The Concise Guide to PHARMACOLOGY 2023/24: Enzymes.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is 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 almost 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/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes 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-2023, and supersedes data presented in the 2021/22, 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.

Oocyte-Specific Deletion of Slc6a9 Encoding the GLYT1 Glycine Transporter Eliminates Glycine Transport in Mouse Preimplantation Embryos and Their Ability to Counter Hypertonic Stress.

Early preimplantation mouse embryos are sensitive to increased osmolarity, which can block their development. To overcome this, they accumulate organic osmolytes to maintain cell volume. The main organic osmolyte used by early mouse embryos is glycine. Glycine is transported during the mature egg and 1-cell to 4-cell embryo stages by a transporter identified as GLYT1, encoded by the Slc6a9 gene. Here, we have produced an oocyte-specific knockout of Slc6a9 by crossing mice that have a segment of the gene flanked by LoxP elements with transgenic mice expressing iCre driven by the oocyte-specific Gdf9 promoter. Slc6a9 null oocytes failed to develop glycine transport activity during meiotic maturation. However, females with these oocytes were fertile. When enclosed in their cumulus-oocyte complex, Slc6a9 null oocytes could accumulate glycine via GLYT1 transport in their coupled cumulus cells, which may support female fertility in vivo. In vitro, embryos derived from Slc6a9 null oocytes displayed a clear phenotype. While glycine rescued complete preimplantation development of wild type embryos from increased osmolarity, embryos derived from null oocytes failed to develop past the 2-cell stage even with glycine. Thus, Slc6a9 is required for glycine transport and protection against increased osmolarity in mouse eggs and early embryos.

Bipolar mania and epilepsy pathophysiology and treatment may converge in purine metabolism: A new perspective on available evidence.

Decreased ATPergic signaling is an increasingly recognized pathophysiology in bipolar mania disease models. In parallel, adenosine deficit is increasingly recognized in epilepsy pathophysiology. Under-recognized ATP and/or adenosine-increasing mechanisms of several antimanic and antiseizure therapies including lithium, valproate, carbamazepine, and ECT suggest a fundamental pathogenic role of adenosine deficit in bipolar mania to match the established role of adenosine deficit in epilepsy. The depletion of adenosine-derivatives within the purine cycle is expected to result in a compensatory increase in oxopurines (uric acid precursors) and secondarily increased uric acid, observed in both bipolar mania and epilepsy. Cortisol-based inhibition of purine conversion to adenosine-derivatives may be reflected in observed uric acid increases and the well-established contribution of cortisol to both bipolar mania and epilepsy pathology. Cortisol-inhibited conversion from IMP to AMP as precursor of both ATP and adenosine may represent a mechanism for treatment resistance common in both bipolar mania and epilepsy. Anti-cortisol therapies may therefore augment other treatments both in bipolar mania and epilepsy. Evidence linking (i) adenosine deficit with a decreased need for sleep, (ii) IMP/cGMP excess with compulsive hypersexuality, and (iii) guanosine excess with grandiose delusions may converge to suggest a novel theory of bipolar mania as a condition characterized by disrupted purine metabolism. The potential for disease-modification and prevention related to adenosine-mediated epigenetic changes in epilepsy may be mirrored in mania. Evaluating the purinergic effects of existing agents and validating purine dysregulation may improve diagnosis and treatment in bipolar mania and epilepsy and provide specific targets for drug development.

Chemobrain: An accelerated aging process linking adenosine A2A receptor signaling in cancer survivors.

Chemotherapy has a significant positive impact in cancer treatment outcomes, reducing recurrence and mortality. However, many cancer surviving children and adults suffer from aberrant chemotherapy neurotoxic effects on learning, memory, attention, executive functioning, and processing speed. This chemotherapy-induced cognitive impairment (CICI) is referred to as "chemobrain" or "chemofog". While the underlying mechanisms mediating CICI are still unclear, there is strong evidence that chemotherapy accelerates the biological aging process, manifesting as effects which include telomere shortening, epigenetic dysregulation, oxidative stress, mitochondrial defects, impaired neurogenesis, and neuroinflammation, all of which are known to contribute to increased anxiety and neurocognitive decline. Despite the increased prevalence of CICI, there exists a lack of mechanistic understanding by which chemotherapy detrimentally affects cognition in cancer survivors. Moreover, there are no approved therapeutic interventions for this condition. To address this gap in knowledge, this review attempts to identify how adenosine signaling, particularly through the adenosine A2A receptor, can be an essential tool to attenuate accelerated aging phenotypes. Importantly, the adenosine A2A receptor uniquely stands at the crossroads of cancer treatment and improved cognition, given that it is widely known to control tumor induced immunosuppression in the tumor microenvironment, while also posited to be an essential regulator of cognition in neurodegenerative disease. Consequently, we propose that the adenosine A2A receptor may provide a multifaceted therapeutic strategy to enhance anticancer activity, while combating chemotherapy induced cognitive deficits, both which are essential to provide novel therapeutic interventions against accelerated aging in cancer survivors.

Ectopic expression of neuronal adenosine kinase, a biomarker in mesial temporal lobe epilepsy without hippocampal sclerosis.

AIMS: Mesial temporal lobe epilepsy without hippocampal sclerosis (no-HS MTLE) refers to those MTLE patients who have neither magnetic resonance imaging (MRI) lesions nor definite pathological evidence of hippocampal sclerosis. They usually have resistance to antiepileptic drugs, difficulties in precise seizure location and poor surgical outcomes. Adenosine is a neuroprotective neuromodulator that acts as a seizure terminator in the brain. The role of adenosine in no-HS MTLE is still unclear. Further research to explore the aetiology and pathogenesis of no-HS MTLE may help to find new therapeutic targets. METHODS: In surgically resected hippocampal specimens, we examined the maladaptive changes of the adenosine system of patients with no-HS MTLE. In order to better understand the dysregulation of the adenosine pathway in no-HS MTLE, we developed a rat model based on the induction of focal cortical lesions through a prenatal freeze injury. RESULTS: We first examined the adenosine system in no-HS MTLE patients who lack hippocampal neuronal loss and found ectopic expression of the astrocytic adenosine metabolising enzyme adenosine kinase (ADK) in hippocampal pyramidal neurons, as well as downregulation of neuronal A1 receptors (A1 Rs) in the hippocampus. In the no-HS MTLE model rats, the transition of ADK from neuronal expression to an adult pattern of glial expression in the hippocampus was significantly delayed. CONCLUSIONS: Ectopic expression of neuronal ADK might be a pathological hallmark of no-HS MTLE. Maladaptive changes in adenosine metabolism might be a novel target for therapeutic intervention in no-HS MTLE.

Aberrant adenosine signaling in patients with focal cortical dysplasia.

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 signaling in surgically resected cortical specimens from patients with FCD type I and type II via immunohistochemistry and immunoblot analysis. Adenosine enzyme signaling was assessed by quantifying the levels of the key enzymes of adenosine metabolism, i.e., ADK, adenosine deaminase (ADA), and ecto-5'-nucleotidase (CD73). Adenosine receptor 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 density, 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.

Astrocyte-neuron circuits in epilepsy.

The epilepsies are a diverse spectrum of disease states characterized by spontaneous seizures and associated comorbidities. Neuron-focused perspectives have yielded an array of widely used anti-seizure medications and are able to explain some, but not all, of the imbalance of excitation and inhibition which manifests itself as spontaneous seizures. Furthermore, the rate of pharmacoresistant epilepsy remains high despite the regular approval of novel anti-seizure medications. Gaining a more complete understanding of the processes that turn a healthy brain into an epileptic brain (epileptogenesis) as well as the processes which generate individual seizures (ictogenesis) may necessitate broadening our focus to other cell types. As will be detailed in this review, astrocytes augment neuronal activity at the level of individual neurons in the form of gliotransmission and the tripartite synapse. Under normal conditions, astrocytes are essential to the maintenance of blood-brain barrier integrity and remediation of inflammation and oxidative stress, but in epilepsy these functions are impaired. Epilepsy results in disruptions in the way astrocytes relate to each other by gap junctions which has important implications for ion and water homeostasis. In their activated state, astrocytes contribute to imbalances in neuronal excitability due to their decreased capacity to take up and metabolize glutamate and an increased capacity to metabolize adenosine. Furthermore, due to their increased adenosine metabolism, activated astrocytes may contribute to DNA hypermethylation and other epigenetic changes that underly epileptogenesis. Lastly, we will explore the potential explanatory power of these changes in astrocyte function in detail in the specific context of the comorbid occurrence of epilepsy and Alzheimer's disease and the disruption in sleep-wake regulation associated with both conditions.

Preclinical pharmacokinetics and tolerability of a novel meglumine-based parenteral solution of topiramate and topiramate combinations for treatment of status epilepticus.

OBJECTIVE: For an antiseizure medication (ASM) to be effective in status epilepticus (SE), the drug should be administered intravenously (i.v.) to provide quick access to the brain. However, poor aqueous solubility is a major problem in the development of parenteral drug solutions. Given its multiple mechanisms of action, topiramate (TPM) is a promising candidate for the treatment of established or refractory SE, as supported by clinical studies using nasogastric tube TPM administration. However, TPM is not clinically available as a solution for i.v. administration, which hampers its use in the treatment of SE. Here, we describe a novel easy-to-use and easy-to-prepare i.v. TPM formulation using the U.S. Food and Drug Administration (FDA)-approved excipient meglumine. METHODS: During formulation development, we compared the solubility of TPM in bi-distilled water with vs without a range of meglumine concentrations. Furthermore, the solubility of combinations of TPM and levetiracetam and TPM, levetiracetam, and atorvastatin in aqueous meglumine concentrations was determined. Subsequently, the pharmacokinetics and tolerability of meglumine-based solutions of TPM and TPM combinations were evaluated in rats, including animals following fluid percussion injury or pilocarpine-induced SE. RESULTS: The amino sugar meglumine markedly enhances the aqueous solubility of TPM. A comparison with data on dissolving TPM using sulfobutylether-ß-cyclodextrin (Captisol) demonstrates that meglumine is much more effective for dissolving TPM. Furthermore, meglumine can be used to prepare drug cocktails where TPM is co-administered with another ASM for SE treatment. The tolerability studies of the meglumine-based TPM solution and meglumine-based TPM combinations in normal rats and the rat fluid percussion injury and pilocarpine-induced SE models demonstrate excellent tolerability of the novel drug solutions. Preclinical studies on antiseizure efficacy in the SE model are underway. SIGNIFICANCE: In conclusion, the novel meglumine-based solution of TPM presented here may be well suited for clinical development.

A randomized controlled trial to evaluate outcomes with Aggrenox in patients with SARS-CoV-2 infection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is an immunoinflammatory and hypercoagulable state that contributes to respiratory distress, multi-organ dysfunction, and mortality. Dipyridamole, by increasing extracellular adenosine, has been postulated to be protective for COVID-19 patients through its immunosuppressive, anti-inflammatory, anti-coagulant, vasodilatory, and anti-viral actions. Likewise, low-dose aspirin has also demonstrated protective effects for COVID-19 patients. This study evaluated the effect of these two drugs formulated together as Aggrenox in hospitalized COVID-19 patients. METHODS: In an open-label, single site randomized controlled trial (RCT), hospitalized COVID-19 patients were assigned to adjunctive Aggrenox (Dipyridamole ER 200mg/ Aspirin 25mg orally/enterally) with standard of care treatment compared to standard of care treatment alone. Primary endpoint was illness severity according to changes on the eight-point COVID ordinal scale, with levels of 1 to 8 where higher scores represent worse illness. Secondary endpoints included all-cause mortality and respiratory failure. Outcomes were measured through days 14, 28, and/or hospital discharge. RESULTS: From October 1, 2020 to April 30, 2021, a total of 98 patients, who had a median [IQR] age of 57 [47, 62] years and were 53.1% (n = 52) female, were randomized equally between study groups (n = 49 Aggrenox plus standard of care versus n = 49 standard of care alone). No clinically significant differences were found between those who received adjunctive Aggrenox and the control group in terms of illness severity (COVID ordinal scale) at days 14 and 28. The overall mortality through day 28 was 6.1% (3 patients, n = 49) in the Aggrenox group and 10.2% (5 patients, n = 49) in the control group (OR [95% CI]: 0.40 [0.04, 4.01], p = 0.44). Respiratory failure through day 28 occurred in 4 (8.3%, n = 48) patients in the Aggrenox group and 7 (14.6%, n = 48) patients in the standard of care group (OR [95% CI]: 0.21 [0.02, 2.56], p = 0.22). A larger decrease in the platelet count and blood glucose levels, and larger increase in creatinine and sodium levels within the first 7 days of hospital admission were each independent predictors of 28-day mortality (p < 0.05). CONCLUSION: In this study of hospitalized patients with COVID-19, while the outcomes of COVID illness severity, odds of mortality, and chance of respiratory failure were better in the Aggrenox group compared to standard of care alone, the data did not reach statistical significance to support the standard use of adjuvant Aggrenox in such patients.

The role of adenosine in alcohol-induced respiratory suppression.

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 A1 receptor antagonist DPCPX (1 mg/kg) and the selective A2A receptor antagonist istradefylline (3.3 mg/kg). Istradefylline, but not DPCPX, reduced alcohol-induced respiratory suppression indicating an A2A receptor mediated effect. In the fifth experiment, alcohol-induced respiratory suppression was evaluated in Adk+/- mice which have impaired adenosine metabolism. Alcohol-induced respiratory suppression was exacerbated in Adk+/- mice. These findings indicate that adenosinergic signaling contributes to alcohol-induced respiratory suppression. Improving our understanding of how alcohol affects breathing may lead to better treatment strategies and better outcomes for patients with severe alcohol poisoning.

Integrin-KCNB1 potassium channel complexes regulate neocortical neuronal development and are implicated in 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 α5ß5 (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.

Diaphragmatic pacing for the prevention of sudden unexpected death in epilepsy.

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 < 0.001) and reduced the likelyhood of seizure-induced death (Fisher's exact test, P = 0.026). Unexpectedly, diaphragmatic pacing did not instantly restore breathing during the postictal period, potentially indicating peripheral airway occlusion by laryngospasm. All diaphragmatically paced animals breathed at some point during the pacing stimulation. Two animals took their first breath prior to the onset of pacing and some animals had significant apnoeas after the pacing stimulation. Sudden unexpected death in epilepsy results in more years of potential life lost than any other neurological condition with the exception of stroke. By demonstrating that seizure-induced respiratory arrest can be prevented by transient diaphragmatic pacing in animal models we hope to inform the development of closed-loop systems capable of detecting and preventing sudden unexpected death in epilepsy.

Astrocytes in the initiation and progression of epilepsy.

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.

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

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

Deep brain stimulation of the anterior thalamus attenuates PTZ kindling with concomitant reduction of adenosine kinase expression in rats.

BACKGROUND: Deep brain stimulation (DBS) of the anterior nucleus of the thalamus (ANT) is an emerging therapy to provide seizure control in patients with refractory epilepsy, although its therapeutic mechanisms remain elusive. OBJECTIVE: We tested the hypothesis that ANT-DBS might interfere with the kindling process using three experimental groups: PTZ, DBS-ON and DBS-OFF. METHODS: 79 male rats were used in two experiments and exposed to chemical kindling with pentylenetetrazole (PTZ, 30 mg/kg i.p.), delivered three times a week for a total of 18 kindling days (KD). These animals were divided into two sets of three groups: PTZ (n = 26), DBS-ON (n = 28) and DBS-OFF (n = 25). ANT-DBS (130 Hz, 90 µs, and 200 µA) was paired with PTZ injections, while DBS-OFF group, although implanted remained unstimulated. After KD 18, the first set of PTZ-treated animals and an additional group of 11 naïve rats were euthanized for brain extraction to study adenosine kinase (ADK) expression. To observe possible long-lasting effects of ANT stimulation, the second set of animals underwent a 1-week treatment and stimulation-free period after KD 18 before a final PTZ challenge. RESULTS: ANT-DBS markedly attenuated kindling progression in the DBS-ON group, which developed seizure scores of 2.4 on KD 13, whereas equivalent seizure scores were reached in the DBS-OFF and PTZ groups as early as KD5 and KD6, respectively. The incidence of animals with generalized seizures following 3 consecutive PTZ injections was 94%, 74% and 21% in PTZ, DBS-OFF and DBS-ON groups, respectively. Seizure scores triggered by a PTZ challenge one week after cessation of stimulation revealed lasting suppression of seizure scores in the DBS-ON group (2.7 ± 0.2) compared to scores of 4.5 ± 0.1 for the PTZ group and 4.3 ± 0.1 for the DBS-OFF group (P = 0.0001). While ANT-DBS protected hippocampal cells, the expression of ADK was decreased in the DBS-ON group compared to both PTZ (P < 0.01) and naïve animals (P < 0.01). CONCLUSIONS: Our study demonstrates that ANT-DBS interferes with the kindling process and reduced seizure activity was maintained after a stimulation free period of one week. Our findings suggest that ANT-DBS might have additional therapeutic benefits to attenuate seizure progression in epilepsy.

ATP and Adenosine Metabolism in Cancer: Exploitation for Therapeutic Gain.

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.

The metabolic basis of epilepsy.

The brain is a highly energy-demanding organ and requires bioenergetic adaptability to balance normal activity with pathophysiological fuelling of spontaneous recurrent seizures, the hallmark feature of the epilepsies. Recurrent or prolonged seizures have long been known to permanently alter neuronal circuitry and to cause excitotoxic injury and aberrant inflammation. Furthermore, pathological changes in bioenergetics and metabolism are considered downstream consequences of epileptic seizures that begin at the synaptic level. However, as we highlight in this Review, evidence is also emerging that primary derangements in cellular or mitochondrial metabolism can result in seizure genesis and lead to spontaneous recurrent seizures. Basic and translational research indicates that the relationships between brain metabolism and epileptic seizures are complex and bidirectional, producing a vicious cycle that compounds the deleterious consequences of seizures. Metabolism-based treatments such as the high-fat, antiseizure ketogenic diet have become mainstream, and metabolic substrates and enzymes have become attractive molecular targets for seizure prevention and recovery. Moreover, given that metabolism is crucial for epigenetic as well as inflammatory changes, the idea that epileptogenesis can be both negatively and positively influenced by metabolic changes is rapidly gaining ground. Here, we review evidence that supports both pathophysiological and therapeutic roles for brain metabolism in epilepsy.

Loss of perivascular aquaporin-4 localization impairs glymphatic exchange and promotes amyloid ß plaque formation in mice.

BACKGROUND: Slowed clearance of amyloid ß (Aß) is believed to underlie the development of Aß plaques that characterize Alzheimer's disease (AD). Aß 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 Aß clearance, and promotes Aß 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 ß 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 Aß 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 ß levels. CONCLUSIONS: These findings suggest that the loss of perivascular AQP4 localization may contribute to the development of AD pathology in human populations.

The impact of methodology on the reproducibility and rigor of DNA methylation data.

Epigenetic modifications are crucial for normal development and implicated in disease pathogenesis. While epigenetics continues to be a burgeoning research area in neuroscience, unaddressed issues related to data reproducibility across laboratories remain. Separating meaningful experimental changes from background variability is a challenge in epigenomic studies. Here we show that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. We examined genome-wide DNA methylation and gene expression profiles of hippocampal tissues from wild-type rats housed in three independent laboratories using nearly identical conditions. Reduced-representation bisulfite sequencing and RNA-seq respectively identified 3852 differentially methylated and 1075 differentially expressed genes between laboratories, even in the absence of experimental intervention. Difficult-to-match factors such as animal vendors and a subset of husbandry and tissue extraction procedures produced quantifiable variations between wild-type animals across the three laboratories. Our study demonstrates that seemingly minor experimental variations, even under normal baseline conditions, can have a significant impact on epigenome outcome measures and data interpretation. This is particularly meaningful for neurological studies in animal models, in which baseline parameters between experimental groups are difficult to control. To enhance scientific rigor, we conclude that strict adherence to protocols is necessary for the execution and interpretation of epigenetic studies and that protocol-sensitive epigenetic changes, amongst naive animals, may confound experimental results.

Genetic variations of adenosine kinase as predictable biomarkers of efficacy of vagus nerve stimulation in patients with pharmacoresistant epilepsy.

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.