Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

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.

Adenosine A2A receptor blockade prevents cisplatin-induced impairments in neurogenesis and cognitive function.

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A2A receptor (A2AR) and its downstream effectors, cAMP and CREB, by cisplatin in the adult mouse hippocampus, a critical brain structure for learning and memory. Notably, A2AR inhibition by the Food and Drug Administration-approved A2AR antagonist KW-6002 prevented cisplatin-induced impairments in neural progenitor proliferation and dendrite morphogenesis of adult-born neurons, while improving memory and anxiety-like behavior, without affecting tumor growth or cisplatin's antitumor activity. Collectively, our study identifies A2AR signaling as a key pathway that can be therapeutically targeted to prevent cisplatin-induced cognitive impairments.

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.

Repeated binge-like eating episodes in female rats alter adenosine A2A and dopamine D2 receptor genes regulation in the brain reward system.

OBJECTIVE: Binge-eating disorder is an eating disorder characterized by recurrent binge-eating episodes, during which individuals consume excessive amounts of highly palatable food (HPF) in a short time. This study investigates the intricate relationship between repeated binge-eating episode and the transcriptional regulation of two key genes, adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R), in selected brain regions of rats. METHOD: Binge-like eating behavior on HPF was induced through the combination of food restrictions and frustration stress (15 min exposure to HPF without access to it) in female rats, compared to control rats subjected to only restriction or only stress or none of these two conditions. After chronic binge-eating episodes, nucleic acids were extracted from different brain regions, and gene expression levels were assessed through real-time quantitative PCR. The methylation pattern on genes' promoters was investigated using pyrosequencing. RESULTS: The analysis revealed A2AAR upregulation in the amygdala and in the ventral tegmental area (VTA), and D2R downregulation in the nucleus accumbens in binge-eating rats. Concurrently, site-specific DNA methylation alterations at gene promoters were identified in the VTA for A2AAR and in the amygdala and caudate putamen for D2R. DISCUSSION: The alterations on A2AAR and D2R genes regulation highlight the significance of epigenetic mechanisms in the etiology of binge-eating behavior, and underscore the potential for targeted therapeutic interventions, to prevent the development of this maladaptive feeding behavior. These findings provide valuable insights for future research in the field of eating disorders. PUBLIC SIGNIFICANCE: Using an animal model with face, construct, and predictive validity, in which cycles of food restriction and frustration stress evoke binge-eating behavior, we highlight the significance of epigenetic mechanisms on adenosine A2A receptor (A2AAR) and dopamine D2 receptor (D2R) genes regulation. They could represent new potential targets for the pharmacological management of eating disorders characterized by this maladaptive feeding behavior.

Adenosine A2A Receptor Blockade Provides More Effective Benefits at the Onset Rather than after Overt Neurodegeneration in a Rat Model of Parkinson's Disease.

Adenosine A2A receptor (A2AR) antagonists are the leading nondopaminergic therapy to manage Parkinson's disease (PD) since they afford both motor benefits and neuroprotection. PD begins with a synaptic dysfunction and damage in the striatum evolving to an overt neuronal damage of dopaminergic neurons in the substantia nigra. We tested if A2AR antagonists are equally effective in controlling these two degenerative processes. We used a slow intracerebroventricular infusion of the toxin MPP+ in male rats for 15 days, which caused an initial loss of synaptic markers in the striatum within 10 days, followed by a neuronal loss in the substantia nigra within 30 days. Interestingly, the initial loss of striatal nerve terminals involved a loss of both dopaminergic and glutamatergic synaptic markers, while GABAergic markers were preserved. The daily administration of the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) in the first 10 days after MPP+ infusion markedly attenuated both the initial loss of striatal synaptic markers and the subsequent loss of nigra dopaminergic neurons. Strikingly, the administration of SCH58261 (0.1 mg/kg, i.p. for 10 days) starting 20 days after MPP+ infusion was less efficacious to attenuate the loss of nigra dopaminergic neurons. This prominent A2AR-mediated control of synaptotoxicity was directly confirmed by showing that the MPTP-induced dysfunction (MTT assay) and damage (lactate dehydrogenase release assay) of striatal synaptosomes were prevented by 50 nM SCH58261. This suggests that A2AR antagonists may be more effective to counteract the onset rather than the evolution of PD pathology.

Genetic Polymorphisms of P2RX7 but Not of ADORA2A Are Associated with the Severity of SARS-CoV-2 Infection.

SARS-CoV-2 infection ranges from mild to severe presentations, according to the intensity of the aberrant inflammatory response. Purinergic receptors dually control the inflammatory response: while adenosine A2A receptors (A2ARs) are anti-inflammatory, ATP P2X7 receptors (P2X7Rs) exert pro-inflammatory effects. The aim of this study was to assess if there were differences in allelic and genotypic frequencies of a loss-of-function SNP of ADORA2A (rs2298383) and a gain-of-function single nucleotide polymorphism (SNP) of P2RX7 (rs208294) in the severity of SARS-CoV-2-associated infection. Fifty-five individuals were enrolled and categorized according to the severity of the infection. Endpoint genotyping was performed in blood cells to screen for both SNPs. The TT genotype (vs. CT + CC) and the T allele (vs. C allele) of P2RX7 SNP were found to be associated with more severe forms of COVID-19, whereas the association between ADORA2A SNP and the severity of infection was not significantly different. The T allele of P2RX7 SNP was more frequent in people with more than one comorbidity and with cardiovascular conditions and was associated with colorectal cancer. Our findings suggest a more prominent role of P2X7R rather than of A2AR polymorphisms in SARS-CoV-2 infection, although larger population-based studies should be performed to validate our conclusions.

Radiosynthesis and In Vitro Evaluation of [11C]tozadenant as Adenosine A2A Receptor Radioligand.

Tozadenant (4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide) is a highly selective adenosine A2A receptor (A2AR) antagonist and a promising lead structure for the development of A2AR-selective positron emission tomography (PET) probes. Although several 18F-labelled tozadenant derivatives showed favorable in vitro properties, recent in vivo PET studies observed poor brain penetration and lower specific binding than anticipated from the in vitro data. While these findings might be attributable to the structural modification associated with 18F-labelling, they could also reflect inherent properties of the parent compound. However, PET studies with radioisotopologues of tozadenant to evaluate its cerebral pharmacokinetics and brain distribution are still lacking. In the present work, we applied N-Boc-O-desmethyltozadenant as a suitable precursor for the preparation of [O-methyl-11C]tozadenant ([11C]tozadenant) by O-methylation with [11C]methyl iodide followed by acidic deprotection. This approach afforded [11C]tozadenant in radiochemical yields of 18 ± 2%, with molar activities of 50-60 GBq/µmol (1300-1600 mCi/µmol) and radiochemical purities of 95 ± 3%. In addition, in vitro autoradiography in pig and rat brain slices demonstrated the expected striatal accumulation pattern and confirmed the A2AR specificity of the radioligand, making it a promising tool for in vivo PET studies on the cerebral pharmacokinetics and brain distribution of tozadenant.

Neural representation and modulation of volitional motivation in response to escalating efforts.

Task-dependent volitional control of the selected neural activity in the cortex is critical to neuroprosthetic learning to achieve reliable and robust control of the external device. The volitional control of neural activity is driven by a motivational factor (volitional motivation), which directly reinforces the target neurons via real-time biofeedback. However, in the absence of motor behaviour, how do we evaluate volitional motivation? Here, we defined the criterion (ΔF/F) of the calcium fluorescence signal in a volitionally controlled neural task, then escalated the efforts by progressively increasing the number of reaching the criterion or holding time after reaching the criterion. We devised calcium-based progressive threshold-crossing events (termed 'Calcium PTE') and calcium-based progressive threshold-crossing holding-time (termed 'Calcium PTH') for quantitative assessment of volitional motivation in response to progressively escalating efforts. Furthermore, we used this novel neural representation of volitional motivation to explore the neural circuit and neuromodulator bases for volitional motivation. As with behavioural motivation, chemogenetic activation and pharmacological blockade of the striatopallidal pathway decreased and increased, respectively, the breakpoints of the 'Calcium PTE' and 'Calcium PTH' in response to escalating efforts. Furthermore, volitional and behavioural motivation shared similar dopamine dynamics in the nucleus accumbens in response to trial-by-trial escalating efforts. In general, the development of a neural representation of volitional motivation may open a new avenue for smooth and effective control of brain-machine interface tasks. KEY POINTS: Volitional motivation is quantitatively evaluated by M1 neural activity in response to progressively escalating volitional efforts. The striatopallidal pathway and adenosine A2A receptor modulate volitional motivation in response to escalating efforts. Dopamine dynamics encode prediction signal for reward in response to repeated escalating efforts during motor and volitional conditioning. Mice learn to modulate neural activity to compensate for repeated escalating efforts in volitional control.

Role of adenosine A2A receptors in the loss of consciousness induced by propofol anesthesia.

The mechanism of propofol-anesthesia-induced loss of consciousness (LOC) remains largely unknown. We speculated that the adenosine A2A receptor serves as a vital molecular target in regulating LOC states under propofol anesthesia. c-Fos staining helped observe the changes in the neuronal activity in the nucleus accumbens (NAc). Initially, the adenosine signals in the NAc were measured under propofol anesthesia using fiber photometry recordings. Then, behavior tests and electrophysiological recordings were used to verify the effect of systemic A2A R agonist or antagonist treatment on propofol anesthesia. Next, the microinjection technique was used to clarify the role of the NAc A2A R under propofol anesthesia. Fiber photometry recordings were applied to assess the effect of A2A R agonist or antagonist systemic treatment on adenosine signal alterations in the NAc during propofol anesthesia. Then, as the GABAergic neurons are the main neurons in the NAc, we further measured the neuronal activity of GABAergic neurons. In our study, propofol anesthesia enhanced the neuronal activity in the NAc, and the adenosine signals were increased in the NAc. SCH58261 reduced the LOC time and sedative depth, while CGS21680 increased those via intraperitoneal injection. Additionally, CGS21680 increased the changes in delta, theta, alpha, beta, and low-gamma oscillations in the NAc. Moreover, microinjection of SCH58261 significantly shortened the LOC time, whereas microinjection of CGS21680 into the NAc significantly prolonged the LOC duration. The results illustrated that after A2A R agonist administration, the level of extracellular adenosine signals in the NAc was decreased and the neuronal activity of GABAergic neurons was enhanced, whereas after A2A R antagonist administration via intraperitoneal injection, the opposite occurred. This study reveals the vital role of the A2A R in propofol-induced LOC and that the A2A R could affect the maintenance of propofol anesthesia.

Adenosine induces IL-31 secretion by T-helper 2 cells: Implication for the effect of adenosine on atopic dermatitis and its therapeutic strategy.

Interleukin (IL)-31 is a recently-identified cytokine with a well-defined role in the pathogenesis of pruritus. Previously, we reported that adenosine upregulates IL-17A secretion by T-helper (Th)17 cells; however, the effect of adenosine on T cell subsets other than Th17 remains unclear. In this report, we show that adenosine upregulated production of IL-31 by cluster of differentiation (CD)4+ T cells. IL-31 was also upregulated by administration of an adenosine A2a receptor (A2aR) agonist (PSB0777), and adenosine-mediated IL-31 production was inhibited by an A2aR antagonist (istradefylline). Production of Th2-related cytokines (IL-4, IL-10, and IL-13) by CD4+ T cells showed the same tendency. Immune subset analyses revealed that adenosine upregulated IL-31 secretion by CD4+ chemokine receptor 3high T cells, and that Th2 cells differentiated from naïve CD4+ T cells. Administration of istradefylline to mice with atopic dermatitis suppressed the symptoms, suggesting that A2aR antagonists are an effective treatment for inflammatory dermatitis. Taken together, the results indicate that adenosine upregulates secretion of Th2-related cytokines by effector T cells in the skin, thereby triggering atopic dermatitis and associated pruritus.

In silico identification of A1 agonists and A2a inhibitors in pain based on molecular docking strategies and dynamics simulations.

Most recently, the adenosine is considered as one of the most promising targets for treating pain, with few side effects. It exists in the central nervous system, and plays a key role in nociceptive afferent pathway. It is reported that the A1 receptor (A1R) could inhibit Ca2+ channels to reduce the pain like analgesic mechanism of morphine. And, A2a receptor (A2aR) was reported to enhance the accumulation of AMP (cAMP) and released peptides from sensory neurons, resulting in constitutive activation of pain. Much evidence showed that A1R and A2aR could be served as the interesting targets for the treatment of pain. Herein, virtual screening was utilized to identify the small molecule compounds towards A1R and A2aR, and top six molecules were considered as candidates via amber scores. The molecular dynamic (MD) simulations and molecular mechanics/generalized born surface area (MM/GBSA) were employed to further analyze the affinity and binding stability of the six molecules towards A1R and A2aR. Moreover, energy decomposition analysis showed significant residues in A1R and A2aR, including His1383, Phe1276, and Glu1277. It provided basics for discovery of novel agonists and antagonists. Finally, the agonists of A1R (ZINC19943625, ZINC13555217, and ZINC04698406) and inhibitors of A2aR (ZINC19370372, ZINC20176051, and ZINC57263068) were successfully recognized. Taken together, our discovered small molecules may serve as the promising candidate agents for future pain research.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

Feedback facilitation by adenosine A2A receptors of ATP release from mouse hippocampal nerve terminals.

The adenosine modulation system is mostly composed by inhibitory A1 receptors (A1R) and the less abundant facilitatory A2A receptors (A2AR), the latter selectively engaged at high frequency stimulation associated with synaptic plasticity processes in the hippocampus. A2AR are activated by adenosine originated from extracellular ATP through ecto-5'-nucleotidase or CD73-mediated catabolism. Using hippocampal synaptosomes, we now investigated how adenosine receptors modulate the synaptic release of ATP. The A2AR agonist CGS21680 (10-100 nM) enhanced the K+-evoked release of ATP, whereas both SCH58261 and the CD73 inhibitor α,ß-methylene ADP (100 µM) decreased ATP release; all these effects were abolished in forebrain A2AR knockout mice. The A1R agonist CPA (10-100 nM) inhibited ATP release, whereas the A1R antagonist DPCPX (100 nM) was devoid of effects. The presence of SCH58261 potentiated CPA-mediated ATP release and uncovered a facilitatory effect of DPCPX. Overall, these findings indicate that ATP release is predominantly controlled by A2AR, which are involved in an apparent feedback loop of A2AR-mediated increased ATP release together with dampening of A1R-mediated inhibition. This study is a tribute to María Teresa Miras-Portugal.

Preservation of vascular endothelial glycocalyx and barrier by activation of adenosine A2A receptor (A2AR) improved renal dysfunction in cirrhotic rats.

Cirrhosis-related hepatic and renal endothelial dysfunction is characterized by macrophage-endothelium adhesion-mediated inflammation, glycocalyx/barrier damage, and impaired vasodilation. Activation of adenosine A2A receptor (A2AR) protects cirrhotic rats from impairment of hepatic microcirculation post hepatectomy. This study evaluates the effects of A2AR activation on the cirrhosis-related hepatic and renal endothelial dysfunction in biliary cirrhotic rats receiving two weeks of A2AR agonist PSB0777 [bile duct ligated (BDL)+PSB0777] treatment. Endothelial dysfunction in cirrhotic liver, renal vessels, and kidney is characterized by downregulation of the A2AR expressions, decreased vascular endothelial vasodilatory (p-eNOS)/anti-inflammatory (IL-10/IL-10R)/barrier [VE-cadherin (CDH5) and ß-catenin (CTNNB1)]/glycocalyx [syndecan-1 (SDC1) and hyaluronan synthase-2 (HAS2)] markers, and increased leukocyte-endothelium adhesion molecules (F4/80, CD68, ICAM-1, and VCAM-1). In BDL rats, PSB0777 treatment improves hepatic and renal endothelial dysfunction, ameliorates portal hypertension, and attenuates renal hypoperfusion by restoring of the vascular endothelial anti-inflammatory, barrier, glycocalyx markers and vasodilatory response as well as inhibiting the leukocyte-endothelium adhesion. In an in vitro study, conditioned medium (CM) of bone marrow-derived macrophage (BMDM) of BDL rats [BMDM-CM (BDL)] induced barrier/glycocalyx damage, which was reversed by the PSB0777 pre-treatment. The A2AR agonist is a potential agent that can simultaneously correct cirrhosis-related hepatic and renal endothelial dysfunction, portal hypertension, renal hypoperfusion, and renal dysfunction.

The adenosine A2A receptor antagonist/inverse agonist, KW-6356 enhances the anti-parkinsonian activity of L-DOPA with a low risk of dyskinesia in MPTP-treated common marmosets.

The adenosine A2A receptor antagonist/inverse agonist, KW-6356 has been shown to be effective in Parkinson's disease (PD) patients as monotherapy and as an adjunct therapy to L-3,4-dihydroxyphenylalanine (L-DOPA)/decarboxylase inhibitor. However, the effects of KW-6356 combined with L-DOPA on anti-parkinsonian activity and established dyskinesia has not been investigated in preclinical experiments. We examined the effects of combination of KW-6356 with L-DOPA in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated common marmosets. Oral administration of KW-6356 (1 mg/kg) enhanced the anti-parkinsonian activities of various doses of L-DOPA (2.5-10 mg/kg). In MPTP-treated common marmosets primed with L-DOPA to show dyskinesia, KW-6356 (1 mg/kg) also enhanced the anti-parkinsonian activities of various doses of L-DOPA (1.25-10 mg/kg) but not dyskinesia. Chronic co-administration of KW-6356 (1 mg/kg) with a low dose of L-DOPA (2.5 mg/kg) for 21 days increased the degree of dyskinesia induced by the low dose of L-DOPA, but the amplitude of dyskinesia induced by combined administration of KW-6356 (1 mg/kg) with L-DOPA (2.5 mg/kg) was lower than that induced by an optimal dose of L-DOPA (10 mg/kg). These results suggest that KW-6356 can be used to potentiate the effects of a wide range of L-DOPA doses with a low risk of dyskinesia for the treatment of PD.

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.

α-Synuclein Selectively Impairs Motor Sequence Learning and Value Sensitivity: Reversal by the Adenosine A2A Receptor Antagonists.

Parkinson's disease (PD) is characterized pathologically by alpha-synuclein (α-Syn) aggregates and clinically by the motor as well as cognitive deficits, including impairments in sequence learning and habit learning. Using intracerebral injection of WT and A53T mutant α-Syn fibrils, we investigate the behavioral mechanism of α-Syn for procedure-learning deficit in PD by critically determining the α-Syn-induced effects on model-based goal-directed behavior, model-free (probability-based) habit learning, and hierarchically organized sequence learning. 1) Contrary to the widely held view of habit-learning deficit in early PD, α-Syn aggregates in the dorsomedial striatum (DMS) and dorsolateral striatum (DLS) did not affect acquisition of habit learning, but selectively impaired goal-directed behavior with reduced value sensitivity. 2) α-Syn in the DLS (but not DMS) and SNc selectively impaired the sequence learning by affecting sequence initiation with the reduced first-step accuracy. 3) Adenosine A2A receptor (A2AR) antagonist KW6002 selectively improved sequence learning by preferentially improving sequence initiation and shift of sequence learning as well as behavioral reactivity. These findings established a casual role of α-Syn in the SN-DLS pathway in sequence-learning deficit and DMS α-Syn in goal-directed behavior deficit and suggest a novel therapeutic strategy to improve sequence-learning deficit in PD with enhanced sequence initiation by A2AR antagonists.

Biglycan Involvement in Heart Fibrosis: Modulation of Adenosine 2A Receptor Improves Damage in Immortalized Cardiac Fibroblasts.

Cardiac fibrosis is a common pathological feature of different cardiovascular diseases, characterized by the aberrant deposition of extracellular matrix (ECM) proteins in the cardiac interstitium, myofibroblast differentiation and increased fibrillar collagen deposition stimulated by transforming growth factor (TGF)-ß activation. Biglycan (BGN), a small leucine-rich proteoglycan (SLRPG) integrated within the ECM, plays a key role in matrix assembly and the phenotypic control of cardiac fibroblasts. Moreover, BGN is critically involved in pathological cardiac remodeling through TGF-ß binding, thus causing myofibroblast differentiation and proliferation. Adenosine receptors (ARs), and in particular A2AR, may play a key role in stimulating fibrotic damage through collagen production/deposition, as a consequence of cyclic AMP (cAMP) and AKT activation. For this reason, A2AR modulation could be a useful tool to manage cardiac fibrosis in order to reduce fibrotic scar deposition in heart tissue. Therefore, the aim of the present study was to investigate the possible crosstalk between A2AR and BGN modulation in an in vitro model of TGF-ß-induced fibrosis. Immortalized human cardiac fibroblasts (IM-HCF) were stimulated with TGF-ß at the concentration of 10 ng/mL for 24 h to induce a fibrotic phenotype. After applying the TGF-ß stimulus, cells were treated with two different A2AR antagonists, Istradefylline and ZM241385, for an additional 24 h, at the concentration of 10 µM and 1 µM, respectively. Both A2AR antagonists were able to regulate the oxidative stress induced by TGF-ß through intracellular reactive oxygen species (ROS) reduction in IM-HCFs. Moreover, collagen1a1, MMPs 3/9, BGN, caspase-1 and IL-1ß gene expression was markedly decreased following A2AR antagonist treatment in TGF-ß-challenged human fibroblasts. The results obtained for collagen1a1, SMAD3, α-SMA and BGN were also confirmed when protein expression was evaluated; phospho-Akt protein levels were also reduced following Istradefylline and ZM241385 use, thus suggesting that collagen production involves AKT recruited by the A2AR. These results suggest that A2AR modulation might be an effective therapeutic option to reduce the fibrotic processes involved in heart pathological remodeling.

α-Synuclein Aggregates in the Nigro-Striatal Dopaminergic Pathway Impair Fine Movement: Partial Reversal by the Adenosine A2A Receptor Antagonist.

Parkinson's disease (PD) is characterized pathologically by abnormal aggregation of alpha-synuclein (α-Syn) in the brain and clinically by fine movement deficits at the early stage, but the roles of α-Syn and associated neural circuits and neuromodulator bases in the development of fine movement deficits in PD are poorly understood, in part due to the lack of appropriate behavioral testing paradigms and PD models without motor confounding effects. Here, we coupled two unique behavioral paradigms with two PD models to reveal the following: (i) Focally injecting α-Syn fibrils into the dorsolateral striatum (DLS) and the transgenic expression of A53T-α-Syn in the dopaminergic neurons in the substantia nigra (SN, PITX3-IRES2-tTA/tetO-A53T mice) selectively impaired forelimb fine movements induced by the single-pellet reaching task. (ii) Injecting α-Syn fibers into the SN suppressed the coordination of cranial and forelimb fine movements induced by the sunflower seed opening test. (iii) Treatments with the adenosine A2A receptor (A2AR) antagonist KW6002 reversed the impairment of forelimb and cranial fine movements induced by α-Syn aggregates in the SN. These findings established a causal role of α-Syn in the SNc-DLS dopaminergic pathway in the development of forelimb and cranial fine movement deficits and suggest a novel therapeutic strategy to improve fine movements in PD by A2AR antagonists.