Molecular pathways mediating systemic inflammation entering the brain parenchyma to induce sepsis-associated encephalopathy (SAE) remain elusive. Here, we report that in mice during the first 6 hours of peripheral lipopolysaccharide (LPS)-evoked systemic inflammation (6 hpi), the plasma level of adenosine quickly increased and enhanced the tone of central extracellular adenosine which then provoked neuroinflammation by triggering early astrocyte reactivity. Specific ablation of astrocytic Gi protein-coupled A1 adenosine receptors (A1ARs) prevented this early reactivity and reduced the levels of inflammatory factors (e.g., CCL2, CCL5, and CXCL1) in astrocytes, thereby alleviating microglial reaction, ameliorating blood-brain barrier disruption, peripheral immune cell infiltration, neuronal dysfunction, and depression-like behaviour in the mice. Chemogenetic stimulation of Gi signaling in A1AR-deficent astrocytes at 2 and 4 hpi of LPS injection could restore neuroinflammation and depression-like behaviour, highlighting astrocytes rather than microglia as early drivers of neuroinflammation. Our results identify early astrocyte reactivity towards peripheral and central levels of adenosine as an important pathway driving SAE and highlight the potential of targeting A1ARs for therapeutic intervention.

The study of protein function and dynamics in their native cellular environment is essential for progressing fundamental science. To overcome the requirement of genetic modification of the protein or the limitations of dissociable fluorescent ligands, ligand-directed (LD) chemistry has most recently emerged as a complementary, bioorthogonal approach for labeling native proteins. Here, we describe the rational design, development, and application of the first ligand-directed chemistry approach for labeling the A1AR in living cells. We pharmacologically demonstrate covalent labeling of A1AR expressed in living cells while the orthosteric binding site remains available. The probes were imaged using confocal microscopy and fluorescence correlation spectroscopy to study A1AR localization and dynamics in living cells. Additionally, the probes allowed visualization of the specific localization of A1ARs endogenously expressed in dorsal root ganglion (DRG) neurons. LD probes developed here hold promise for illuminating ligand-binding, receptor signaling, and trafficking of the A1AR in more physiologically relevant environments.

Xanthohumol (Xn) is an antioxidant flavonoid mainly extracted from hops (Humulus lupulus), one of the main ingredients of beer. As with other bioactive compounds, their therapeutic potential against different diseases has been tested, one of which is Alzheimer\'s disease (AD). Adenosine is a neuromodulatory nucleoside that acts through four different G protein-coupled receptors: A1 and A3, which inhibit the adenylyl cyclases (AC) pathway, and A2A and A2B, which stimulate this activity, causing either a decrease or an increase, respectively, in the release of excitatory neurotransmitters such as glutamate. This adenosinergic pathway, which is altered in AD, could be involved in the excitotoxicity process. Therefore, the aim of this work is to describe the effect of Xn on the adenosinergic pathway using cell lines. For this purpose, two different cellular models, rat glioma C6 and human neuroblastoma SH-SY5Y, were exposed to a non-cytotoxic 10 µM Xn concentration. Adenosine A1 and A2A, receptor levels, and activities related to the adenosine pathway, such as adenylate cyclase, protein kinase A, and 5\'-nucleotidase, were analyzed. The adenosine A1 receptor was significantly increased after Xn exposure, while no changes in A2A receptor membrane levels or AC activity were reported. Regarding 5\'-nucleotidases, modulation of their activity by Xn was noted since CD73, the extracellular membrane attached to 5\'-nucleotidase, was significantly decreased in the C6 cell line. In conclusion, here we describe a novel pathway in which the bioactive flavonoid Xn could have potentially beneficial effects on AD as it increases membrane A1 receptors while modulating enzymes related to the adenosine pathway in cell cultures.

Evidence has shown that both sleep loss and daily caffeine intake can induce changes in grey matter (GM). Caffeine is frequently used to combat sleepiness and impaired performance caused by insufficient sleep. It is unclear (1) whether daily use of caffeine could prevent or exacerbate the GM alterations induced by 5-day sleep restriction (i.e. chronic sleep restriction, CSR), and (2) whether the potential impact on GM plasticity depends on individual differences in the availability of adenosine receptors, which are involved in mediating effects of caffeine on sleep and waking function. Thirty-six healthy adults participated in this double-blind, randomized, controlled study (age = 28.9 ± 5.2 y/; F:M = 15:21; habitual level of caffeine intake < 450 mg; 29 homozygous C/C allele carriers of rs5751876 of ADORA2A, an A2A adenosine receptor gene variant). Each participant underwent a 9-day laboratory visit consisting of one adaptation day, 2 baseline days (BL), 5-day sleep restriction (5 h time-in-bed), and a recovery day (REC) after an 8-h sleep opportunity. Nineteen participants received 300 mg caffeine in coffee through the 5 days of CSR (CAFF group), while 17 matched participants received decaffeinated coffee (DECAF group). We examined GM changes on the 2nd BL Day, 5th CSR Day, and REC Day using magnetic resonance imaging and voxel-based morphometry. Moreover, we used positron emission tomography with [18F]-CPFPX to quantify the baseline availability of A1 adenosine receptors (A1R) and its relation to the GM plasticity. The results from the voxel-wise multimodal whole-brain analysis on the Jacobian-modulated T1-weighted images controlled for variances of cerebral blood flow indicated a significant interaction effect between caffeine and CSR in four brain regions: (a) right temporal-occipital region, (b) right dorsomedial prefrontal cortex (DmPFC), (c) left dorsolateral prefrontal cortex (DLPFC), and (d) right thalamus. The post-hoc analyses on the signal intensity of these GM clusters indicated that, compared to BL, GM on the CSR day was increased in the DECAF group in all clusters  but decreased in the thalamus, DmPFC, and DLPFC in the CAFF group. Furthermore, lower baseline subcortical A1R availability predicted a larger GM reduction in the CAFF group after CSR of all brain regions except for the thalamus. In conclusion, our data suggest an adaptive GM upregulation after 5-day CSR, while concomitant use of caffeine instead leads to a GM reduction. The lack of consistent association with individual A1R availability may suggest that CSR and caffeine affect thalamic GM plasticity predominantly by a different mechanism. Future studies on the role of adenosine A2A receptors in CSR-induced GM plasticity are warranted.

UNASSIGNED: Levetiracetam (LEV) has been found to have an antihyperalgesic effect via acting on the adenosine system. However, the effects of LEV on the modulation of the adenosine system in the brain have not been elucidated in the prevention of seizures and epilepsy. The present study aimed to explore the possible LEV mechanisms of action in the adenosine signaling systems in an animal model of epilepsy.
UNASSIGNED: A docking study was initially performed to determine the possible interaction of LEV with adenosine A1 receptors (A1Rs) and equilibrative nucleoside transporters-1 (ENT1). The experimental study was divided into an acute seizure test (32 mice distributed into 4 groups) and a chronic kindling model study (40 mice distributed into 5 groups), followed by gene expression analysis and immunohistochemistry. The kindling model lasted 26 days and took 13 subconvulsive doses of pentylenetetrazole (PTZ) to completely kindle the mice in the PTZ control group. Gene expression changes in the A1Rs, potassium inwardly-rectifying channel 3.2 (Kir3.2), and ENT1 in the brain tissue samples of the mice following treatment with LEV were analyzed using reverse transcription-quantitative polymerase chain reaction, and immunohistochemistry was performed for the A1R protein expression.
UNASSIGNED: Docking studies predicted a significant interaction of LEV with A1Rs and ENT1 proteins. Results from the acute testing revealed that caffeine (100 mg/kg) and 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg) significantly reversed the antiseizure effects of LEV by reversing the percent protection and shortening the onset of the first myoclonic jerk (FMJ) and generalized clonic seizures (GCSs). In the PTZ-induced kindling, LEV demonstrated an increased gene expression of A1Rs and Kir3.2 in the brain. LEV also significantly reduced the gene expression of ENT1. Furthermore, the immunohistochemical analysis showed that LEV increased the protein expression of A1Rs in the brain.
UNASSIGNED: Based on these results, it can be concluded that LEV modulates epileptogenesis by acting on the adenosine pathway in the central nervous system.

Metabolic (dysfunction)-associated steatohepatitis (MASH) is the advanced stage of metabolic (dysfunction)-associated fatty liver disease (MAFLD) lacking approved clinical drugs. Adenosine A1 receptor (A1R), 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 A1R in MAFLD remains unclear. Here, we report that liver-specific depletion of A1R aggravates while overexpression attenuates diet-induced metabolic-associated fatty liver (MAFL)/MASH in mice. Mechanistically, activation of hepatic A1R 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 A1R 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 A1R agonists attenuate MAFL/MASH in an A1R-dependent manner. These results highlight that hepatic A1R is a potential target for MAFL/MASH therapy.

Acute alcoholic hepatitis (AAH) from binge drinking is a serious disease. It is associated with a high mortality rate, especially among young adults. Apoptosis is known to be a primary cause of liver damage, and it can be induced by either intrinsic signaling pathways or by reactive oxygen species (ROS). Adenosine A1 receptors (ADORA1) are known to be involved in ethanol metabolism; however, underlying mechanism is not well understood. For investigating how the intrinsic ADORA1 function in ethanol metabolism in normal human hepatocytes without interference by extrinsic molecules, primary hepatocytes pose a challenge, due to unavoidable contamination by other kinds of cells in the liver. Also, they are difficult to culture stably. As a novel alternative, hepatocytes derived from human-induced pluripotent stem cells were employed because they display similar function to primary hepatocytes and they can be stably cultured. The dynamics and integrity of signal transduction mechanisms were investigated by following chronological changes in gene expression. This shed light on how and when the ADORA1 function and on causal relationships between the pathways and clinical symptoms. The findings of the present study shows that ADORA1 are most activated soon after exposure to ethanol, and transfection of small interfering RNA targeting ADORA1-messenger-RNA (ADORA1-siRNA) into the hepatocytes significantly suppresses production of actin protein and ROS. It suggests that ADORA1 in the liver contribute to apoptosis in acute alcoholism through both intrinsic pathway and ROS activity. Also, actin that is abundant in the cells could be an appropriate biomarker evaluating hepatic function status.

The cholinergic pathway plays a crucial role in improving inflammatory end-organ damage. Given the interplay between cholinergic and adenosinergic neurotransmission, we tested the hypothesis that central adenosine A1 receptors (A1ARs) modulate the nicotine counteraction of cardiovascular and inflammatory insults induced by sepsis in rats. Sepsis was induced by cecal ligation and puncture (CLP) 24-h before cardiovascular measurements. Nicotine (25-100 µg/kg i.v.) dose-dependently reversed septic manifestations of hypotension and impaired heart rate variability (HRV) and cardiac sympathovagal balance. Like nicotine, intracisternal (i.c.) administration of N(6)-cyclopentyladenosine (CPA, A1AR agonist) to CLP rats increased indices of HRV and sympathovagal balance. Moreover, greater surges in these parameters were noted upon simultaneous nicotine/CPA administration. The favorable influences of nicotine on blood pressure and HRV in sepsis were diminished after central blockade of A1ARs by i.c. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX). Molecular studies revealed that (i) septic rises in myocardial and brainstem nucleus of solitary tract (NTS) NFκB expression were abrogated by nicotine and largely reinstated after blockade of A1ARs, and (ii) A1AR expression in the same areas was reduced by DPCPX. It is concluded that myocardial and medullary A1ARs facilitate the cholinergic counteraction of cardiac and neuroinflammation induced by sepsis and interrelated cardiomyopathic and neuropathic hitches.

G protein-coupled receptors (GPCRs) represent the target for approximately a third of FDA-approved small molecule drugs. The adenosine A1 receptor (A1R), one of four adenosine GPCR subtypes, has important (patho)physiological roles in humans. A1R has well-established roles in the regulation of the cardiovascular and nervous systems, where it has been identified as a potential therapeutic target for a number of conditions, including cardiac ischemia-reperfusion injury, cognition, epilepsy, and neuropathic pain. A1R small molecule drugs, typically orthosteric ligands, have undergone clinical trials. To date, none have progressed into the clinic, predominantly due to dose-limiting unwanted effects. The development of A1R allosteric modulators that target a topographically distinct binding site represent a promising approach to overcome current limitations. Pharmacological parameters of allosteric ligands, including affinity, efficacy and cooperativity, can be optimized to regulate A1R activity with high subtype, spatial and temporal selectivity. This review aims to offer insights into the A1R as a potential therapeutic target and highlight recent advances in the structural understanding of A1R allosteric modulation.

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.