory than wild-type mice did. In addition, bTBI-induced cortical and hippocampal lesions, as well as proinflammatory cytokine expression, glutamate release, edema, cell loss, and gliosis in both early and prolonged phases of the injury, were significantly attenuated in A2AR KO mice. The results suggest that early injury and chronic neuropathological damages are important mechanisms of bTBI-induced cognitive impairment, and that the impairment can be attenuated by preventing A2AR activation. These findings suggest that A2AR antagonism is a potential therapeutic strategy for mild-to-moderate bTBI and consequent cognitive impairment.

The vesicular monoamine transporter (VMAT-2) inhibitor tetrabenazine induces tremulous jaw movements in rodents: Implications for pharmacological models of parkinsonian tremor

Tetrabenazine (TBZ) is a reversible inhibitor of vesicular monoamine storage that is used to treat Huntington’s disease. TBZ preferentially depletes striatal dopamine (DA), and patients being treated with TBZ often experience parkinsonian side effects. The present studies were conducted to investigate the ability of TBZ to induce tremulous jaw movements (TJMs), which are a rodent model of parkinsonian tremor, and to determine if interference with adenosine A2A receptor transmission can attenuate TJMs and other motor effects of TBZ. In rats, TBZ (0.25–2.0mg/kg) significantly induced TJMs, which primarily occurred in the 3.0–7.5-Hz frequency range. The adenosine A2A antagonist MSX-3 (1.25–10.0mg/kg) significantly attenuated the TJMs induced by 2.0mg/kg TBZ in rats, and also significantly reduced the display of catalepsy and locomotor suppression induced by TBZ. In mice, TBZ (2.5–10.0mg/kg) dose dependently induced TJMs, and adenosine A2A receptor knockout mice showed significantly fewer TJMs compared to wild-type controls. MSX-3 (2.5–10.0mg/kg) also significantly reduced TBZ-induced TJMs in CD1 mice. To provide a cellular marker of these pharmacological conditions, we examined c-Fos expression in the ventrolateral neostriatum (VLS). TBZ (2.0mg/kg) significantly increased the number of c-Fos-positive cells in the VLS, which is indicative of reduced DA D2 receptor transmission, and 10.0mg/kg MSX-3 significantly attenuated the TBZ-induced c-Fos expression. These results indicate that TBZ induces tremor as measured by the TJM model, and that pharmacological antagonism and genetic deletion of adenosine A2A receptors are capable of attenuating this oral tremor.

Adenosine A2A receptor: a target for regulating renal interstitial fibrosis in obstructive nephropathy.

Renal interstitial fibrosis (RIF) is the common pathological process of chronic kidney diseases leading inevitably to renal function deterioration. RIF and its preceding epithelial-mesenchymal transition (EMT) are commonly triggered by an early occurring renal inflammation. However, an effective approach to prevent EMT and RIF is still lacking and of urgent need. Recently, the adenosine A2A receptor (A2AR) emerges as a novel inflammation regulator, therefore manipulation of A2AR may suppress the EMT process and as such protect against RIF. To test this hypothesis we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR knockout mice and their wild-type littermates, combined with the intervention of a selective A2AR agonist, CGS 21680. On days 3, 7 and 14 post-UUO we evaluated the effects of A2AR manipulation on the molecular pathological progresses of RIF, including the cellular component of interstitial infiltration, expression of profibrotic factors, cellular biomarkers of EMT, and collagen deposition of extracellular matrix. Our data demonstrated that activation of A2AR significantly suppressed the deposition of collagen types I and III, reduced the infiltration of CD4+ T lymphocytes, and attenuated the expression of TGF-β1 and ROCK1, which in turn inhibited and postponed the EMT progress. Conversely, genetic inactivation of A2AR exacerbated the aforementioned pathological processes of UUO-induced RIF. Together, activation of A2AR effectively alleviated EMT and RIF in mice, suggesting A2AR as a potential therapeutic target for the treatment of RIF.

The effects of adenosine A2A receptor knockout on renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction

Adenosine A2A receptor (A2AR) plays an important regulatory role in the processes of inflammation and fibrosis. However, it is unknown whether A2AR can mediate renal interstitial fibrosis (RIF). To evaluate the effect of genetic A2AR knockout (KO) on the pathological progress of RIF, we applied a unilateral ureteral obstruction (UUO) model of RIF on A2AR KO mice and their wild-type (WT) littermates. Renal pathological assessment was performed at different post-UUO stages using hematoxylin and eosin (H&E) and Masson's trichrome staining as well as quantitative morphological analysis. Our data demonstrated that: (i) the extent of RIF was determined by the development of UUO in a time-dependent manner; (ii) A2AR KO exacerbated the pathological progress of RIF in mice at the early post-UUO stage, i.e. day 3 and day 7; (iii) the profibrotic effect of A2AR KO was prominent until the late post-UUO stage, i.e. day 14, at which RIF reached a similar severity level in A2AR KO and WT mice. Our findings revealed that A2AR KO significantly exacerbated the progression of UUO-induced RIF in mice, prominently at the initial stage.

Conditional neural knockout of the adenosine A2A receptor and pharmacological A2A antagonism reduce pilocarpine-induced tremulous jaw movements: Studies with a mouse model of parkinsonian tremor

Tremulous jaw movements are rapid vertical deflections of the lower jaw that resemble chewing but are not directed at any particular stimulus. In rats, tremulous jaw movements can be induced by a number of conditions that parallel those seen in human parkinsonism, including dopamine depletion, dopamine antagonism, and cholinomimetic drugs. Moreover, tremulous jaw movements in rats can be attenuated using antiparkinsonian agents such as L-DOPA, dopamine agonists, muscarinic antagonists, and adenosine A2A antagonists. In the present studies, a mouse model of tremulous jaw movements was established to investigate the effects of adenosine A2A antagonism, and a conditional neuronal knockout of adenosine A2A receptors, on cholinomimetic-induced tremulous jaw movements. The muscarinic agonist pilocarpine significantly induced tremulous jaw movements in a dose-dependent manner (0.25–1.0mg/kg IP). These movements occurred largely in the 3–7.5Hz local frequency range. Administration of the adenosine A2A antagonist MSX-3 (2.5–10.0mg/kg IP) significantly attenuated pilocarpine-induced tremulous jaw movements. Furthermore, adenosine A2A receptor knockout mice showed a significant reduction in pilocarpine-induced tremulous jaw movements compared to littermate controls. These results demonstrate the feasibility of using the tremulous jaw movement model in mice, and indicate that adenosine A2A receptor antagonism and deletion are capable of reducing cholinomimetic-induced tremulous jaw movements in mice. Future studies should investigate the effects of additional genetic manipulations using the mouse tremulous jaw movement model.