Adenosine A2A Receptor Antagonist SCH Research Articles

Adenosine A2A receptor antagonist SCH58261 improves the cognitive function in Alzheimer's disease model mice through activation of Nrf2 via an autophagy-dependent pathway.

Adenosine, an important endogenous neuromodulator, contributes to a broad set of several neurodegenerative diseases. The adenosine A2A receptor (A2AR) is the most involved in neuropathological effects and plays an important role in the pathogenesis of Alzheimer's disease (AD). However, the effect of A2AR antagonist and the underlying mechanism in AD model mice remains unclear. The amyloid beta (Aβ)1-42-induced mice AD models were used in this study. Several behavioral experiments were performed to evaluate the improvement of AD mice treat with A2AR receptor antagonist. For mechanism analysis, autophagy-related proteins, Kelch-like ECH-associated protein1(Keap1)- nuclear factor erythroid-derived factor 2-related factor (Nrf2) pathway activation and synaptic function were studied by using western blot, immunofluorescence (IF), immunohistochemistry (IHC), transmission electron microscope (TEM), real-time quantitative (PCR) and patch clamp. Pharmacological blockade of adenosine A2AR by SCH58261 (SCH) ameliorated cognitive deficits and decreased expression levels of several AD biomarkers, including Aβ and hyperphosphorylation of Tau. Moreover, SCH activated the Nrf2 pathway through autophagy mediated Keap1 degradation, resulting in the improvement of neurons autophagy dysfunction, synaptic plasticity, and synaptic transmission. Our data clarified that the SCH58261 (an antagonist of adenosine A2A receptor) could increase the level of autophagy, promote the ability of anti-oxidative stress by the activation of Keap1-Nrf2 pathway, and improve the synaptic function in Aβ1-42-induced Alzheimer's disease mice or cell model, which provided a potential therapeutic strategy for AD. A2AR antagonism represents a promising strategy for the anti-AD agents development through autophagy-dependent pathway.

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.

The effect of extracorporeal shock wave on joint capsule fibrosis based on A2AR-Nrf2/HO-1 pathway in a rat extending knee immobilization model

Joint capsule fibrosis, a common complication of joint immobilization, is mainly characterized by abnormal collagen deposition. The present study aimed to investigate the effect of extracorporeal shock wave therapy (ESWT) on reduced collagen deposition in the joint capsule during immobilization-induced joint capsule fibrosis. Additionally, the potential involvement of the adenosine A2A receptor (A2AR)-Neurotrophic factor e2-related factor 2 (Nrf2)/Haem oxygenase-1 (HO-1) pathway was explored. Thirty 3-month-old male Sprague–Dawley rats were randomly assigned to five groups: control (C), immobilization model (IM), natural recovery (NR), ESWT intervention (EI), and ESWT combined with A2AR antagonist SCH 58261 intervention (CI). After the left knee joints of rats in the IM, NR, EI and CI groups were immobilized using a full-extension fixation brace for 4 weeks, the EI and CI groups received ESWT twice a week for 4 weeks. The CI group was also treated with ESWT following intraperitoneal injection of SCH 58261 (0.01 mg/kg) for 4 weeks. The range of motion of the left knee joint was measured, and the protein levels of collagens I and III, A2AR, phosphorylated-protein kinase A/protein kinase A (p-PKA/PKA), p-Nrf2/Nrf2, and HO-1 were analysed by Western blotting. The IM and NR groups showed significantly greater arthrogenic contracture than the C group (P < 0.05). Compared to the NR group, the EI and CI groups exhibited significant improvement in arthrogenic contracture (P < 0.05). Conversely, the EI group showed lower contracture than the CI group (P < 0.05). Similar results were observed for collagen deposition and the protein levels of collagens I and III. The intervention groups (EI and CI groups) showed higher levels of p-Nrf2/Nrf2 and HO-1 than the NR group (P < 0.05). Moreover, the EI group exhibited higher levels of p-PKA/PKA, p-Nrf2/Nrf2, and HO-1 than the CI group (P < 0.05). However, no significant difference was found in the A2AR levels among the five groups (P > 0.05). ESWT may activate A2AR, leading to the phosphorylation of PKA. Subsequently, Nrf2 may be activated, resulting in the upregulation of HO-1, which then reduces collagen deposition and alleviates immobilization-induced joint capsule fibrosis.

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 30μM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300μM, 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 > 300µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37µM), 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.

Adenosine A2A Receptor Antagonist Improves Cognitive Impairment by Inhibiting Neuroinflammation and Excitatory Neurotoxicity in Chronic Periodontitis Mice.

The adenosine A2A receptor antagonist SCH58261 has been reported to have anti-inflammatory effects. However, its role in chronic periodontitis (CP)-induced cognitive impairment, which is associated with Porphyromonas gingivalis lipopolysaccharide (P. gingivalis LPS), remains unclear. This study investigated the role of SCH58261 in mice with CP-induced cognitive impairment. C57BL/6J mice were used to develop CP model by injecting 0.5 mg/kg P. gingivalis LPS into the palatal gingival sulcus of maxillary first molars twice a week for four weeks. The mice were divided into control, P. gingivalis LPS (P-LPS), P-LPS + SCH58261, and SCH58261 groups. The passive avoidance test (PAT) and Morris water maze (MWM) were used to assess cognition in mice. Furthermore, CD73/adenosine, neuroinflammation, glutamate transporters, and glutamate were assessed. Compared with the P-LPS group, 0.1 and 0.5 mg/kg SCH58261 increased latency and decreased error times in PAT, but increased platform crossing number in MWM. SCH58261 inhibited microglial activation, and decreased pro-inflammatory cytokines and glutamate levels, but increased GLT-1 and PSD95 expression in the hippocampus. This was the first report of SCH58261 treatment for CP-induced cognitive impairment, which may be related to its anti-inflammatory activities and anti-glutamate excitatory neurotoxicity. This suggests that SCH58261 can be used as a novel agent to treat cognitive impairment.

Adenosine A2A receptors blockade attenuates dexamethasone-induced alterations in cultured astrocytes.

Anxiety involves abnormal glucocorticoid signalling and altered glia-neuron communication in brain regions processing emotional responses. Adenosine A2A receptor (A2AR) blockade ameliorates mood and memory impairments by preventing synaptic dysfunction and astrogliosis. Since the glucocorticoid dexamethasone (DEX) can mimic early life-stress conditions, leading to anxiety-like behaviours, we now tested if A2AR blockade prevents alterations in the morphology and function of astrocytes exposed to DEX. Cultured astrocytes exposed to DEX exhibited an up-regulation of astrocytic markers (GFAP, connexin-43 and glutamine synthetase), as well as of A2AR. Moreover, DEX enhanced ATP and glutamate release and increased basal astrocytic Ca2+ levels. The selective A2AR antagonist SCH58261 prevented DEX-induced alterations in ATP release and basal Ca2+ levels but did not affect DEX-induced alteration of glutamate release and astrocytic markers. These findings suggest that alterations in astrocytes function, which might contribute to abnormal glucocorticoid brain signalling, are controlled by A2AR, and therefore, reinforce the relevance of A2AR as a potential therapeutic target to manage mood disorders.

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.

Electroacupuncture improves neuronal plasticity through the A2AR/cAMP/PKA signaling pathway in SNL rats

Improvements in neuronal plasticity are considered to be conducive to recovery from neuropathic pain. Electroacupuncture (EA) is regarded as an effective rehabilitation method for neuropathic pain. However, the effects and potential mechanism associated with EA-induced repair of hyperesthesia are not fully understood. Evidence has suggested that the adenosine A2A receptor (A2AR) and the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway play an important role in improving neuropathic pain. Here, we examined the function of EA in promoting neuronal plasticity in spinal nerve ligation (SNL) rats. The A2AR antagonist SCH58261, A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamido adenosine HCl (CGS21680) and A2AR siRNA were used to confirm the relationship between A2AR and the cAMP/PKA pathway as well as the effects of A2AR on EA-induced improvements in neurobehavioral state and neuronal plasticity. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), HE staining, Western blotting, RT-PCR, immunofluorescence, enzyme-linked immunosorbent assay, Nissl staining, silver staining, Golgi-Cox staining and transmission electron microscopy were used to evaluate the changes in neurobehavioral performance, protein expression, neuronal structure and dendrites/synapses. The results showed that EA and CGS21680 improved the behavioral performance, neuronal structure and dendritic/synaptic morphology of SNL rats, consistent with higher expression levels of A2AR, cAMP and PKA. In contrast to the positive effects of EA, SCH58261 inhibited dendritic growth and promoted dendritic spine/synaptic remodeling. In addition, the EA-induced improvement in neuronal plasticity was inhibited by SCH58261 and A2AR siRNA, consistent with lower expression levels of A2AR, cAMP and PKA, and worse behavioral performance. These results indicate that EA suppresses SNL-induced neuropathic pain by improving neuronal plasticity via upregulating the A2AR/cAMP/PKA signaling pathway.

Motor Deficits Coupled to Cerebellar and Striatal Alterations in Ube3am-/p+ Mice Modelling Angelman Syndrome Are Attenuated by Adenosine A2A Receptor Blockade.

Angelman syndrome (AS) is a neurogenetic disorder involving ataxia and motor dysfunction, resulting from the absence of the maternally inherited functional Ube3a protein in neurons. Since adenosine A2A receptor (A2AR) blockade relieves synaptic and motor impairments in Parkinson's or Machado-Joseph's diseases, we now tested if A2AR blockade was also effective in attenuating motor deficits in an AS (Ube3am-/p+) mouse model and if this involved correction of synaptic alterations in striatum and cerebellum. Chronic administration of the A2AR antagonist SCH58261 (0.1 mg/kg/day, ip) promoted motor learning of AS mice in the accelerating-rotarod task and rescued the grip strength impairment of AS animals. These motor impairments were accompanied by synaptic alterations in cerebellum and striatum typified by upregulation of synaptophysin and vesicular GABA transporters (vGAT) in the cerebellum of AS mice along with a downregulation of vGAT, vesicular glutamate transporter 1 (vGLUT1) and the dopamine active transporter in AS striatum. Notably, A2AR blockade prevented the synaptic alterations found in AS mice cerebellum as well as the downregulation of striatal vGAT and vGLUT1. This provides the first indications that A2AR blockade may counteract the characteristic motor impairments and synaptic changes of AS, although more studies are needed to unravel the underlying mechanisms.

Inhibition of adenosine A1 receptors abolished the nutritional ketosis-evoked delay in the onset of isoflurane-induced anesthesia in Wistar Albino Glaxo Rijswijk rats

BackgroundIt has been demonstrated that administration of exogenous ketone supplement ketone salt (KS) and ketone ester (KE) increased blood ketone level and delayed the onset of isoflurane-induced anesthesia in different rodent models, such as Wistar Albino Glaxo Rijswijk (WAG/Rij) rats. The modulatory effect of adenosinergic system may have a role in the ketone supplementation-evoked effects on isoflurane-generated anesthesia. Thus, we investigated whether adenosine receptor antagonists can modulate the effect of exogenous ketone supplements on the onset of akinesia induced by isoflurane.MethodsTo investigate the effect of exogenous ketone supplements on anesthetic induction we used ketone supplement KE, KS, KEKS (1:1 mix of KE and KS), KSMCT and KEMCT (1:1 mix of KS and KE with medium chain triglyceride/MCT oil, respectively) in WAG/Rij rats. Animals were fed with standard diet (SD), which was supplemented by oral gavage of different ketone supplements (2.5 g/kg/day) for 1 week. After 7 days, isoflurane (3%) was administered for 5 min and the time until onset of isoflurane-induced anesthesia (time until immobility; light phase of anesthesia: loss of consciousness without movement) was measured. Changes in levels of blood β-hydroxybutyrate (βHB), blood glucose and body weight of animals were also recorded. To investigate the putative effects of adenosine receptors on ketone supplements-evoked influence on isoflurane-induced anesthesia we used a specific adenosine A1 receptor antagonist DPCPX (intraperitoneally/i.p. 0.2 mg/kg) and a selective adenosine A2A receptor antagonist SCH 58261 (i.p. 0.5 mg/kg) alone as well as in combination with KEKS.ResultsSignificant increases were demonstrated in both blood βHB levels and the number of seconds required before isoflurane-induced anesthesia (immobility) after the final treatment by all exogenous ketone supplements. Moreover, this effect of exogenous ketone supplements positively correlated with blood βHB levels. It was also demonstrated that DPCPX completely abolished the effect of KEKS on isoflurane-induced anesthesia (time until immobility), but not SCH 58261.ConclusionsThese findings strengthen our previous suggestion that exogenous ketone supplements may modulate the isoflurane-induced onset of anesthesia (immobility), likely through A1Rs.

The adenosine A2A receptor antagonist SCH58261 reduces macrophage/microglia activation and protects against experimental autoimmune encephalomyelitis in mice

Multiple sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS) affecting more than 2.5 million individuals worldwide. In the present study, myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice were treated with adenosine receptor A2A antagonist SCH58261 at different periods of EAE development. The administration of SCH58261 at 11–28 days post-immunization (d.p.i.) with MOG improved the neurological deficits. This time window corresponds to the therapeutic time window for MS treatment. SCH58261 significantly reduced the CNS neuroinflammation including reduced local infiltration of inflammatory cells, demyelination, and the numbers of macrophage/microglia in the spinal cord. Importantly, SCH58261 ameliorated the EAE-induced neurobehavioral deficits. By contrast, the SCH58261 treatment was ineffective when administered at the beginning of the onset of EAE (i.e., 1–10 d.p.i). The identification of the effective therapeutic window of A2A receptor antagonist provide insight into the role of A2A receptor signaling in EAE, and support SCH58261 as a candidate for the treatment of MS in human.

Blockade of microglial adenosine A2A receptor suppresses elevated pressure-induced inflammation, oxidative stress, and cell death in retinal cells.

Glaucoma is a retinal degenerative disease characterized by the loss of retinal ganglion cells and damage of the optic nerve. Recently, we demonstrated that antagonists of adenosine A2A receptor (A2AR) control retinal inflammation and afford protection to rat retinal cells in glaucoma models. However, the precise contribution of microglia to retinal injury was not addressed, as well as the effect of A2AR blockade directly in microglia. Here we show that blocking microglial A2AR prevents microglial cell response to elevated pressure and it is sufficient to protect retinal cells from elevated pressure‐induced death. The A2AR antagonist SCH 58261 or the knockdown of A2AR expression with siRNA in microglial cells prevented the increase in microglia response to elevated hydrostatic pressure. Furthermore, in retinal neural cell cultures, the A2AR antagonist decreased microglia proliferation, as well as the expression and release of pro‐inflammatory mediators. Microglia ablation prevented neural cell death triggered by elevated pressure. The A2AR blockade recapitulated the effects of microglia depletion, suggesting that blocking A2AR in microglia is able to control neurodegeneration in glaucoma‐like conditions. Importantly, in human organotypic retinal cultures, A2AR blockade prevented the increase in reactive oxygen species and the morphological alterations in microglia triggered by elevated pressure. These findings place microglia as the main contributors for retinal cell death during elevated pressure and identify microglial A2AR as a therapeutic target to control retinal neuroinflammation and prevent neural apoptosis elicited by elevated pressure.

Balance between dopamine and adenosine signals regulates the PKA/Rap1 pathway in striatal medium spiny neurons

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.

Guanosine Attenuates Behavioral Deficits After Traumatic Brain Injury by Modulation of Adenosinergic Receptors.

Traumatic brain injury (TBI) is a leading cause of disability worldwide, triggering chronic neurodegeneration underlying cognitive and mood disorder still without therapeutic prospects. Based on our previous observations that guanosine (GUO) attenuates short-term neurochemical alterations caused by TBI, this study investigated the effects of chronical GUO treatment in behavioral, molecular, and morphological disturbances 21days after trauma. Rats subject to TBI displayed mood (anxiety-like) and memory dysfunction. This was accompanied by a decreased expression of both synaptic (synaptophysin) and plasticity proteins (BDNF and CREB), a loss of cresyl violet-stained neurons, and increased astrogliosis and microgliosis in the hippocampus. Notably, chronic GUO treatment (7.5mg/kgi.p. daily starting 1h after TBI) prevented all these TBI-induced long-term behavioral, neurochemical, and morphological modifications. This neuroprotective effect of GUO was abrogated in the presence of the adenosine A1 receptor antagonist DPCPX (1mg/kg) but unaltered by the adenosine A2A receptor antagonist SCH58261 (0.05mg/kg). These findings show that a chronic GUO treatment prevents the long-term mood and memory dysfunction triggered by TBI, which involves adenosinergic receptors.

Single and combined effects of plant-derived and synthetic cannabinoids on cognition and cannabinoid-associated withdrawal signs in mice.

It has been suggested that the non-euphorogenic phytocannabinoid cannabidiol (CBD) can ameliorate adverse effects of Δ9 -tetrahydrocannabinol (THC). We determined whether CBD ameliorates cognitive deficits and withdrawal signs induced by cannabinoid CB1 /CB2 receptor agonists or produces these pharmacological effects on its own. The effects of THC or the CB1 /CB2 receptor full agonist WIN55212 alone, CBD alone or their combination were tested across a range of doses. Cognitive effects were assessed in C57BL/6 mice in a conditional discrimination task and in the Barnes maze. Cannabinoid withdrawal signs were assessed following precipitated withdrawal by acute administration of the CB1 receptor antagonist SR141716, the 5-HT1A receptor antagonist WAY100635, the TRPV1 receptor antagonist capsazepine or the adenosine A2A receptor antagonist SCH58261. THC produced significant motor and cognitive impairment in the Barnes maze task, none of which were attenuated by the addition of CBD. CBD alone did not affect cognitive performance. Precipitation of withdrawal signs by SR141716 occurred in mice chronically treated with THC or WIN55,212. These withdrawal signs were not attenuated by addition of chronic CBD. Chronic treatment with CBD alone did not induce withdrawal signs precipitated by SR141716 or WAY100635. Chronic CBD treatment also produced anxiolysis, which was not altered by attempting to precipitate withdrawal-induced anxiety with a range of antagonists. CBD as a monotherapy may prove to be a safer pharmacological agent, than CB1 receptor agonists alone or in combination with CBD, for the treatment of several disorders. This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Adenosine A2A receptor signaling affects IL-21/IL-22 cytokines and GATA3/T-bet transcription factor expression in CD4+ T cells from a BTBR T+ Itpr3tf/J mouse model of autism

Autism is a complex heterogeneous neurodevelopmental disorder; previous studies have identified altered immune responses among individuals diagnosed with autism. An imbalance in the production of pro- and anti-inflammatory cytokines and transcription factors plays a role in neurodevelopmental behavioral and autism disorders. BTBR T+ Itpr3tf/J (BTBR) mice are used as a model for autism, as they exhibit social deficits, communication deficits, and repetitive behaviors compared with C57BL/6J (B6) mice. The adenosine A2A receptor (A2AR) appears to be a potential target for the improvement of behavioral, inflammatory, immune, and neurological disorders. We investigated the effects of the A2AR antagonist SCH 5826 (SCH) and agonist CGS 21680 (CGS) on IL-21, IL-22, T-bet, T-box transcription factor (T-bet), GATA3 (GATA Binding Protein 3), and CD152 (CTLA-4) expression in BTBR mice. Our results showed that BTBR mice treated with SCH had increased CD4+IL-21+, CD4+IL-22+, CD4+GATA3+, and CD4+T-bet+ and decreased CD4+CTLA-4+ expression in spleen cells compared with BTBR control mice. Moreover, CGS efficiently decreased CD4+IL-21+, CD4+IL-22+, CD4+GATA3+, and CD4+T-bet+ and increased CD4+CTLA-4 production in spleen cells compared with SCH-treated and BTBR control mice. Additionally, SCH treatment significantly increased the mRNA and protein expression levels of IL-21, IL-22, GATA3, and T-bet in brain tissue compared with CGS-treated and BTBR control mice. The augmented levels of IL-21/IL-22 and GATA3/T-bet could be due to altered A2AR signaling. Our results indicate that A2AR agonists may represent a new class of compounds that can be developed for use in the treatment of autistic and neuroimmune dysfunctions.

Toll-like receptors, NF-κB, and IL-27 mediate adenosine A2A receptor signaling in BTBR T+ Itpr3tf/J mice

Autism is a predominant neurodevelopmental disorder characterized by impaired communication, social deficits, and repetitive behaviors. Recent research has proposed that the impairment of innate immunity may play an important role in autism. Toll-like receptors (TLRs) are potential therapeutic targets against neuroinflammation. The BTBR T+ Itpr3tf/J (BTBR) mouse is a well-known model of autism, showing repetitive behaviors such as cognitive inflexibility and increased grooming as compared to C57BL/6 (B6) mice. Adenosine A2A receptor (A2AR) signaling is involved in inflammation, brain injury, and lymphocyte infiltration into the CNS, but the role of A2AR in autism remains unknown. We investigated the effect of A2AR antagonist SCH 5826 (SCH) and agonist CGS 21680 (CGS) on the expression levels of TLRs, IL-27, NF-κB p65, and IκBα in BTBR mice. Treatment of BTBR mice with SCH increased the percentage of splenic CD14+TLR2+ cells, CD14+TLR3+ cells, CD14+TLR4+ cells, and decreased the percentage of CD14+IL-27+ cells, as compared to the untreated BTBR mice. Our results reveal that BTBR mice treated with CGS had reversal of SCH-induced immunological responses. Moreover, mRNA and protein expression analyses confirmed increased expression of TLR2, TLR3, TLR4, and NF-κB p65 in brain tissue, and decreased IL-27 and IκBα expression following SCH treatment, as compared to the untreated-BTBR and CGS-treated BTBR mice. Together, these results suggest that the A2AR agonist corrects neuroimmune dysfunction observed in BTBR mice, and thus has the potential as a therapeutic approach in autism.

Adenosine A2 receptor activation ameliorates mitochondrial oxidative stress upon reperfusion through the posttranslational modification of NDUFV2 subunit of complex I in the heart

While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5′-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I via the mitochondrial Src tyrosine kinase. Phosphorylation of Tyr118 residue in NDUFV2 subunit may account for the inhibitory effect of NECA on complex I.

Selective A2A receptor antagonist prevents microglia-mediated neuroinflammation and protects retinal ganglion cells from high intraocular pressure–induced transient ischemic injury

Glaucoma is a leading cause of vision loss and blindness worldwide, characterized by chronic and progressive neuronal loss. Reactive microglial cells have been recognized as a neuropathologic feature, contributing to local inflammation and retinal neurodegeneration. In a recent in vitro work (organotypic cultures), we demonstrated that blockade of adenosine A2A receptor (A2AR) prevents the neuroinflammatory response and affords protection to retinal ganglion cells (RGCs) against exposure to elevated hydrostatic pressure (EHP), to mimic elevated intraocular pressure (IOP), the main risk factor for glaucoma development. Herein, we investigated whether a selective A2AR antagonist (SCH 58261) could modulate retinal microglia reactivity and their inflammatory response. Furthermore, we took advantage of the high IOP-induced transient ischemia (ischemia-reperfusion, I-R) animal model to evaluate the protective role of A2AR blockade in the control of retinal neuroinflammation and neurodegeneration. Primary microglial cell cultures were challenged either with lipopolysaccharide or with EHP, in the presence or absence of A2AR antagonist SCH 58261 (50 nM). In addition, I-R injury was induced in adult Wistar rats after intravitreal administration of SCH 58261 (100 nM, 5 μL). Our results showed that SCH 58261 attenuated microglia reactivity and the increased expression and release of proinflammatory cytokines. Moreover, intravitreal administration of SCH 58261 prevented I-R-induced cell death and RGC loss, by controlling microglial-mediated neuroinflammatory response. These results prompt the proposal that A2AR blockade may have great potential in the management of retinal neurodegenerative diseases characterized by microglia reactivity and RGC death, such as glaucoma and ischemic diseases.

Adenosine A2AR blockade prevents neuroinflammation-induced death of retinal ganglion cells caused by elevated pressure.

BackgroundElevated intraocular pressure (IOP) is a major risk factor for glaucoma, a degenerative disease characterized by the loss of retinal ganglion cells (RGCs). There is clinical and experimental evidence that neuroinflammation is involved in the pathogenesis of glaucoma. Since the blockade of adenosine A2A receptor (A2AR) confers robust neuroprotection and controls microglia reactivity in the brain, we now investigated the ability of A2AR blockade to control the reactivity of microglia and neuroinflammation as well as RGC loss in retinal organotypic cultures exposed to elevated hydrostatic pressure (EHP) or lipopolysaccharide (LPS).MethodsRetinal organotypic cultures were either incubated with LPS (3 μg/mL), to elicit a pro-inflammatory response, or exposed to EHP (+70 mmHg), to mimic increased IOP, for 4 or 24 h, in the presence or absence of the A2AR antagonist SCH 58261 (50 nM). A2AR expression, microglial reactivity and neuroinflammatory response were evaluated by immunohistochemistry, quantitative PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA). RGC loss was assessed by immunohistochemistry. In order to investigate the contribution of pro-inflammatory mediators to RGC loss, the organotypic retinal cultures were incubated with rabbit anti-tumour necrosis factor (TNF) (2 μg/mL) and goat anti-interleukin-1β (IL-1β) (1 μg/mL) antibodies.ResultsWe report that the A2AR antagonist (SCH 58261) prevented microglia reactivity, increase in pro-inflammatory mediators as well as RGC loss upon exposure to either LPS or EHP. Additionally, neutralization of TNF and IL-1β prevented RGC loss induced by LPS or EHP.ConclusionsThis work demonstrates that A2AR blockade confers neuroprotection to RGCs by controlling microglia-mediated retinal neuroinflammation and prompts the hypothesis that A2AR antagonists may be a novel therapeutic option to manage glaucomatous disorders.