The astrocytic enzyme adenosine kinase (ADK) is a key negative regulator of the brain's endogenous anticonvulsant adenosine. Astrogliosis with concomitant upregulation of ADK is part of the epileptogenic cascade and contributes to seizure generation. To molecularly dissect the respective roles of astrogliosis and ADK-expression for seizure generation, we used a transgenic approach to uncouple ADK-expression from astrogliosis: in Adk-tg mice the endogenous Adk-gene was deleted and replaced by a ubiquitously expressed Adk-transgene with novel ectopic expression in pyramidal neurons, resulting in spontaneous seizures. Here, we followed a unique approach to selectively injure the CA3 of these Adk-tg mice. Using this strategy, we had the opportunity to study astrogliosis and epileptogenesis in the absence of the endogenous astrocytic Adk-gene. After triggering epileptogenesis we demonstrate astrogliosis without upregulation of ADK, but lack of seizures, whereas matching wild-type animals developed astrogliosis with upregulation of ADK and spontaneous recurrent seizures. By uncoupling ADK-expression from astrogliosis, we demonstrate that global expression levels of ADK rather than astrogliosis per se contribute to seizure generation.

Clinical radiation therapy for the treatment of CNS cancers leads to unintended and debilitating impairments in cognition. Radiation-induced cognitive dysfunction is long lasting; however, the underlying molecular and cellular mechanisms are still not well established. Since ionizing radiation causes microglial and astroglial activation, we hypothesized that maladaptive changes in astrocyte function might be implicated in radiation-induced cognitive dysfunction. Among other gliotransmitters, astrocytes control the availability of adenosine, an endogenous neuroprotectant and modulator of cognition, via metabolic clearance through adenosine kinase (ADK). Adult rats exposed to cranial irradiation (10 Gy) showed significant declines in performance of hippocampal-dependent cognitive function tasks [novel place recognition, novel object recognition (NOR), and contextual fear conditioning (FC)] 1 month after exposure to ionizing radiation using a clinically relevant regimen. Irradiated rats spent less time exploring a novel place or object. Cranial irradiation also led to reduction in freezing behavior compared to controls in the FC task. Importantly, immunohistochemical analyses of irradiated brains showed significant elevation of ADK immunoreactivity in the hippocampus that was related to astrogliosis and increased expression of glial fibrillary acidic protein (GFAP). Conversely, rats treated with the ADK inhibitor 5-iodotubercidin (5-ITU, 3.1 mg/kg, i.p., for 6 days) prior to cranial irradiation showed significantly improved behavioral performance in all cognitive tasks 1 month post exposure. Treatment with 5-ITU attenuated radiation-induced astrogliosis and elevated ADK immunoreactivity in the hippocampus. These results confirm an astrocyte-mediated mechanism where preservation of extracellular adenosine can exert neuroprotection against radiation-induced pathology. These innovative findings link radiation-induced changes in cognition and CNS functionality to altered purine metabolism and astrogliosis, thereby linking the importance of adenosine homeostasis in the brain to radiation injury.

Experimental research over the past decade has supported the critical role of astrocytes activated by different types of injury and the pathophysiological processes that underlie the development of epilepsy. In both experimental and human epileptic tissues astrocytes undergo complex changes in their physiological properties, which can alter glio-neuronal communication, contributing to seizure precipitation and recurrence. In this context, understanding which of the molecular mechanisms are crucially involved in the regulation of glio-neuronal interactions under pathological conditions associated with seizure development is important to get more insight into the role of astrocytes in epilepsy. This article reviews current knowledge regarding the role of glial adenosine kinase as a neuropathological marker of the epileptic brain. Both experimental findings in clinically relevant models, as well as observations in drug-resistant human epilepsies will be discussed, highlighting the link between astrogliosis, dysfunction of adenosine homeostasis and seizure generation and therefore suggesting new strategies for targeting astrocyte-mediated epileptogenesis.

This study was undertaken to determine the role of adenosine signalling in the development of age-related hearing loss (ARHL). We and others have shown previously that adenosine signalling via A(1) receptors is involved in cochlear protection from noise-induced cochlear injury. Here we demonstrate that enhanced adenosine signalling in the cochlea provides partial protection from ARHL in C57BL/6J mice. We targeted adenosine kinase (ADK), the key enzyme in adenosine metabolism, using a treatment regime with the selective ADK inhibitor ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the age of three months or six months. This treatment, intended to increase free adenosine levels in the cochlea, was maintained until the age of nine months and hearing thresholds were evaluated monthly using auditory brainstem responses (ABR). At nine months, when C57BL/6J mice normally exhibit significant ARHL, both groups treated with ABT-702 showed lower ABR threshold shifts at 10 and 16kHz compared to control animals receiving the vehicle solution. The better thresholds of the ABT-702-treated mice at these frequencies were supported by increased survival of hair cells in the apical region of the cochlea. This study provides the first evidence that ARHL can be mitigated by enhancing adenosine signalling in the cochlea.

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.

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.

Adenosine A2A receptors (A2AR) are located postsynaptically in striatopallidal GABAergic neurons, antagonizing dopamine D2 receptor functions, and are also located presynaptically at corticostriatal terminals, facilitating glutamate release. To address the hypothesis that these two A2AR populations differently control the action of psychostimulants, we characterized A2AR modulation of cocaine-induced effects at the level of DARPP-32 phosphorylation at Thr-34 and Thr-75, c-Fos expression, and psychomotor activity using two lines of cell-type selective A2AR knockout (KO) mice with selective A2AR deletion in GABAergic neurons (striatum-A2AR-KO mice), or with A2AR deletion in both striatal GABAergic neurons and projecting cortical glutamatergic neurons (forebrain-A2AR-KO mice). We demonstrated that striatum-A2AR KO mice lacked A2ARs exclusively in striatal GABAergic terminals whereas forebrain-A2AR KO mice lacked A2ARs in both striatal GABAergic and glutamatergic terminals leading to a blunted A2AR-mediated facilitation of synaptosomal glutamate release. The inactivation of A2ARs in GABAergic neurons reduced striatal DARPP-32 phosphorylation at Thr-34 and increased its phosphorylation at Thr-75. Conversely, the additional deletion of corticostriatal glutamatergic A2ARs produced opposite effects on DARPP-32 phosphorylation at Thr-34 and Thr-75. This distinct modulation of DARPP-32 phosphorylation was associated with opposite responses to cocaine-induced striatal c-Fos expression and psychomotor activity in striatum-A2AR KO (enhanced) and forebrain-A2AR KO mice (reduced). Thus, A2ARs in glutamatergic corticostriatal terminals and in GABAergic striatal neurons modulate the action of psychostimulants and DARPP-32 phosphorylation in opposite ways. We conclude that A2ARs in glutamatergic terminals prominently control the action of psychostimulants and define a novel mechanism by which A2ARs fine-tune striatal activity by integrating GABAergic, dopaminergic and glutamatergic signaling.

A high-fat low-carbohydrate ketogenic diet (KD) is an effective treatment for refractory epilepsy, yet myriad metabolic effects in vivo have not been reconciled clearly with neuronal effects. A KD limits blood glucose and produces ketone bodies from β-oxidation of lipids. Studies have explored changes in ketone bodies and/or glucose in the effects of the KD, and glucose is increasingly implicated in neurological conditions. To examine the interaction between altered glucose and the neural effects of a KD, we fed rats and mice a KD and restricted glucose in vitro while examining the seizure-prone CA3 region of acute hippocampal slices. Slices from KD-fed animals were sensitive to small physiological changes in glucose, and showed reduced excitability and seizure propensity. Similar to clinical observations, reduced excitability depended on maintaining reduced glucose. Enhanced glucose sensitivity and reduced excitability were absent in slices obtained from KD-fed mice lacking adenosine A1 receptors (A1Rs); in slices from normal animals effects of the KD could be reversed with blockers of pannexin-1 channels, A1Rs, or KATP channels. Overall, these studies reveal that a KD sensitizes glucose-based regulation of excitability via purinergic mechanisms in the hippocampus and thus link key metabolic and direct neural effects of the KD.

Adenosine acts as a neuromodulator and metabolic regulator of the brain through receptor dependent and independent mechanisms. In the brain, adenosine is tightly controlled through its metabolic enzyme adenosine kinase (ADK), which exists in a cytoplasmic (ADK-S) and nuclear (ADK-L) isoform. We recently discovered that ADK-L contributes to adult hippocampal neurogenesis regulation. Although the cerebellum (CB) is a highly plastic brain area with a delayed developmental trajectory, little is known about the role of ADK. Here, we investigated the developmental profile of ADK expression in C57BL/6 mice CB and assessed its role in developmental and proliferative processes. We found high levels of ADK-L during cerebellar development, which was maintained into adulthood. This pattern contrasts with that of the cerebrum, in which ADK-L expression is gradually downregulated postnatally and largely restricted to astrocytes in adulthood. Supporting a functional role in cell proliferation, we found that the ADK inhibitor 5-iodotubericine (5-ITU) reduced DNA synthesis of granular neuron precursors in a concentration-dependent manner in vitro In the developing CB, immunohistochemical studies indicated ADK-L is expressed in immature Purkinje cells and granular neuron precursors, whereas in adulthood, ADK is absent from Purkinje cells, but widely expressed in mature granule neurons and their molecular layer (ML) processes. Furthermore, ADK-L is expressed in developing and mature Bergmann glia in the Purkinje cell layer, and in astrocytes in major cerebellar cortical layers. Together, our data demonstrate an association between neuronal ADK expression and developmental processes of the CB, which supports a functional role of ADK-L in the plasticity of the CB.

Adenosine A2A receptors (A2ARs) are enriched in the striatum but are also present at lower levels in the extrastriatal forebrain (i.e., hippocampus, cortex), integrating dopamine, glutamate, and brain-derived neurotrophic factor (BDNF) signaling, and are thus essential for striatal neuroplasticity and fear and anxiety behavior.

The ubiquitous metabolic intermediary and nucleoside adenosine is a "master regulator" in all living systems. Under baseline conditions adenosine kinase (ADK) is the primary enzyme for the metabolic clearance of adenosine. By regulating the availability of adenosine, ADK is a critical upstream regulator of complex homeostatic and metabolic networks. Not surprisingly, ADK dysfunction is involved in several pathologies, including diabetes, epilepsy, and cancer. ADK protein exists in the two isoforms nuclear ADK-L, and cytoplasmic ADK-S, which are subject to dynamic expression changes during brain development and in response to brain injury; however, gene expression changes of the Adk gene as well as regulatory mechanisms that direct the cell-type and isoform specific expression of ADK have never been investigated. Here we analyzed potential gene regulatory mechanisms that may influence Adk expression including DNA promoter methylation, histone modifications and transcription factor binding. Our data suggest binding of transcription factor SP1 to the Adk promoter influences the regulation of Adk expression.

Adenosine kinase (ADK) deficiency in human patients (OMIM:614300) disrupts the methionine cycle and triggers hypermethioninemia, hepatic encephalopathy, cognitive impairment, and seizures. To identify whether this neurological phenotype is intrinsically based on ADK deficiency in the brain or if it is secondary to liver dysfunction, we generated a mouse model with a brain-wide deletion of ADK by introducing a Nestin-Cre transgene into a line of conditional ADK deficient Adkfl/fl mice. These AdkΔbrain mice developed a progressive stress-induced seizure phenotype associated with spontaneous convulsive seizures and profound deficits in hippocampus-dependent learning and memory. Pharmacological, biochemical, and electrophysiological studies suggest enhanced adenosine levels around synapses resulting in an enhanced adenosine A1 receptor (A1R)-dependent protective tone despite lower expression levels of the receptor. Theta-burst-induced LTP was enhanced in the mutants and this was dependent on adenosine A2A receptor (A2AR) and tropomyosin-related kinase B signaling, suggesting increased activation of these receptors in synaptic plasticity phenomena. Accordingly, reducing adenosine A2A receptor activity in AdkΔbrain mice restored normal associative learning and contextual memory and attenuated seizure risk. We conclude that ADK deficiency in the brain triggers neuronal adaptation processes that lead to dysregulated synaptic plasticity, cognitive deficits, and increased seizure risk. Therefore, ADK mutations have an intrinsic effect on brain physiology and may present a genetic risk factor for the development of seizures and learning impairments. Furthermore, our data show that blocking A2AR activity therapeutically can attenuate neurological symptoms in ADK deficiency.

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.

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 produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by in vivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

The ATP-gated ionotropic P2X7 receptor (P2X7R) modulates glial activation, cytokine production and neurotransmitter release following brain injury. Levels of the P2X7R are increased in experimental and human epilepsy but the mechanisms controlling P2X7R expression remain poorly understood. Here we investigated P2X7R responses after focal-onset status epilepticus in mice, comparing changes in the damaged, ipsilateral hippocampus to the spared, contralateral hippocampus. P2X7R-gated inward currents were suppressed in the contralateral hippocampus and P2rx7 mRNA was selectively uploaded into the RNA-induced silencing complex (RISC), suggesting microRNA targeting. Analysis of RISC-loaded microRNAs using a high-throughput platform, as well as functional assays, suggested the P2X7R is a target of microRNA-22. Inhibition of microRNA-22 increased P2X7R expression and cytokine levels in the contralateral hippocampus after status epilepticus and resulted in more frequent spontaneous seizures in mice. The major pro-inflammatory and hyperexcitability effects of microRNA-22 silencing were prevented in P2rx7(-/-) mice or by treatment with a specific P2X7R antagonist. Finally, in vivo injection of microRNA-22 mimics transiently suppressed spontaneous seizures in mice. The present study supports a role for post-transcriptional regulation of the P2X7R and suggests therapeutic targeting of microRNA-22 may prevent inflammation and development of a secondary epileptogenic focus in the brain.

Area II of the 2014 Epilepsy Research Benchmarks aims to establish goals for preventing the development and progression of epilepsy. In this review, we will highlight key advances in Area II since the last summary of research progress and opportunities was published in 2016. We also highlight areas of investigation that began to develop before 2016 and in which additional progress has been made more recently.

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.

Traumatic brain injury (TBI) is a major public health concern and remains a leading cause of disability and socio-economic burden. To date, there is no proven therapy that promotes brain repair following an injury to the brain. In this study, we explored the role of an isoform of adenosine kinase expressed in the cell nucleus (ADK-L) as a potential regulator of neural stem cell proliferation in the brain. The rationale for this hypothesis is based on coordinated expression changes of ADK-L during foetal and postnatal murine and human brain development indicating a role in the regulation of cell proliferation and plasticity in the brain. We first tested whether the genetic disruption of ADK-L would increase neural stem cell proliferation after TBI. Three days after TBI, modelled by a controlled cortical impact, transgenic mice, which lack ADK-L (ADKΔneuron) in the dentate gyrus (DG) showed a significant increase in neural stem cell proliferation as evidenced by significant increases in doublecortin and Ki67-positive cells, whereas animals with transgenic overexpression of ADK-L in dorsal forebrain neurons (ADK-Ltg) showed an opposite effect of attenuated neural stem cell proliferation. Next, we translated those findings into a pharmacological approach to augment neural stem cell proliferation in the injured brain. Wild-type C57BL/6 mice were treated with the small molecule adenosine kinase inhibitor 5-iodotubercidin for 3 days after the induction of TBI. We demonstrate significantly enhanced neural stem cell proliferation in the DG of 5-iodotubercidin-treated mice compared to vehicle-treated injured animals. To rule out the possibility that blockade of ADK-L has any effects in non-injured animals, we quantified baseline neural stem cell proliferation in ADKΔneuron mice, which was not altered, whereas baseline neural stem cell proliferation in ADK-Ltg mice was enhanced. Together these findings demonstrate a novel function of ADK-L involved in the regulation of neural stem cell proliferation after TBI.