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Epilepsy affects ~65 million people worldwide. First-line treatment options include >20 antiseizure medications, but seizure control is not achieved in approximately one-third of patients. Antiseizure medications act primarily on neurons and can provide symptomatic control of seizures, but do not alter the onset and progression of epilepsy and can cause serious adverse effects. Therefore, medications with new cellular and molecular targets and mechanisms of action are needed. Accumulating evidence indicates that astrocytes are crucial to the pathophysiological mechanisms of epilepsy, raising the possibility that these cells could be novel therapeutic targets. In this Review, we discuss how dysregulation of key astrocyte functions — gliotransmission, cell metabolism and immune function — contribute to the development and progression of hyperexcitability in epilepsy. We consider strategies to mitigate astrocyte dysfunction in each of these areas, and provide an overview of how astrocyte activation states can be monitored in vivo not only to assess their contribution to disease but also to identify markers of disease processes and treatment effects. Improved understanding of the roles of astrocytes in epilepsy has the potential to lead to novel therapies to prevent the initiation and progression of epilepsy.

Potassium (K+) channels are robustly expressed during prenatal brain development, including in progenitor cells and migrating neurons, but their function is poorly understood. Here, we investigate the role of voltage-gated K+ channel KCNB1 (Kv2.1) in neocortical development. Neuronal migration of glutamatergic neurons was impaired in the neocortices of KCNB1 null mice. Migratory defects persisted into the adult brains, along with disrupted morphology and synaptic connectivity. Mice developed seizure phenotype, anxiety, and compulsive behavior. To determine whether defective KCNB1 can give rise to developmental channelopathy, we constructed Knock In (KI) mice, harboring the gene variant Kcnb1R312H (R312H mice) found in children with developmental and epileptic encephalopathies (DEEs). The R312H mice exhibited a similar phenotype to the null mice. Wild type (WT) and R312H KCNB1 channels made complexes with integrins α5β5 (Integrin_K+ channel_Complexes, IKCs), whose biochemical signaling was impaired in R312H brains. Treatment with Angiotensin II in vitro, an agonist of Focal Adhesion kinase, a key component of IKC signaling machinery, corrected the neuronal abnormalities. Thus, a genetic mutation in a K+ channel induces severe neuromorphological abnormalities through non-conducting mechanisms, that can be rescued by pharmacological intervention. This underscores a previously unknown role of IKCs as key players in neuronal development, and implicate developmental channelopathies in the etiology of DEEs.

Adenosine is an evolutionary ancient metabolic regulator linking energy state to physiologic processes, including immunomodulation and cell proliferation. Tumors create an adenosine-rich immunosuppressive microenvironment through the increased release of ATP from dying and stressed cells and its ectoenzymatic conversion into adenosine. Therefore, the adenosine pathway becomes an important therapeutic target to improve the effectiveness of immune therapies. Prior research has focused largely on the two major ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase 1/cluster of differentiation (CD)39 and ecto-5'-nucleotidase/CD73, which catalyze the breakdown of extracellular ATP into adenosine, and on the subsequent activation of different subtypes of adenosine receptors with mixed findings of antitumor and protumor effects. New findings, needed for more effective therapeutic approaches, require consideration of redundant pathways controlling intratumoral adenosine levels, including the alternative NAD-inactivating pathway through the CD38-ectonucleotide pyrophosphatase phosphodiesterase (ENPP)1-CD73 axis, the counteracting ATP-regenerating ectoenzymatic pathway, and cellular adenosine uptake and its phosphorylation by adenosine kinase. This review provides a holistic view of extracellular and intracellular adenosine metabolism as an integrated complex network and summarizes recent data on the underlying mechanisms through which adenosine and its precursors ATP and ADP control cancer immunosurveillance, tumor angiogenesis, lymphangiogenesis, cancer-associated thrombosis, blood flow, and tumor perfusion. Special attention is given to differences and commonalities in the purinome of different cancers, heterogeneity of the tumor microenvironment, subcellular compartmentalization of the adenosine system, and novel roles of purine-converting enzymes as targets for cancer therapy. SIGNIFICANCE STATEMENT: The discovery of the role of adenosine as immune checkpoint regulator in cancer has led to the development of novel therapeutic strategies targeting extracellular adenosine metabolism and signaling in multiple clinical trials and preclinical models. Here we identify major gaps in knowledge that need to be filled to improve the therapeutic gain from agents targeting key components of the adenosine metabolic network and, on this basis, provide a holistic view of the cancer purinome as a complex and integrated network.

Focal cortical dysplasia (FCD), a common malformation of cortical development, is frequently associated with pharmacoresistant epilepsy in both children and adults. Adenosine is an inhibitory modulator of brain activity and a prospective anti-seizure agent with potential for clinical translation. Our previous results demonstrated that the major adenosine-metabolizing enzyme adenosine kinase (ADK) was upregulated in balloon cells (BCs) within FCD type IIB lesions, suggesting that dysfunction of the adenosine system is implicated in the pathophysiology of FCD. In our current study, we therefore performed a comprehensive analysis of adenosine metabolism and signaling in surgically resected cortical specimens from patients with FCD type I and type II via immunohistochemistry and immunoblot analysis. Adenosine metabolism was assessed by quantifying the levels of the key enzymes of adenosine metabolism, i.e., ADK, adenosine deaminase (ADA), and 5’-ectonucleotidase (CD73). Adenosine signaling was assessed by quantifying the levels of adenosine A2A receptor (A2AR) and putative downstream mediators of adenosine, namely, glutamate transporter-1 (GLT-1) and mammalian target of rapamycin (mTOR). Within lesions in FCD specimens, we found that the adenosine-metabolizing enzymes ADK and ADA, as well as the adenosine-producing enzyme CD73, were upregulated. We also observed an increase in A2AR expression, as well as a decrease in GLT-1 levels and an increase in mTOR levels, in FCD specimens compared with control tissue. These results suggest that dysregulation of the adenosine system is a common pathologic feature of both FCD type I and type II. The adenosine system might therefore be a therapeutic target for the treatment of epilepsy associated with FCD.

A multifunctional, ubiquitous molecule, adenosine acts at cell-surface G protein-coupled receptors, as well as numerous enzymes, including protein kinases and adenylyl cyclase. Extracellular adenosine is thought to be produced either by export or by metabolism, predominantly through ecto-5’-nucleotidase activity (also producing inorganic phosphate). It is inactivated either by extracellular metabolism via adenosine deaminase (also producing ammonia) or, following uptake by nucleoside transporters, via adenosine deaminase or adenosine kinase (requiring ATP as co-substrate). Intracellular adenosine may be produced by cytosolic 5’-nucleotidases or through S-adenosylhomocysteine hydrolase (also producing L-homocysteine).

Post traumatic trigeminal neuropathy, presenting frequently as a complication of dental procedures, is often detrimental to the patient, with psychological and socioeconomic ramifications. Following a neuronal injury, numerous changes occur in both the peripheral (PNS) and central nervous systems (CNS) contributing to neuronal plasticity. Over the years, many studies have documented neuronal mechanisms involved in neuropathic pain, whereas non neuronal mechanisms, such as those based on glia have gained increased focus only recently. While the question of whether chronic neuropathic pain is a gliopathy remains enigmatic, activation and proliferation of glial cells and their interaction with neurons are believed to be key mechanisms underlying chronic neuropathic pain following a traumatic trigeminal nerve injury. Glial cells such as microglia, astrocytes and satellite glial cells have been extensively studied based on spinal neuropathic pain models, but the available literature on trigeminal pain models is very limited. In this review we aim to discuss i) available evidence on the role of central and peripheral glial cells in trigeminal neuropathic pain (TNP); and ii) gaps in knowledge between spinal and trigeminal pain models with regards to glial plasticity

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A

Chemotherapy-induced cognitive impairment (CICI) has emerged as a significant medical problem without therapeutic options. Using the platinum-based chemotherapy cisplatin to model CICI, we revealed robust elevations in the adenosine A 2A receptor (A 2A R) and its downstream effectors, cAMP and CREB, by cisplatin in the adult mouse hippocampus, a critical brain structure for learning and memory. Notably, A 2A R inhibition by the Food and Drug Administration–approved A 2A R antagonist KW-6002 prevented cisplatin-induced impairments in neural progenitor proliferation and dendrite morphogenesis of adult-born neurons, while improving memory and anxiety-like behavior, without affecting tumor growth or cisplatin’s antitumor activity. Collectively, our study identifies A 2A R signaling as a key pathway that can be therapeutically targeted to prevent cisplatin-induced cognitive impairments.

[Image: see text] DNA methylation has a major role in cancer, and its inhibitors are used therapeutically. DNA methylation depends on methyl group flux through the transmethylation pathway, which forms adenosine. We hypothesized that an adenosine kinase isoform with nuclear expression (ADK-L) determines global DNA methylation in cancer cells. We quantified ADK-L expression (Western Blot) and global DNA methylation as percent 5-methyldeoxycytidine (5mdC, LC-MS/MS) in three cancer lines (HeLa, HepG2, and U373). ADK-L expression and global DNA methylation correlated positively with the highest levels in HeLa cells compared to U373 and HepG2 cells. To determine whether ADK increases global DNA methylation and to validate its potential therapeutics, we treated HeLa cells with potent ADK inhibitors MRS4203 and MRS4380 (IC(50) 88 and 140 nM, respectively). Both nucleosides, but not a structurally related poor ADK inhibitor, significantly reduced global DNA methylation in HeLa cells in a concentration-dependent manner. Thus, ADK-L is a potential target for the therapeutic manipulation of DNA methylation levels in cancer.

Sudden unexpected death in epilepsy is the leading cause of epilepsy related death. Currently, there are no reliable methods for preventing sudden unexpected death in epilepsy. The precise pathophysiology of sudden unexpected death in epilepsy is unclear; however, convergent lines of evidence suggest that seizure-induced respiratory arrest plays a central role. It is generally agreed that sudden unexpected death in epilepsy could be averted if the patient could be rapidly ventilated following the seizure. The diaphragm is a muscle in the chest which contracts to draw air into the lungs. Diaphragmatic pacing is a surgical intervention which facilitates normal ventilation in situations, such as spinal cord injury and sleep apnoea, in which endogenous respiration would be inadequate or non-existent. In diaphragmatic pacing, electrodes are implanted directly onto diaphragm or adjacent to the phrenic nerves which innervate the diaphragm. These electrodes are then rhythmically stimulated, thereby eliciting contractions of the diaphragm which emulate endogenous breathing. The goal of this study was to test the hypothesis that seizure-induced respiratory arrest and death can be prevented with diaphragmatic pacing. Our approach was to induce respiratory arrest using maximal electroshock seizures in adult, male, C57BL6 mice outfitted with EEG and diaphragmatic electrodes (n = 8 mice). In the experimental group, the diaphragm was stimulated to exogenously induce breathing. In the control group, no stimulation was applied. Breathing and cortical electrographic activity were monitored using whole body plethysmography and EEG, respectively. A majority of the animals that did not receive the diaphragmatic pacing intervention died of seizure-induced respiratory arrest. Conversely, none of the animals that received the diaphragmatic pacing intervention died. Diaphragmatic pacing improved postictal respiratory outcomes (two-way ANOVA, P

Clinical and public interests in the ketogenic diet and metabolic treatments continue to spur research into mechanisms underlying their anticonvulsant, neuroprotective, and antiepileptogenic efficacy. Opportunities beyond seizure control are being explored and discovered in basic, translational, and clinical research paradigms. The range of specific mechanisms includes transcription factors, enzymes, energy molecules, ion channels, and more. Clinical opportunities discussed in this section include neurodegenerative disease, seizure disorders, and psychiatric disorders, as well as pan-disease benefits, such as reduced inflammation and even increased longevity. Many of these benefits are likely due to a combination of acute and long-lasting effects of metabolic therapy.

Approximately 60% of all epilepsy cases occur as a consequence of acute insults to the brain, such as traumatic brain injury, cerebrovascular insult, or infections. After an insult, the brain enters a period during which progressive neurobiologic alterations convert a non-epileptic brain into a brain capable of generating spontaneous and recurrent seizures, which are defined as epilepsy. The series of events is known as epileptogenesis. Epigenetic (DNA methylation) changes may affect several genes thought to represent risk factors for epilepsy; epigenetic changes are potentially reversible and may constitute a novel target for therapeutic intervention. DNA hypermethylation related to adenosine deficiency results in a vicious cycle associated with the onset of epileptogenesis and leading to chronic pharmacoresistant epilepsy. DNA hypermethylation is restored by the ketogenic diet (KD) via adenosine augmentation, a shift in the S-adenosylhomocysteine and S-adenosylmethionine homeostasis, and aiding DNA methylation by DNA methyltransferases. The KD-induced increase in ketone bodies inhibits histone deacetylases and prevents histone deacetylation. A clearer understanding of how KD therapy affects adenosine metabolism and its epigenetic sequelae may guide the development of therapies designed to directly restore adenosine homeostasis.

The brain is a highly energy-demanding organ and requires bioenergetics adaptability to balance normal activity with pathophysiologic fueling of spontaneous recurrent seizures, the hallmark feature of the epilepsies. For decades, it has been accepted that recurrent or prolonged seizures can permanently alter neuronal circuitry and cause excitotoxic injury and aberrant inflammation. Further, pathological changes in bioenergetics and metabolism have been considered downstream consequences of epileptic seizures that may begin at the synaptic level. Less appreciated is the fact that primary derangements in cellular or mitochondrial metabolism, in reverse fashion, can result in seizure genesis, and lead to spontaneous recurrent seizures. Increasingly, however, basic-translational research has demonstrated that the relationships between brain metabolism and epileptic seizures are both complex, bidirectional and produce a vicious cycle that compounds the deleterious consequences. From a therapeutic standpoint, metabolism-based treatments such as the high-fat, anti-seizure ketogenic diet have become mainstream, and metabolic substrates and enzymes (such as ketone bodies, certain fatty acids and adenosine) have become attractive molecular targets to prevent seizures and to aid in recovery processes. Moreover, given that metabolism is critical for epigenetic as well as inflammatory changes, the thesis that epileptogenesis – the molecular and cellular processes that produce a chronic epileptic brain – can be both negatively and positively influenced by metabolic changes is rapidly gaining ground. Here, we present the current evidence supporting the view that brain metabolism is both a pathophysiological and therapeutic paradigm for epilepsy.

Background: Lethal apnea is a significant cause of acute mortality following a severe traumatic brain injury (TBI). TBI is associated with a surge of adenosine, which also suppresses respiratory function in the brainstem. Methods and Materials: This study examined the acute and chronic effects of caffeine, an adenosine receptor antagonist, on acute mortality and morbidity after fluid percussion injury. Results: We demonstrate that, regardless of preinjury caffeine exposure, an acute bolus of caffeine given immediately following the injury dosedependently prevented lethal apnea and has no detrimental effects on motor performance following sublethal injuries. Finally, we demonstrate that chronic caffeine treatment after injury, but not caffeine withdrawal, impairs recovery of motor function. Conclusions: Preexposure of the injured brain to caffeine does not have a major impact on acute and delayed outcome parameters; more importantly, a single acute dose of caffeine after the injury can prevent lethal apnea regardless of chronic caffeine preexposure.

Alcohol-related poisoning is the foremost cause of death resulting from excessive acute alcohol consumption. Respiratory failure is crucial to the pathophysiology of fatal alcohol poisoning. Alcohol increases accumulation of extracellular adenosine. Adenosine suppresses breathing. The goal of this investigation was to test the hypothesis that adenosine signaling contributes to alcohol-induced respiratory suppression. In the first experiment, the breathing of mice was monitored following an injection of the non-selective adenosine receptor antagonist caffeine (40 mg/kg), alcohol (5 g/kg), or alcohol and caffeine combined. Caffeine reduced alcohol-induced respiratory suppression suggesting that adenosine contributes to the effects of alcohol on breathing. The second experiment utilized the same experimental design, but with the blood brain barrier impermeant non-selective adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT, 60 mg/kg) instead of caffeine. 8-SPT did not reduce alcohol-induced respiratory suppression suggesting that adenosine is contributing to alcohol-induced respiratory suppression in the central nervous system. The third and fourth experiments used the same experimental design as the first, but with the selective A

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15542. Enzymes are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Background Slowed clearance of amyloid β (Aβ) is believed to underlie the development of Aβ plaques that characterize Alzheimer’s disease (AD). Aβ is cleared in part by the glymphatic system, a brain-wide network of perivascular pathways that supports the exchange of cerebrospinal and brain interstitial fluid. Glymphatic clearance, or perivascular CSF-interstitial fluid exchange, is dependent on the astroglial water channel aquaporin-4 (AQP4) as deletion of Aqp4 in mice slows perivascular exchange, impairs Aβ clearance, and promotes Aβ plaque formation. Methods To define the role of AQP4 in human AD, we evaluated AQP4 expression and localization in a human post mortem case series. We then used the α-syntrophin (Snta1) knockout mouse model which lacks perivascular AQP4 localization to evaluate the effect that loss of perivascular AQP4 localization has on glymphatic CSF tracer distribution. Lastly, we crossed this line into a mouse model of amyloidosis (Tg2576 mice) to evaluate the effect of AQP4 localization on amyloid β levels. Results In the post mortem case series, we observed that the perivascular localization of AQP4 is reduced in frontal cortical gray matter of subjects with AD compared to cognitively intact subjects. This decline in perivascular AQP4 localization was associated with increasing Aβ and neurofibrillary pathological burden, and with cognitive decline prior to dementia onset. In rodent studies, Snta1 gene deletion slowed CSF tracer influx and interstitial tracer efflux from the mouse brain and increased amyloid β levels. Conclusions These findings suggest that the loss of perivascular AQP4 localization may contribute to the development of AD pathology in human populations.

A multifunctional, ubiquitous molecule, adenosine acts at cell-surface G protein-coupled receptors, as well as numerous enzymes, including protein kinases and adenylyl cyclase. Extracellular adenosine is thought to be produced either by export or by metabolism, predominantly through ecto-5’-nucleotidase activity (also producing inorganic phosphate). It is inactivated either by extracellular metabolism via adenosine deaminase (also producing ammonia) or, following uptake by nucleoside transporters, via adenosine deaminase or adenosine kinase (requiring ATP as co-substrate). Intracellular adenosine may be produced by cytosolic 5’-nucleotidases or through S-adenosylhomocysteine hydrolase (also producing L-homocysteine).

The adenosine hypothesis of schizophrenia posits that reduced availability of the neuromodulator adenosine contributes to dysregulation of dopamine and glutamate transmission and the symptoms associated with schizophrenia. It has been proposed that increased expression of the enzyme adenosine kinase (ADK) may drive hypofunction of the adenosine system. While animal models of ADK overexpression support such a role for altered ADK, the expression of ADK in schizophrenia has yet to be examined. In this study, we assayed ADK gene and protein expression in frontocortical tissue from schizophrenia subjects. In the dorsolateral prefrontal cortex (DLPFC), ADK-long and -short splice variant expression was not significantly altered in schizophrenia compared to controls. There was also no significant difference in ADK splice variant expression in the frontal cortex of rats treated chronically with haloperidol-decanoate, in a study to identify the effect of antipsychotics on ADK gene expression. ADK protein expression was not significantly altered in the DLPFC or anterior cingulate cortex (ACC). There was no significant effect of antipsychotic medication on ADK protein expression in the DLPFC or ACC. Overall, our results suggest that increased ADK expression does not contribute to hypofunction of the adenosine system in schizophrenia and that alternative mechanisms are involved in dysregulation of this system in schizophrenia.

Rasmussen encephalitis (RE) is a severe pediatric inflammatory brain disease characterized by unilateral inflammation and atrophy of the cerebral cortex, drug‐resistant focal epilepsy and progressive neurological and cognitive deterioration. The etiology and pathogenesis of RE remain unclear. Our previous results demonstrated that the adenosine A1 receptor (A1R) and the major adenosine‐removing enzyme adenosine kinase play an important role in the etiology of RE. Because the downstream pathways of inhibitory A1R signaling are modulated by stimulatory A2AR signaling, which by itself controls neuro‐inflammation, glial activation and glial glutamate homeostasis through interaction with glutamate transporter GLT‐1, we hypothesized that maladaptive changes in adenosine A2A receptor (A2AR) expression are associated with RE. We used immunohistochemistry and Western blot analysis to examine the expression of A2ARs, glutamate transporter‐I (GLT‐1) and the apoptotic marker Bcl‐2 in surgically resected cortical specimens from RE patients (n = 18) in comparison with control cortical tissue. In lesions of the RE specimen we found upregulation of A2ARs, downregulation of GLT‐1 and increased apoptosis of both neurons and astroglia. Double staining revealed colocalization of A2ARs and Bcl‐2 in RE lesions. These results suggest that maladaptive changes in A2AR expression are associated with a decrease in GLT‐I expression as a possible precipitator for apoptotic cell loss in RE. Because A2AR antagonists are already under clinical evaluation for Parkinson's disease, the A2AR might likewise be a tractable target for the treatment of RE.

OBJECTIVE Vagus nerve stimulation (VNS) is an alternative treatment option for individuals with refractory epilepsy, with nearly 40% of patients showing no benefit after VNS and only 6%–8% achieving seizure freedom. It is presently unclear why some patients respond to treatment and others do not. Therefore, identification of biomarkers to predict efficacy of VNS is of utmost importance. The objective of this study was to explore whether genetic variations in genes involved in adenosine kinase (ADK), ecto-5′-nucleotidase (NT5E), and adenosine A1 receptor (A1R) are linked to outcome of VNS in patients with refractory epilepsy. METHODS Thirty single-nucleotide polymorphisms (SNPs), including 9 in genes encoding ADK, 3 in genes encoding NT5E, and 18 in genes encoding A1R, were genotyped in 194 refractory epilepsy patients who underwent VNS. The chi-square test and binary logistic regression were used to determine associations between genetic differences and VNS efficacy. RESULTS A significant association between ADK SNPs rs11001109, rs7899674, and rs946185 and seizure reduction with VNS was found. Regardless of sex, age, seizure frequency and type, antiseizure drug use, etiology, and prior surgical history, all patients (10/10 patients [100%]) with minor allele homozygosity at rs11001109 (genotype AA) or rs946185 (AA) achieved > 50% seizure reduction and 4 patients (4/10 [40%]) achieved seizure freedom. VNS therapy demonstrated higher efficacy among carriers of minor allele rs7899674 (CG + GG) (68.3% vs 48.8% for patients with major allele homozygosity). CONCLUSIONS Homozygous ADK SNPs rs11001109 (AA) and rs946185 (AA), as well as minor allele rs7899674 (CG + GG), may serve as useful biomarkers for prediction of VNS therapy outcome.

Ketogenic diet is a high-fat, low-carbohydrate metabolic intervention that has been in use for the treatment of epilepsy for almost a hundred years. This metabolic intervention affects epilepsy through multiple mechanisms, which combine to exert potent anti-inflammatory activity. Thereby, ketogenic diet not only affects seizures per se but also has the potential to attenuate or prevent the development of epilepsy and its progression. This chapter will focus on novel disease-modifying properties of ketogenic diet therapy and will discuss therapeutic challenges and opportunities.

Adenosine kinase (ADK) is the key regulator of adenosine and catalyzes the metabolism of adenosine to 5'-adenosine monophosphate. The enzyme exists in two isoforms: a long isoform (ADK-long, ADK-L) and a short isoform (ADK-short, ADK-S). The two isoforms are developmentally regulated and are differentially expressed in distinct subcellular compartments with ADK-L localized in the nucleus and ADK-S localized in the cytoplasm. The nuclear localization of ADK-L and its biochemical link to the transmethylation pathway suggest a specific role for gene regulation via epigenetic mechanisms. Recent evidence reveals an adenosine receptor-independent role of ADK in determining the global methylation status of DNA and thereby contributing to epigenomic regulation. Here we summarize recent progress in understanding the biochemical interactions between adenosine metabolism by ADK-L and epigenetic modifications linked to transmethylation reactions. This review will provide a comprehensive overview of ADK-associated changes in DNA methylation in developmental, as well as in pathological conditions including brain injury, epilepsy, vascular diseases, cancer, and diabetes. Challenges in investigating the epigenetic role of ADK for therapeutic gains are briefly discussed.

Sudden unexpected death in epilepsy (SUDEP) is a leading cause of death in patients with refractory epilepsy. Centrally-mediated respiratory dysfunction has been identified as one of the principal mechanisms responsible for SUDEP. Seizures generate a surge in adenosine release. Elevated adenosine levels suppress breathing. Insufficient metabolic clearance of a seizure-induced adenosine surge might be a precipitating factor in SUDEP. In order to deliver targeted therapies to prevent SUDEP, reliable biomarkers must be identified to enable prompt intervention. Because of the integral role of the phrenic nerve in breathing, we hypothesized that suppression of phrenic nerve activity could be utilized as predictive biomarker for imminent SUDEP. We used a rat model of kainic acid-induced seizures in combination with pharmacological suppression of metabolic adenosine clearance to trigger seizure-induced death in tracheostomized rats. Recordings of EEG, blood pressure, and phrenic nerve activity were made concomitant to the seizure. We found suppression of phrenic nerve burst frequency to 58.9% of baseline (p < 0.001, one-way ANOVA) which preceded seizure-induced death; importantly, irregularities of phrenic nerve activity were partly reversible by the adenosine receptor antagonist caffeine. Suppression of phrenic nerve activity may be a useful biomarker for imminent SUDEP. The ability to reliably detect the onset of SUDEP may be instrumental in the timely administration of potentially lifesaving interventions.

Epileptogenesis is a common consequence of brain insults, however, the prevention or delay of the epileptogenic process remains an important unmet medical challenge. Overexpression of glycine transporter 1 (GlyT1) is proposed as a pathological hallmark in the hippocampus of patients with temporal lobe epilepsy (TLE), and we previously demonstrated in rodent epilepsy models that augmentation of glycine suppressed chronic seizures and altered acute seizure thresholds. In the present study we evaluated the effect of the GlyT1 inhibitor, sarcosine (aka N-methylglycine), on epileptogenesis and also investigated possible mechanisms. We developed a modified rapid kindling model of epileptogenesis in rats combined with seizure score monitoring to evaluate the antiepileptogenic effect of sarcosine. We used immunohistochemistry and Western blot analysis for the evaluation of GlyT1 expression and epigenetic changes of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the epileptogenic hippocampi of rats, and further evaluated expression changes in enzymes involved in the regulation of DNA methylation, ten-eleven translocation methylcytosine dioxygenase 1 (TET1), DNA-methyltransferase 1 (DNMT1), and DNMT3a. Our results demonstrated: (i) experimental evidence that sarcosine (3 g/kg, i.p. daily) suppressed kindling epileptogenesis in rats; (ii) the sarcosine-induced antiepileptogenic effect was accompanied by a suppressed hippocampal GlyT1 expression as well as a reduction of hippocampal 5mC levels and a corresponding increase in 5hmC; and (iii) sarcosine treatment caused differential expression changes of TET1 and DNMTs. Together, these findings suggest that sarcosine has unprecedented disease-modifying properties in a kindling model of epileptogenesis in rats, which was associated with altered hippocampal DNA methylation. Thus, manipulation of the glycine system is a potential therapeutic approach to attenuate the development of epilepsy.

© Mary Ann Liebert, Inc., Adenosine is an endogenous anticonvulsant and neuroprotectant of the brain. Seizure activity produces large quantities of adenosine, and it is this seizure-induced adenosine surge that normally stops a seizure. However, within the context of epilepsy, adenosine plays a wide spectrum of different roles. It not only controls seizures (ictogenesis), but also plays a major role in processes that turn a normal brain into an epileptic brain (epileptogenesis). It is involved in the control of abnormal synaptic plasticity and neurodegeneration and plays a major role in the expression of comorbid symptoms and complications of epilepsy, such as sudden unexpected death in epilepsy (SUDEP). Given the important role of adenosine in epilepsy, therapeutic strategies are in development with the goal to utilize adenosine augmentation not only for the suppression of seizures but also for disease modification and epilepsy prevention, as well as strategies to block adenosine A2A receptor overfunction associated with neurodegeneration. This review provides a comprehensive overview of the role of adenosine in epilepsy., The authors acknowledge grant support from the National Institutes of Health to D.B. (NINDS: NS103740, NS065957); support to A.M.S. by LISBOA-01-0145-FEDER-007391, project cofunded by Fundo Europeu Para o Desenvolvimento Regional through POR Lisboa 2020 (Programa Operacional Regional de Lisboa) from PORTUGAL 2020 and Fundação para a Ciência e Tecnologia (FCT), by an FCT project (PTDC/MED-FAR/30933/2017) and by Twinning action (SynaNet) from the EU H2020 programme (project number: 692340); and support to R.A.C. and A.R.T. by Fundacion LaCaixa (LCF/PR/HP17/52190001), Centro 2020 (CENTRO-01-0145-FEDER-000008: BrainHealth 2020 and CENTRO-01-0246-FEDER-000010) and FCT (POCI-01-0145-FEDER-03127).

Aim Adenosine receptors and extracellular adenosine have been demonstrated to modulate vascular smooth muscle cell (VSMC) proliferation and neointima formation. Adenosine kinase (ADK) is a major enzyme regulating intracellular adenosine levels, but is function in VSMC remains unclear. Here, we investigated the role of ADK in vascular injury-induced smooth muscle proliferation and delineated the mechanisms underlying its action. Methods and results We found that ADK expression was higher in the neointima of injured vessels and in PDGF-treated VSMCs. Genetic and pharmacological inhibition of ADK was enough to attenuate arterial injury-induced neointima formation due to inhibition of VSMC proliferation. Mechanistically, using infinium methylation assays and bisulfite sequencing, we showed that ADK metabolized the intracellular adenosine and potentiated the transmethylation pathway, then induced the aberrant DNA hypermethylation. Pharmacological inhibition of aberrant DNA hypermethylation increased KLF4 expression and suppressed VSMC proliferation as well as the neointima formation. Importantly, in human femoral arteries, we observed increased ADK expression and DNA hypermethylation as well as decreased KLF4 expression in neointimal VSMCs of stenotic vessels suggesting that our findings in mice are relevant for human disease and may hold translational significance. Conclusions Our study unravels a novel mechanism by which ADK promotes VSMC proliferation via inducing aberrant DNA hypermethylation, thereby downregulating KLF4 expression and promoting neointima formation. These findings advance the possibility of targeting ADK as an epigenetic modulator to combat vascular injury. Translational perspective Abnormal proliferation of vascular smooth muscle cell (VSMC) is key to abundant occlusive vascular diseases in humans, such as atherosclerosis and intimal hyperplasia associated with restenosis. Adenosine has been shown to combat abnormal smooth muscle proliferation. Here, we demonstrate that increased catabolism of adenosine by adenosine kinase (ADK) promotes abnormal VSMC proliferation. The pathological ADK overexpression in both mice and humans with vascular disease promotes VSMC proliferation via inducing aberrant DNA hypermethylation and KLF4 downregulation. Our study suggests that pharmacological augmentation of endogenous adenosine by targeting ADK represents a promising therapeutic strategy for occlusive vascular diseases.

Extracellular adenosine mediates diverse anti-inflammatory, angiogenic and vasoactive effects and becomes an important therapeutic target for cancer, which has been translated into clinical trials. This study was designed to comprehensively assess adenosine metabolism in prostate and breast cancer cells. We identified cellular adenosine turnover as a complex cascade, comprised of (a) the ectoenzymatic breakdown of ATP via sequential nucleotide pyrophosphatase/phosphodiesterase-1, ecto-5’-nucleotidase/CD73 and adenosine deaminase reactions, and ATP re-synthesis through counteracting adenylate kinase and nucleoside diphosphokinase; (b) the uptake of nucleotide-derived adenosine via equilibrative nucleoside transporters; and (c) the intracellular adenosine phosphorylation into ATP by adenosine kinase and other nucleotide kinases. The exposure of cancer cells to 1% O2 for 24 hours triggered ∼2-fold up-regulation of CD73, without affecting nucleoside transporters, adenosine kinase activity and cellular ATP content. The ability of adenosine to inhibit the tumor-initiating potential of breast cancer cells via receptor-independent mechanism was confirmed in vivo using a xenograft mouse model. The existence of redundant pathways controlling extracellular and intracellular adenosine provides a sufficient justification for reexamination of the current concepts of cellular purine homeostasis and signaling in cancer.