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We previously found that chronic adenosine A1 receptor stimulation with N6-Cyclopentyladenosine increased α-synuclein misfolding and neurodegeneration in a novel α-synucleinopathy model, a hallmark of Parkinson's disease. Here, we aimed to synthesize a dimer caffeine-indan linked by a 6-carbon chain to cross the blood–brain barrier and tested its ability to bind α-synuclein, reducing misfolding, behavioral abnormalities, and neurodegeneration in our rodent model. Behavioral tests and histological stains assessed neuroprotective effects of the dimer compound. A rapid synthesis of the 18F-labeled analogue enabled Positron Emission Tomography and Computed Tomography imaging for biodistribution measurement. Molecular docking analysis showed that the dimer binds to α-synuclein N- and C-termini and the non-amyloid-β-component (NAC) domain, similar to 1-aminoindan, and this binding promotes a neuroprotective α-synuclein "loop" conformation. The dimer also binds to the orthosteric binding site for adenosine within the adenosine A1 receptor. Immunohistochemistry and confocal imaging showed the dimer abolished α-synuclein upregulation and aggregation in the substantia nigra and hippocampus, and the dimer mitigated cognitive deficits, anxiety, despair, and motor abnormalities. The 18F-labeled dimer remained stable post-injection and distributed in various organs, notably in the brain, suggesting its potential as a Positron Emission Tomography tracer for α-synuclein and adenosine A1 receptor in Parkinson's disease therapy. [ABSTRACT FROM AUTHOR]

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABAA receptors subunits α2, β2, γ1, and, γ2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration. [ABSTRACT FROM AUTHOR]

A significant proportion of the world's population suffers from insomnia, a disorder characterized by complications in initiating and maintaining sleep. Many medications used to treat insomnia target the γ-aminobutyric acid (GABA) neurotransmitter system. However, these substances, such as benzodiazepines, induce significant adverse consequences, including dependence and memory impairment, after prolonged use. Thus, current studies are aimed at developing therapeutic hypnotics derived from natural sources that may cause less severe side effects. Heukharang is a variety of lettuce from Korea that was discovered to contain sleep-promoting compounds. Therefore, we investigated the potential effects of sub-chronic administration of Heukharang extract (FSD-LS) on sleep behavior (pentobarbital-induced sleeping test), brain wave activity and sleep architecture (electroencephalography), and physiological behavior (open-field test and rota-rod) in mice, along with radioligand binding assays (GABAA, adenosine A1 and A2A receptors). We found that FSD-LS prolonged the total sleep duration and reduced the onset time of sleep, and enhanced delta wave power and non-rapid eye movement (NREM) sleep duration, all indicating persistent sleep-enhancing effects. FSD-LS lacked adverse effects on the spontaneous locomotor activity and motor coordination of mice, unlike diazepam. Pharmacological blocking using caffeine and bicuculline supported the possible involvement of adenosine receptors in the sleep-promoting effects of FSD-LS, with partial contribution from GABA receptor activity. Overall, our study recommends FSD-LS as a potential source for the development of sleep-aiding therapeutics. [ABSTRACT FROM AUTHOR]

L-Glutamate (L-Glu) is an amino acid present in the diet that plays a fundamental role in the central nervous system, as the main excitatory neurotransmitter participating in learning and memory processes. In addition, the nucleoside adenosine has a crucial role in L-Glu metabolism, by regulating the liberation of this neurotransmitter through four different receptors: A1, A2A, A2B and A3, which activate (A2A and A2B) or inhibit (A1 and A3) adenylate cyclase pathway. L-Glu at high concentrations can act as a neurotoxin and induce oxidative stress. The study of the oxidative stress correlated with an excess of L-Glu consumption during maternity is key to understand its effects on foetuses and neonates. Previous studies have shown that there is a change in the receptor levels in the brain of pregnant rats and their foetuses when mothers are administered L-Glu during gestation; however, its effect on the cerebellum is unknown. Cerebellum is known to be responsible for motor, cognitive and emotional functions, so its possible involvement after L-Glu consumption is an important issue to study. Therefore, the aim of the present work was to study the effect of L-Glu exposure during gestation and lactation on oxidative stress biomarkers and neurotransmitter receptors from the cerebellum of foetuses and neonates. After maternal L-Glu intake during gestation, oxidative stress was increased, as the ionotropic L-Glu receptors, and GluR1 AMPA subunit levels were altered in foetuses. A1 adenosine receptor suffered changes after L-Glu treatment during gestation, lactation or both, in lactating neonate cerebellum, while adenylate cyclase activity remain unaltered. Further studies will be necessary to elucidate the importance of L-Glu intake and its possible excitotoxicity in the cerebellum of Wistar rats during the pregnancy period and their involvement in long-term neurodegeneration. [ABSTRACT FROM AUTHOR]

Perilla frutescens (PF) is a plant with various biological activities, including antioxidant and antidepressant activities; however, the efficacy of its fermented product has not been evaluated. Herein, we found that the Bacillus subtilis-fermented PF (FPF) increased non-rapid eye movement sleep and slow wave activity. FPF improved the sleep onset latency and sleep duration in a pentobarbital-induced sleep model, and this efficacy was abolished by the adenosine A1 receptor (A1R) antagonist. In addition, FPF increased and decreased the neuronal activity in sleep-activating and inhibiting brain regions, respectively, and these effects were also blocked by A1R antagonist 8-cyclopentyl-1,3-dipropylxanthine. Furthermore, FPF exhibited direct binding and agonistic activities for A1R. HPLC/NMR analysis revealed uracil, protocatechuic acid, apigenin-7-O-diglucuronide, and luteolin-7-O-glucuronide to be the major active components responsible for the sleep-promoting efficacy of FPF. In conclusion, this study suggests that FPF has A1R agonistic activity and this novel activity may be critical for its sleep-promoting effect.

Chronic inflammation plays an important role in the development of neurodegenerative diseases, such as Parkinson's disease (PD). In the present study, we synthesized 25 novel xanthine derivatives with variable substituents at the N1-, N3- and C8-position as adenosine receptor antagonists with potential anti-inflammatory activity. The compounds were investigated in radioligand binding studies at all four human adenosine receptor subtypes, A1, A2A, A2B and A3. Compounds showing nanomolar A2A and dual A1/A2A affinities were obtained. Three compounds, 19, 22 and 24, were selected for further studies. Docking and molecular dynamics simulation studies indicated binding poses and interactions within the orthosteric site of adenosine A1 and A2A receptors. In vitro studies confirmed the high metabolic stability of the compounds, and the absence of toxicity at concentrations of up to 12.5 µM in various cell lines (SH-SY5Y, HepG2 and BV2). Compounds 19 and 22 showed anti-inflammatory activity in vitro. In vivo studies in mice investigating carrageenan- and formalin-induced inflammation identified compound 24 as the most potent anti-inflammatory derivative. Future studies are warranted to further optimize the compounds and to explore their therapeutic potential in neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

OBJECTIVES: The goal of the research is to investigate the protocatechuic acid (PCA) potential action, a phenolic acid derivative, on pain induced by neuropathy and to determine its efficacy on activation of KATP type channels and A1 receptors. MATERIALS AND METHODS: Neuropathic pain by cause of sciatic nerve damage was induced in Sprague-Dawley rats. Anti-allodynic and anti-hyperalgesic effects were evaluated with von Frey apparatus and Hargreave's plantar test apparatus, respectively. The effects of PCA at the doses of 75, 150 and 300 mg/kg, carbamazepine at the doses of 50 and 100 mg/kg, combination of low effective doses of PCA and carbamazepine were tested. Pretreatments 3 µg/kg DPCPX as adenosine A1 receptor antagonist and 60.7 nmol glibenclamide as KATP channel blocker were applied for mechanistic studies. RESULTS: PCA showed anti-allodynic and anti-hyperalgesic effects without impairing locomotor activity. In addition, the combination treatment was found to be more effective than the separate individual treatments of drugs. KATP channel activation related with A1 receptor stimulation makes a significant contribution to the anti-allodynia and anti-hyperalgesia induced by PCA. CONCLUSIONS: It can be said that PCA has similar effects with carbamazepine, which is used in clinical practice, and that PCA can take place as an adjuvant drug in neuropathic pain with the combination group. In addition, it is seen that the undesirable effects that drugs can cause alone can be avoided and a more effective treatment potential can be created with multiple mechanisms. [ABSTRACT FROM AUTHOR]

Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep-wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated ß-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor. Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of ß-arrestin 2. Stimulation of A1AR results in the recruitment of ß-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay. Results: The assay gives highly reproducible results with a very good signal-tonoise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of ß-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1ARmediated recruitment of ß-arrestin 2 by stimulation with a valerian extract. Conclusion: The presented assay is a useful tool for the quantitative study of A1ARmediated ß-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract. [ABSTRACT FROM AUTHOR]

The accumulation of tau is a hallmark of several neurodegenerative diseases and is associated with neuronal hypoactivity and presynaptic dysfunction. Oral administration of the adenosine A1 receptor antagonist rolofylline (KW-3902) has previously been shown to reverse spatial memory deficits and to normalize the basic synaptic transmission in a mouse line expressing full-length pro-aggregant tau (TauΔK) at low levels, with late onset of disease. However, the efficacy of treatment remained to be explored for cases of more aggressive tauopathy. Using a combination of behavioral assays, imaging with several PET-tracers, and analysis of brain tissue, we compared the curative reversal of tau pathology by blocking adenosine A1 receptors in three mouse models expressing different types and levels of tau and tau mutants. We show through positron emission tomography using the tracer [18F]CPFPX (a selective A1 receptor ligand) that intravenous injection of rolofylline effectively blocks A1 receptors in the brain. Moreover, when administered to TauΔK mice, rolofylline can reverse tau pathology and synaptic decay. The beneficial effects are also observed in a line with more aggressive tau pathology, expressing the amyloidogenic repeat domain of tau (TauRDΔK) with higher aggregation propensity. Both models develop a progressive tau pathology with missorting, phosphorylation, accumulation of tau, loss of synapses, and cognitive decline. TauRDΔK causes pronounced neurofibrillary tangle assembly concomitant with neuronal death, whereas TauΔK accumulates only to tau pretangles without overt neuronal loss. A third model tested, the rTg4510 line, has a high expression of mutant TauP301L and hence a very aggressive phenotype starting at ~3 months of age. This line failed to reverse pathology upon rolofylline treatment, consistent with a higher accumulation of tau-specific PET tracers and inflammation. In conclusion, blocking adenosine A1 receptors by rolofylline can reverse pathology if the pathological potential of tau remains below a threshold value that depends on concentration and aggregation propensity. [ABSTRACT FROM AUTHOR]

G-protein coupled receptors (GPCRs) present specific activation pathways and signaling among receptor subtypes. Hence, an extensive knowledge of the structural dynamics of the receptor is critical for the development of therapeutics. Here, we target the adenosine A1 receptor (A1R), for which a negligible number of drugs have been approved. We combine molecular dynamics simulations, enhanced sampling techniques, network theory, and pocket detection to decipher the activation pathway of A1R, decode the allosteric networks, and identify transient pockets. The A1R activation pathway reveals hidden intermediate and pre-active states together with the inactive and fully-active states observed experimentally. The protein energy networks computed throughout these conformational states successfully unravel the extra and intracellular allosteric centers and the communication pathways that couple them. We observe that the allosteric networks are dynamic, being increased along activation and fine-tuned in the presence of the trimeric G-proteins. Overlap of transient pockets and energy networks uncovers how the allosteric coupling between pockets and distinct functional regions of the receptor is altered along activation. Through an in-depth analysis of the bridge between the activation pathway, energy networks, and transient pockets, we provide a further understanding of A1R. This information can be useful to ease the design of allosteric modulators for A1R.

Background: Adenosine A1 receptor (A1AR) plays a prominent role in neurological and cardiac diseases and inflammatory processes. Its endogenous ligand adenosine is known to be one of the key players in the sleep–wake cycle. Like other G protein-coupled receptors (GPCRs), stimulation of A1AR leads to the recruitment of arrestins in addition to the activation of G proteins. So far, little is known about the role of these proteins in signal transduction and regulation of A1AR compared to the activation of G proteins. In this work, we characterized a live cell assay for A1AR-mediated β-arrestin 2 recruitment. We have applied this assay to a set of different compounds that interact with this receptor.Methods: Based on NanoBit® technology, a protein complementation assay was developed in which the A1AR is coupled to the large part of the nanoluciferase (LgBiT), whereas its small part (SmBiT) is fused to the N-terminus of β-arrestin 2. Stimulation of A1AR results in the recruitment of β-arrestin 2 and subsequent complementation of a functional nanoluciferase. For comparison, corresponding data on the effect of receptor stimulation on intracellular cAMP levels were collected for some data sets using the GloSensor™ assay.Results: The assay gives highly reproducible results with a very good signal-to-noise ratio. Capadenoson, in contrast to adenosine, CPA, or NECA, shows only partial agonism in this assay with respect to the recruitment of β-arrestin 2, whereas it shows full agonism in the case of the inhibitory effect of A1AR on cAMP production. By using a GRK2 inhibitor, it becomes clear that the recruitment is at least partially dependent on the phosphorylation of the receptor by this kinase. Interestingly, this was also the first time that we demonstrate the A1AR-mediated recruitment of β-arrestin 2 by stimulation with a valerian extract.Conclusion: The presented assay is a useful tool for the quantitative study of A1AR-mediated β-arrestin 2 recruitment. It allows data collection for stimulatory, inhibitory, and modulatory substances and is also suitable for more complex substance mixtures such as valerian extract.

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. [ABSTRACT FROM AUTHOR]

Hibernation is an adaptation that allows animals such as the Arctic ground squirrel (AGS) to survive the absence of food or water during the winter season. Understanding mechanisms of metabolic suppression during hibernation torpor promises new therapies for critical care. The activation of the Adenosine A1 receptor (A1AR) has been shown to be necessary and sufficient for entrance into hibernation with a winter season sensitization to the agonist, but the role of the A1AR in seasonal sensitization is unknown. In the current study, we characterize the A1AR in the forebrain, hippocampus and hypothalamus of summer and torpid AGS. For the first time, we define the pharmacological characteristics of the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in the AGS brain. In addition, we test the hypothesis that increased A1AR agonist efficacy is responsible for sensitization of the A1AR during the torpor season. The resulting 35S-GTPγS binding data indicate an increase in agonist potency during torpor in two out of three brain regions. In addition to 35S-GTPγS binding, [3H]DPCPX saturation and competition assays establish for the first-time pharmacological characteristics for the A1AR agonist, N6-cyclohexyladenosine and the A1AR antagonist dipropylcyclopentylxanthine (DPCPX) in AGS brain. [ABSTRACT FROM AUTHOR]

Abstract Background Accumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson’s disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). Methods Nanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague–Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting. Results Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a “knot” conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a “loop” conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus. Conclusions The results indicate that A1R agonists and drugs promoting a “knot” conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a “loop” conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD.

[INLINE:1] Previous studies have suggested that miR-324-3p is related to the pathophysiology of cerebral ischemia, but the mechanism underlying this relationship is unclear. In this study, we found that miR-324-3p expression was decreased in patients with acute ischemic stroke and in in vitro and in vivo models of ischemic stroke. miR-324-3p agomir potentiated ischemic brain damage in rats subjected to middle cerebral artery occlusion, as indicated by increased infarct volumes and cell apoptosis rates and greater neurological deficits. In a PC12 cell oxygen-glucose deprivation/reoxygenation model, a miR-324-3p mimic decreased cell viability and expression of the anti-apoptotic protein BCL2 and increased expression of the pro-apoptotic protein BAX and rates of cell apoptosis, whereas treatment with a miR-324-3p inhibitor had the opposite effects. Silencing miR-324-3p increased adenosine A1 receptor (A1R) expression through regulation of GATA binding protein 2 (GATA2). These findings suggest that silencing miR-324-3p reduces ischemic brain damage via the GATA2/A1R axis.

The acute and long-term consequences of perinatal asphyxia have been extensively investigated, but only a few studies have focused on postnatal asphyxia. In particular, electrophysiological changes induced in the motor cortex by postnatal asphyxia have not been examined so far, despite the critical involvement of this cortical area in epilepsy. In this study, we exposed primary motor cortex slices obtained from infant rats in an age window (16–18 day-old) characterized by high incidence of hypoxia-induced seizures associated with epileptiform motor behavior to 10 min of hypoxia. Extracellular field potentials evoked by horizontal pathway stimulation were recorded in layers II/III of the primary motor cortex before, during, and after the hypoxic event. The results show that hypoxia reversibly depressed glutamatergic synaptic transmission and neuronal excitability. Data obtained in the presence of specific blockers suggest that synaptic depression was mediated by adenosine acting on pre-synaptic A1 receptors to decrease glutamate release, and by a nitric oxide (NO)/cGMP postsynaptic pathway. These effects are neuroprotective because they limit energy failure. The present findings may be helpful in the preclinical search for therapeutic strategies aimed at preventing acute and long-term neurological consequences of postnatal asphyxia. [ABSTRACT FROM AUTHOR]

We report the design, synthesis, and validation of the novel compound photocaged N6-cyclopentyladenosine (cCPA) to achieve precisely localized and timed release of the parent adenosine A1 receptor agonist CPA using 405 nm light. Gi protein-coupled A1 receptors (A1Rs) modulate neurotransmission via pre- and post-synaptic routes. The dynamics of the CPA-mediated effect on neurotransmission, characterized by fast activation and slow recovery, make it possible to implement a closed-loop control paradigm. The strength of neurotransmission is monitored as the amplitude of stimulus-evoked local field potentials. It is used for feedback control of light to release CPA. This system makes it possible to regulate neurotransmission to a pre-defined level in acute hippocampal brain slices incubated with 3 µM cCPA. This novel approach of closed-loop photopharmacology holds therapeutic potential for fine-tuned control of neurotransmission in diseases associated with neuronal hyperexcitability. [ABSTRACT FROM AUTHOR]

[Display omitted] • Fermented Perilla frutescens var. acuta (FPF) has a sleep-promoting effect. • FPF increases neuronal activity in sleep-promoting brain regions. • FPF decreases neuronal activity in wake-promoting brain regions. • FPF has a direct binding and agonistic activity for adenosine A 1 receptor (A 1 R). Perilla frutescens (PF) is a plant with various biological activities, including antioxidant and antidepressant activities; however, the efficacy of its fermented product has not been evaluated. Herein, we found that the Bacillus subtilis -fermented PF (FPF) increased non-rapid eye movement sleep and slow wave activity. FPF improved the sleep onset latency and sleep duration in a pentobarbital-induced sleep model, and this efficacy was abolished by the adenosine A 1 receptor (A 1 R) antagonist. In addition, FPF increased and decreased the neuronal activity in sleep-activating and inhibiting brain regions, respectively, and these effects were also blocked by A 1 R antagonist 8-cyclopentyl-1,3-dipropylxanthine. Furthermore, FPF exhibited direct binding and agonistic activities for A 1 R. HPLC/NMR analysis revealed uracil, protocatechuic acid, apigenin-7-O-diglucuronide, and luteolin-7-O-glucuronide to be the major active components responsible for the sleep-promoting efficacy of FPF. In conclusion, this study suggests that FPF has A 1 R agonistic activity and this novel activity may be critical for its sleep-promoting effect. [ABSTRACT FROM AUTHOR]

Alcohol-related liver fibrosis (ALF) is a form of alcohol-related liver disease (ALD) that generally occurs in response to heavy long-term drinking. Ecto-5′-nucleotidase (NT5E), also known as CD73, is a cytomembrane protein linked to the cell membrane via a GPI anchor that regulates the conversion of extracellular ATP to adenosine. Adenosine and its receptors are important regulators of the cellular response. Previous studies showed that CD73 and adenosine A1 receptor (A1R) were important in alcohol-related liver disease, however the exact mechanism is unclear. The aim of this study was to elucidate the role and mechanism of the CD73-A1R axis in both a murine model of alcohol and carbon tetrachloride (CCl4) induced ALF and in an in vitro model of fibrosis induced by acetaldehyde. The degree of liver injury was determined by measuring serum AST and ALT levels, H & E staining, and Masson's trichrome staining. The expression levels of fibrosis indicators and PLC-IP3-Ca2+/DAG-PKC signaling pathway were detected by quantitative real-time PCR, western blotting, ELISA, and calcium assay. Hepatic stellate cell (HSC) apoptosis was detected using the Annexin V-FITC/PI cell apoptosis detection kit. Knockdown of CD73 significantly attenuated the accumulation of α-SMA and COL1a1 damaged the histological architecture of the mouse liver induced by alcohol and CCl4. In vitro , CD73 inhibition attenuated acetaldehyde-induced fibrosis and downregulated A1R expression in HSC-T6 cells. Inhibition of CD73/A1R downregulated the expression of the PLC-IP3-Ca2+/DAG-PKC signaling pathway. In addition, silencing of CD73/A1R promoted apoptosis in HSC-T6 cells. In conclusion, the CD73-A1R axis can regulate the activation and apoptosis of HSCs through the PLC-IP3-Ca2+/DAG-PKC signaling pathway. [ABSTRACT FROM AUTHOR]

Overexpression of the adenosine A1 receptor (A1AR) has been detected in various cancer cell lines. However, the role of A1AR in tumor development is still unclear. Thirteen A1AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. We have investigated the pharmacology of the mutations located at the 7-transmembrane domain using a yeast system. Concentration–growth curves were obtained with the full agonist CPA and compared to the wild type hA1AR. H78L3.23 and S246T6.47 showed increased constitutive activity, while only the constitutive activity of S246T6.47 could be reduced to wild type levels by the inverse agonist DPCPX. Decreased constitutive activity was observed on five mutant receptors, among which A52V2.47 and W188C5.46 showed a diminished potency for CPA. Lastly, a complete loss of activation was observed in five mutant receptors. A selection of mutations was also investigated in a mammalian system, showing comparable effects on receptor activation as in the yeast system, except for residues pointing toward the membrane. Taken together, this study will enrich the view of the receptor structure and function of A1AR, enlightening the consequences of these mutations in cancer. Ultimately, this may provide an opportunity for precision medicine for cancer patients with pathological phenotypes involving these mutations. [ABSTRACT FROM AUTHOR]

Alcohol-related liver fibrosis (ALF) is a form of alcohol-related liver disease (ALD) that generally occurs in response to heavy long-term drinking. Ecto-5′-nucleotidase (NT5E), also known as CD73, is a cytomembrane protein linked to the cell membrane via a GPI anchor that regulates the conversion of extracellular ATP to adenosine. Adenosine and its receptors are important regulators of the cellular response. Previous studies showed that CD73 and adenosine A1 receptor (A1R) were important in alcohol-related liver disease, however the exact mechanism is unclear. The aim of this study was to elucidate the role and mechanism of the CD73-A1R axis in both a murine model of alcohol and carbon tetrachloride (CCl4) induced ALF and in an in vitro model of fibrosis induced by acetaldehyde. The degree of liver injury was determined by measuring serum AST and ALT levels, H & E staining, and Masson’s trichrome staining. The expression levels of fibrosis indicators and PLC-IP3-Ca2+/DAG-PKC signaling pathway were detected by quantitative real-time PCR, western blotting, ELISA, and calcium assay. Hepatic stellate cell (HSC) apoptosis was detected using the Annexin V-FITC/PI cell apoptosis detection kit. Knockdown of CD73 significantly attenuated the accumulation of α-SMA and COL1a1 damaged the histological architecture of the mouse liver induced by alcohol and CCl4. In vitro, CD73 inhibition attenuated acetaldehyde-induced fibrosis and downregulated A1R expression in HSC-T6 cells. Inhibition of CD73/A1R downregulated the expression of the PLC-IP3-Ca2+/DAG-PKC signaling pathway. In addition, silencing of CD73/A1R promoted apoptosis in HSC-T6 cells. In conclusion, the CD73-A1R axis can regulate the activation and apoptosis of HSCs through the PLC-IP3-Ca2+/DAG-PKC signaling pathway.

Background: Accumulating α-synuclein (α-syn) aggregates in neurons and glial cells are the staples of many synucleinopathy disorders, such as Parkinson's disease (PD). Since brain adenosine becomes greatly elevated in ageing brains and chronic adenosine A1 receptor (A1R) stimulation leads to neurodegeneration, we determined whether adenosine or A1R receptor ligands mimic the action of known compounds that promote α-syn aggregation (e.g., the amphetamine analogue 2-aminoindan) or inhibit α-syn aggregation (e.g., Rasagiline metabolite 1-aminoindan). In the present study, we determined whether adenosine, A1R receptor agonist N6-Cyclopentyladenosine (CPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) could directly interact with α-syn to modulate α-syn aggregation and neurodegeneration of dopaminergic neurons in the substantia nigra (SN). Methods: Nanopore analysis and molecular docking were used to test the binding properties of CPA and DPCPX with α-syn in vitro. Sprague–Dawley rats were administered with 7-day intraperitoneal injections of the A1R ligands and 1- and 2-aminoindan, and levels of α-syn aggregation and neurodegeneration were examined in the SN pars compacta and hippocampal regions using confocal imaging and Western blotting. Results: Using nanopore analysis, we showed that the A1R agonists (CPA and adenosine) interacted with the N-terminus of α-syn, similar to 2-aminoindan, which is expected to promote a "knot" conformation and α-syn misfolding. In contrast, the A1R antagonist DPCPX interacted with the N- and C-termini of α-syn, similar to 1-aminoindan, which is expected to promote a "loop" conformation that prevents α-syn misfolding. Molecular docking studies revealed that adenosine, CPA and 2-aminoindan interacted with the hydrophobic core of α-syn N-terminus, whereas DPCPX and 1-aminoindan showed direct binding to the N- and C-terminal hydrophobic pockets. Confocal imaging and Western blot analyses revealed that chronic treatments with CPA alone or in combination with 2-aminoindan increased α-syn expression/aggregation and neurodegeneration in both SN pars compacta and hippocampus. In contrast, DPCPX and 1-aminoindan attenuated the CPA-induced α-syn expression/aggregation and neurodegeneration in SN and hippocampus. Conclusions: The results indicate that A1R agonists and drugs promoting a "knot" conformation of α-syn can cause α-synucleinopathy and increase neuronal degeneration, whereas A1R antagonists and drugs promoting a "loop" conformation of α-syn can be harnessed for possible neuroprotective therapies to decrease α-synucleinopathy in PD. [ABSTRACT FROM AUTHOR]

We investigated the effects of Lactuca sativa L. extracts (Lactuc) on pentobarbital-induced sleep in mice to elucidate the mechanisms underlying its impact on sleep quality. Mice were randomly assigned to five groups: control, positive control (diazepam 2 mg/kg b.w.), and three groups orally administered with Lactuc (50, 100, and 200 mg/kg b.w.). After 2 weeks of oral administration and intraperitoneal injections, the mice were killed. We found that the Lactuc-administered groups had significantly reduced sleep latency and increased sleep duration compared with the control group. Furthermore, the oral administration of Lactuc induced a significant increase in mRNA expression and protein expression of adenosine A1 receptor in the brains compared with the expressions in the control group. In addition, the Lactuc-administered groups exhibited significantly higher levels of mRNA expressions of GABA A receptors subunits α 2, β 2, γ 1, and, γ 2 in the brain tissue. Therefore, we suggest that Lactuc could be used to develop natural products that effectively improve sleep quality and duration.

The acute and long-term consequences of perinatal asphyxia have been extensively investigated, but only a few studies have focused on postnatal asphyxia. In particular, electrophysiological changes induced in the motor cortex by postnatal asphyxia have not been examined so far, despite the critical involvement of this cortical area in epilepsy. In this study, we exposed primary motor cortex slices obtained from infant rats in an age window (16–18 day-old) characterized by high incidence of hypoxia-induced seizures associated with epileptiform motor behavior to 10 min of hypoxia. Extracellular field potentials evoked by horizontal pathway stimulation were recorded in layers II/III of the primary motor cortex before, during, and after the hypoxic event. The results show that hypoxia reversibly depressed glutamatergic synaptic transmission and neuronal excitability. Data obtained in the presence of specific blockers suggest that synaptic depression was mediated by adenosine acting on pre-synaptic A1 receptors to decrease glutamate release, and by a nitric oxide (NO)/cGMP postsynaptic pathway. These effects are neuroprotective because they limit energy failure. The present findings may be helpful in the preclinical search for therapeutic strategies aimed at preventing acute and long-term neurological consequences of postnatal asphyxia.

Previous studies suggest that adenosine A1 receptors (A1R) modulate the processing of pain. The aim of this study was to characterize the distribution of A1R in nociceptive tissues and to evaluate whether targeting A1R with the partial agonist capadenoson may reduce neuropathic pain in mice. The cellular distribution of A1R in dorsal root ganglia (DRG) and the spinal cord was analyzed using fluorescent in situ hybridization. In behavioral experiments, neuropathic pain was induced by spared nerve injury or intraperitoneal injection of paclitaxel, and tactile hypersensitivities were determined using a dynamic plantar aesthesiometer. Whole-cell patch-clamp recordings were performed to assess electrophysiological properties of dissociated DRG neurons. We found A1R to be expressed in populations of DRG neurons and dorsal horn neurons involved in the processing of pain. However, administration of capadenoson at established in vivo doses (0.03–1.0 mg/kg) did not alter mechanical hypersensitivity in the spared nerve injury and paclitaxel models of neuropathic pain, whereas the standard analgesic pregabalin significantly inhibited the pain behavior. Moreover, capadenoson failed to affect potassium currents in DRG neurons, in contrast to a full A1R agonist. Despite expression of A1R in nociceptive neurons, our data do not support the hypothesis that pharmacological intervention with partial A1R agonists might be a valuable approach for the treatment of neuropathic pain. [ABSTRACT FROM AUTHOR]

Huntington’s disease (HD) is a life-threatening neurodegenerative disorder. Altered levels and functions of the purinergic ionotropic P2X7 receptors (P2X7Rs) have been found in animal and cellular models of HD, suggesting their possible role in the pathogenesis of the disease; accordingly, the therapeutic potential of P2X7R antagonists in HD has been proposed. Here we further investigated the effects of P2X7R ligands in in vitro and ex vivo HD experimental models. In ST14A/Q120 rat striatal cells, we found a reduction of P2X7R expression; however, the P2X7R agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) induced cellular death, and this effect was fully reversed by the antagonist periodate-oxidized adenosine 5′-triphosphate (OxATP). Moreover, in corticostriatal slices from symptomatic R6/2 mice, BzATP reduced the synaptic transmission to a larger extent than in wild-type (WT) mice. Such an effect was accompanied by a concomitant increase of the paired-pulse ratio, suggesting a presynaptic inhibitory action. This was confirmed to be the case, since while the effects of BzATP were unaffected by the P2X7R antagonist OxATP, they were blocked by the adenosine A1 receptor (A1R) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), suggesting possible BzATP hydrolysis to 2′(3′)-O-(4-benzoylbenzoyl)adenosine (Bz-adenosine) and consequent activation of A1Rs as a mechanism. Taken together, these data point out that 1) P2X7R expression and activity are confirmed to be altered in the presence of HD mutation; 2) in some experimental settings, such an abnormal functioning can be ascribed to presynaptic A1Rs activation.

Overexpression of the adenosine A1 receptor (A1AR) has been detected in various cancer cell lines. However, the role of A1AR in tumor development is still unclear. Thirteen A1AR mutations were identified in the Cancer Genome Atlas from cancer patient samples. We have investigated the pharmacology of the mutations located at the 7-transmembrane domain using a yeast system. Concentration–growth curves were obtained with the full agonist CPA and compared to the wild type hA1AR. H78L3.23 and S246T6.47 showed increased constitutive activity, while only the constitutive activity of S246T6.47 could be reduced to wild type levels by the inverse agonist DPCPX. Decreased constitutive activity was observed on five mutant receptors, among which A52V2.47 and W188C5.46 showed a diminished potency for CPA. Lastly, a complete loss of activation was observed in five mutant receptors. A selection of mutations was also investigated in a mammalian system, showing comparable effects on receptor activation as in the yeast system, except for residues pointing toward the membrane. Taken together, this study will enrich the view of the receptor structure and function of A1AR, enlightening the consequences of these mutations in cancer. Ultimately, this may provide an opportunity for precision medicine for cancer patients with pathological phenotypes involving these mutations.

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a breathing disorder associated with cognitive impairment. However, the mechanisms leading to cognitive deficits in OSAHS remain uncertain. In this study, a mouse model of chronic intermittent hypoxia (CIH) exposures were applied for simulating the deoxygenation-reoxygenation events occurring in OSAHS. The conventional adenosine A1 receptor gene (A1R) knockout mice and the A1R agonist CCPA- or antagonist DPCPX-administrated mice were utilized to determine the precise function of A1R signaling in the process of OSAHS-relevant cognitive impairment. We demonstrated that CIH induced morphological changes and apoptosis in hippocampal neurons. Further, CIH blunted hippocampal long-term potentiation (LTP) and resulted in learning/memory impairment. Disruption of adenosine A1R exacerbated morphological, cellular, and functional damage induced by CIH. In contrast, activation of adenosine A1R signaling reduced morphological changes and apoptosis of hippocampal neurons, promoted LTP, and enhanced learning and memory. A1Rs may up-regulate protein kinase C (PKC) and its subtype PKC-ζ through the activation of Gα(i) improve spatial learning and memory disorder induced by CIH in mice. Taken together, A1R signaling plays a neuroprotective role in CIH-induced cognitive dysfunction and pathological changes in the hippocampus.

Brain iron deficiency (BID) constitutes a primary pathophysiological mechanism in restless legs syndrome (RLS). BID in rodents has been widely used as an animal model of RLS, since it recapitulates key neurochemical changes reported in RLS patients and shows an RLS-like behavioral phenotype. Previous studies with the BID-rodent model of RLS demonstrated increased sensitivity of cortical pyramidal cells to release glutamate from their striatal nerve terminals driving striatal circuits, a correlative finding of the cortical motor hyperexcitability of RLS patients. It was also found that BID in rodents leads to changes in the adenosinergic system, a downregulation of the inhibitory adenosine A1 receptors (A1Rs) and upregulation of the excitatory adenosine A2A receptors (A2ARs). It was then hypothesized, but not proven, that the BID-induced increased sensitivity of cortico-striatal glutamatergic terminals could be induced by a change in A1R/A2AR stoichiometry in favor of A2ARs. Here, we used a newly developed FACS-based synaptometric analysis to compare the relative abundance on A1Rs and A2ARs in cortico-striatal and thalamo-striatal glutamatergic terminals (labeled with vesicular glutamate transporters VGLUT1 and VGLUT2, respectively) of control and BID rats. It could be demonstrated that BID (determined by measuring transferrin receptor density in the brain) is associated with a selective decrease in the A1R/A2AR ratio in VGLUT1 positive-striatal terminals.

Here, we tested the hypothesis that TNAP (tissue nonspecific alkaline phosphatase) modulates vascular responsiveness to norepinephrine. In the isolated, Tyrode's-perfused rat mesentery, 50 µmol/L of L-p-bromotetramisole (L-p-BT; selective TNAP inhibitor, Ki=56 µmol/L) significantly reduced TNAP activity and caused a significant 9.0-fold rightward-shift in the norepinephrine concentration versus vasoconstriction relationship. At 100 µmol/L, L-p-BT further reduced mesenteric TNAP activity and caused an additional significant right-shift of the norepinephrine concentration versus vasoconstriction relationship. A higher concentration (200 µmol/L) of L-p-BT had no further effect on either mesenteric TNAP activity or norepinephrine-induced vasoconstriction. L-p-BT did not alter vascular responses to vasopressin, thus ruling-out nonspecific suppression of vascular reactivity. Since in the rat mesenteric vasculature α1-adrenoceptors mediate norepinephrine-induced vasoconstriction, these finding indicate that TNAP inhibition selectively interferes with α1-adrenoceptor signaling. Additional experiments showed that the effects of TNAP inhibition on norepinephrine-induced vasoconstriction were not mediated by accumulation of pyrophosphate or ATP (TNAP substrates) nor by reduced adenosine levels (TNAP product). TNAP inhibition significantly reduced the Hillslope of the norepinephrine concentration versus vasoconstriction relationship from 1.8±0.2 (consistent with positive cooperativity of α1-adrenoceptor signaling) to 1.0±0.1 (no cooperativity). Selective activation of A1-adenosine receptors, which are known to participate in coincident signaling with α1-adrenoceptors, reversed the suppressive effects of L-p-BT on norepinephrine-induced vasoconstriction. In vivo, L-p-BT administration achieved plasma levels of ≈60 µmol/L and inhibited mesenteric vascular responses to exogenous norepinephrine and sympathetic nerve stimulation. TNAP modulates vascular responses to norepinephrine likely by affecting positive cooperativity of α1-adrenoceptor signaling via a mechanism involving A1 receptor signaling. [ABSTRACT FROM AUTHOR]

The function of zfp91 is mainly studied in vitro, but there is no study in vivo. Accumulative data suggest that zfp91 may be an important gene to regulate all aspects of human response. However, there are no data to date about the function of zfp91 on cardiac homeostasis. Thus, we aimed to observe the role of zfp91 gene in mouse cardiomyocytes on myocardial homeostasis and related mechanisms under pressure overload. In the study, zfp91 mRNA and protein levels were significantly reduced in TAC‐operated WT mice as compared with controls. Genetic ablation of zfp91 dramatically led to pathological cardiac dysfunction and hypertrophy after transverse aortic constriction (TAC). Adenosine A1 receptor (Adora1) mRNA and protein expressions were significantly down‐regulated in the heart of zfp91‐deletion mice with TAC. Zfp91 overexpression reversed isoproterenol‐induced cardiomyocyte hypertrophy, which was abolished by selective Adora1 antagonist. Dual‐luciferase reporter and ChIP‐qPCR assays indicated that zfp91 acted on Adora1 promoter through its binding site. Last, Adora1 agonist rescued heart dysfunction and cardiac hypertrophy in zfp91 loss mice after TAC. Zfp91 may transcriptionally regulate Adora1 expression in the heart, which mainly maintained cardiac homeostasis under pressure overload status. It will provide a new approach to treat cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Obstructive sleep apnea-hypopnea syndrome (OSAHS) is a breathing disorder associated with cognitive impairment. However, the mechanisms leading to cognitive deficits in OSAHS remain uncertain. In this study, a mouse model of chronic intermittent hypoxia (CIH) exposures were applied for simulating the deoxygenation-reoxygenation events occurring in OSAHS. The conventional adenosine A1 receptor gene (A1R) knockout mice and the A1R agonist CCPA- or antagonist DPCPX-administrated mice were utilized to determine the precise function of A1R signaling in the process of OSAHS-relevant cognitive impairment. We demonstrated that CIH induced morphological changes and apoptosis in hippocampal neurons. Further, CIH blunted hippocampal long-term potentiation (LTP) and resulted in learning/memory impairment. Disruption of adenosine A1R exacerbated morphological, cellular, and functional damage induced by CIH. In contrast, activation of adenosine A1R signaling reduced morphological changes and apoptosis of hippocampal neurons, promoted LTP, and enhanced learning and memory. A1Rs may up-regulate protein kinase C (PKC) and its subtype PKC-ζ through the activation of Gα(i) improve spatial learning and memory disorder induced by CIH in mice. Taken together, A1R signaling plays a neuroprotective role in CIH-induced cognitive dysfunction and pathological changes in the hippocampus. [ABSTRACT FROM AUTHOR]

Background/Aims: The adenosine A1 receptor (A1R) has been reported to be involved in the pathogenesis of various cancers, and the effects of A1R on different cancers are pleiotropic. However, the role of A1R in renal cell carcinoma (RCC) remains not well-known. Methods: The expression of A1R in RCC cells was detected by quantitative real-time PCR and Western blotting analysis. Cell proliferation was detected using an MTT assay and a colony formation assay. Tumor growth was also evaluated in nude mice. Cell invasion and migration were evaluated using a wound healing assay and a transwell assay. Cell cycle distribution and apoptosis rates were analyzed by flow cytometry. Results: A1R was the main subtype of ARs and was up-regulated in 786-O and ACHN cells. Functionally, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an A1R antagonist, inhibited RCC cell proliferation in vitro and tumor growth in vivo. Furthermore, DPCPX inhibited RCC cell migration, while N6-Cyclopentyladenosine (CPA), a selective A1 agonist, was able to rescue RCC cell migration. In addition, DPCPX promoted 786-O and ACHN cell apoptosis and induced an S phase cell cycle arrest. Finally, we demonstrated that DPCPX inhibited tumor progression in part via the ERK/JNK pathway. Conclusion: These findings suggest the potentially important role of DPCPX in the control of RCC cell proliferation and migration by regulating the ERK/JNK signaling pathway.

The activation of adenosine A1 receptors is important for protecting against ischemic brain injury and pretreatment with electroacupuncture has been shown to mitigate ischemic brain insult. The aim of this study was to test whether the adenosine A1 receptor mediates electroacupuncture pretreatment-induced neuroprotection against ischemic brain injury. We first performed 30 minutes of electroacupuncture pretreatment at the Baihui acupoint (GV20), delivered with a current of 1 mA, a frequency of 2/15 Hz, and a depth of 1 mm. High-performance liquid chromatography found that adenosine triphosphate and adenosine levels peaked in the cerebral cortex at 15 minutes and 120 minutes after electroacupuncture pretreatment, respectively. We further examined the effect of 15 or 120 minutes electroacupuncture treatment on ischemic brain injury in a rat middle cerebral artery-occlusion model. We found that at 24 hours reperfusion,120 minutes after electroacupuncture pretreatment, but not for 15 minutes, significantly reduced behavioral deficits and infarct volumes. Last, we demonstrated that the protective effect gained by 120 minutes after electroacupuncture treatment before ischemic injury was abolished by pretreatment with the A1-receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mg/kg, intraperitoneally). Our results suggest that pretreatment with electroacupuncture at the Baihui acupoint elicits protection against transient cerebral ischemia via action at adenosine A1 receptors.

Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (A 1 R), belonging to the G-protein-coupled receptors (GPCRs) superfamily, is mainly distributed in the central nervous system and major peripheral organs with wide-ranging physiological functions; however, the exact role of hepatic A 1 R in MAFLD remains unclear. Here, we report that liver-specific depletion of A 1 R aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic A 1 R promotes the competitive binding of sterol-regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) to sequestosome 1 (SQSTM1), rather than protein kinase A (PKA) leading to SCAP degradation in lysosomes. Reduced SCAP hinders SREBP1c/2 maturation and thus suppresses de novo lipogenesis and inflammation. Higher hepatic A 1 R expression is observed in patients with MAFL/MASH and high-fat diet (HFD)-fed mice, which is supposed to be a physiologically adaptive response because A 1 R agonists attenuate MAFL/MASH in an A 1 R-dependent manner. These results highlight that hepatic A 1 R is a potential target for MAFL/MASH therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cordycepin exerts neuroprotective effects against excitotoxic neuronal death. However, its direct electrophysiological evidence in Alzheimer's disease (AD) remains unclear. This study aimed to explore the electrophysiological mechanisms underlying the protective effect of cordycepin against the excitotoxic neuronal insult in AD using whole-cell patch clamp techniques. ß-Amyloid (Aß) and ibotenic acid (IBO)-induced injury model in cultured hippocampal neurons was used for the purpose. The results revealed that cordycepin significantly delayed Aß + IBO-induced excessive neuronal membrane depolarization. It increased the onset time/latency, extended the duration, and reduced the slope in both slow and rapid depolarization. Additionally, cordycepin reversed the neuronal hyperactivity in Aß + IBO-induced evoked action potential (AP) firing, including increase in repetitive firing frequency, shortening of evoked AP latency, decrease in the amplitude of fast afterhyperpolarization, and increase in membrane depolarization. Further, the suppressive effect of cordycepin against Aß + IBO-induced excessive neuronal membrane depolarization and neuronal hyperactivity was blocked by DPCPX (8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor-specific blocker). Collectively, these results revealed the suppressive effect of cordycepin against the Aß + IBO-induced excitotoxic neuronal insult by attenuating excessive neuronal activity and membrane depolarization, and the mechanism through the activation of A1R is strongly recommended, thus highlighting the therapeutic potential of cordycepin in AD. [ABSTRACT FROM AUTHOR]

The balance and smooth shift between flexible, goal-directed behaviors and repetitive, habitual actions are critical to optimal performance of behavioral tasks. The striatum plays an essential role in control of goal-directed versus habitual behaviors through a rich interplay of the numerous neurotransmitters and neuromodulators to modify the input, processing and output functions of the striatum. The adenosine receptors (namely A2AR and A1R), with their high expression pattern in the striatum and abilities to interact and integrate dopamine, glutamate and cannabinoid signals in the striatum, may represent novel therapeutic targets for modulating instrumental behavior. In this study, we examined the effects of pharmacological blockade of the A2ARs and A1Rs on goal-directed versus habitual behaviors in different information processing phases of instrumental learning using a satiety-based instrumental behavior procedure. We found that A2AR antagonist acts at the coding, consolidation and expression phases of instrumental learning to modulate animals’ sensitivity to goal-directed valuation without modifying action-outcome contingency. However, pharmacological blockade and genetic knockout of A1Rs did not affect acquisition or sensitivity to goal-valuation of instrumental behavior. These findings provide pharmacological evidence for a potential therapeutic strategy to control abnormal instrumental behaviors associated with drug addiction and obsessive-compulsive disorder by targeting the A2AR.

Adenosine signaling plays a complex role in multiple physiological processes in the brain, and its dysfunction has been implicated in pathophysiology of neuropsychiatric diseases such as schizophrenia and affective disorders. In the present study, the coupling between adenosine A1 receptor and G-protein was assessed by means of two [35S]GTPγS binding assays, i.e., conventional filtration method and [35S]GTPγS binding/immunoprecipitation in rat and human brain membranes. The latter method provides information about adenosine A1 receptor-mediated Gαi-3 activation in rat as well as human brain membranes. On the other hand, adenosine-stimulated [35S]GTPγS binding determined with conventional assay derives from functional activation of Gαi/o proteins (not restricted only to Gαi-3) coupled to adenosine A1 receptors. The determination of adenosine concentrations in the samples used in the present study indicates the possibility that the assay mixture under our experimental conditions contains residual endogenous adenosine at nanomolar concentrations, which was also suggested by the results on the effects of adenosine receptor antagonists on basal [35S]GTPγS binding level. The effects of adenosine deaminase (ADA) on basal binding also support the presence of adenosine. Nevertheless, the varied patterns of ADA discouraged us from adding ADA into assay medium routinely. The concentration-dependent increases elicited by adenosine were determined in 40 subjects without any neuropsychiatric disorders. The increases in %Emax values determined by conventional assay according to aging and postmortem delay should be taken into account in future studies focusing on the effects of psychiatric disorders on adenosine A1 receptor/G-protein interaction in postmortem human brain tissue. [ABSTRACT FROM AUTHOR]

The balance and smooth shift between flexible, goal-directed behaviors and repetitive, habitual actions are critical to optimal performance of behavioral tasks. The striatum plays an essential role in control of goal-directed versus habitual behaviors through a rich interplay of the numerous neurotransmitters and neuromodulators to modify the input, processing and output functions of the striatum. The adenosine receptors (namely A2AR and A1R), with their high expression pattern in the striatum and abilities to interact and integrate dopamine, glutamate and cannabinoid signals in the striatum, may represent novel therapeutic targets for modulating instrumental behavior. In this study, we examined the effects of pharmacological blockade of the A2ARs and A1Rs on goal-directed versus habitual behaviors in different information processing phases of instrumental learning using a satiety-based instrumental behavior procedure. We found that A2AR antagonist acts at the coding, consolidation and expression phases of instrumental learning to modulate animal's sensitivity to goal-directed valuation without modifying action-outcome contingency. However, pharmacological blockade and genetic knockout of A1Rs did not affect acquisition or sensitivity to goal-valuation of instrumental behavior. These findings provide pharmacological evidence for a potential therapeutic strategy to control abnormal instrumental behaviors associated with drug addiction and obsessive-compulsive disorder by targeting the A2AR. [ABSTRACT FROM AUTHOR]

The positron emission tomography (PET) radioligand for adenosine A1 receptor (A1R) [1-methyl-11C] 8-dicyclopropylmethyl-1-methyl-3-propylxanthine (MPDX) has recently been developed for human brain imaging. In the present study, we evaluated the alteration of the A1R in patients with diffuse axonal injury (DAI) in chronic stage in vivo. Ten patients with DAI (7 men and 3 women) were included in this study. Three PET examinations were sequentially performed to measure A1R binding with 11C-MPDX, glucose metabolism with 18F-fluorodeoxyglucose (FDG), and central benzodiazepine receptor binding with 11C-flumazenil (FMZ), and decreases of 11C-FMZ uptake indicate neuronal loss. 11C- MPDX did not depict any lesion with significantly decreased nondisplaceable binding potential ( BPND) in comparison to healthy controls (14 men) in region of interest (ROI) analysis. Instead, it showed a significant increase of BPND in the lower frontal and posterior cingulate cortexes and rolandic area ( p < 0.05) in ROI analysis. In 18F-FDG PET, the standardized uptake values ( SUVs) ratio to the whole brain were decreased in anterior and posterior cingulate gyrus compared to controls (14 men and 9 women; p < 0.01). In 11C-FMZ PET, the SUV ratio to the cerebellum was decreased in anterior cingulate gyrus in ROI analysis (controls, 9 men and 6 women; p < 0.01). The area with significantly increased 11C-MPDX binding, lower frontal cortex, rolandic area, and posterior cingulate gyrus, did not overlap with the areas of neuronal loss detected by decreased 11C-FMZ binding and did not completely overlap with area of reduced18F-FDG uptake. We obtained the first 11C-MPDX PET images reflecting the A1R BPND in human DAI brain in vivo. 11C-MPDX depicted increased A1R BPND in the areas surrounding the injured brain, whereas 18F-FDG demonstrated reduction throughout the brain. The results suggested that A1R might continuously confer neuroprotective or neuromodulatory effects in DAI even in the chronic stage. [ABSTRACT FROM AUTHOR]

Cannabinoid receptor 1 (CB1R) is widely distributed in the central nervous system, in excitatory and inhibitory neurons, and in astrocytes. CB1R agonists impair cognition and prevent long-term potentiation (LTP) of synaptic transmission, but the influence of endogenously formed cannabinoids (eCBs) on hippocampal LTP remains ambiguous. Based on the knowledge that eCBs are released upon high frequency neuronal firing, we hypothesized that the influence of eCBs upon LTP could change according to the paradigm of LTP induction. We thus tested the influence of eCBs on hippocampal LTP using two θ-burst protocols that induce either a weak or a strong LTP. LTP induced by a weak-θ-burst protocol is facilitated while preventing the endogenous activation of CB1Rs. In contrast, the same procedures lead to inhibition of LTP induced by the strong-θ-burst protocol, suggestive of a facilitatory action of eCBs upon strong LTP. Accordingly, an inhibitor of the metabolism of the predominant eCB in the hippocampus, 2-arachidonoyl-glycerol (2-AG), facilitates strong LTP. The facilitatory action of endogenous CB1R activation does not require the activity of inhibitory A1 adenosine receptors, is not affected by inhibition of astrocytic metabolism, but involves inhibitory GABAergic transmission. The continuous activation of CB1Rs via exogenous cannabinoids, or by drugs known to prevent metabolism of the non-prevalent hippocampal eCB, anandamide, inhibited LTP. We conclude that endogenous activation of CB1Rs by physiologically formed eCBs exerts a fine-tune homeostatic control of LTP in the hippocampus, acting as a high-pass filter, therefore likely reducing the signal-to-noise ratio of synaptic strengthening.