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The neuromodulator adenosine maintains brain homeostasis and regulates complex behaviour via activation of inhibitory and excitatory adenosine receptors (ARs) in a brain region-specific manner. AR antagonists such as caffeine have been shown to ameliorate cognitive impairments in animal disease models but their effects on learning and memory in normal animals are equivocal. An alternative approach to reduce AR activation is to lower the extracellular tone of adenosine, which can be achieved by up-regulating adenosine kinase (ADK), the key enzyme of metabolic adenosine clearance. However, mice that globally over-express an Adk transgene ('Adk-tg' mice) were devoid of a caffeine-like pro-cognitive profile; they instead exhibited severe spatial memory deficits. This may be mechanistically linked to cortical/hippocampal N-methyl-d-aspartate receptor (NMDAR) hypofunction because the motor response to acute MK-801 was also potentiated in Adk-tg mice. Here, we evaluated the extent to which the behavioural phenotypes of Adk-tg mice might be modifiable by up-regulating adenosine levels in the cortex/hippocampus. To this end, we investigated mutant 'fb-Adk-def' mice in which ADK expression was specifically reduced in the telencephalon leading to a selective increase in cortical/hippocampal adenosine, while the rest of the brain remained as adenosine-deficient as in Adk-tg mice. The fb-Adk-def mice showed an even greater impairment in spatial working memory and a more pronounced motor response to NMDAR blockade than Adk-tg mice. These outcomes suggest that maintenance of cortical/hippocampal adenosine homeostasis is essential for effective spatial memory and deviation in either direction is detrimental with increased expression seemingly more disruptive than decreased expression.

Glycine transporter 1 (GlyT1) is a potential pharmacological target to ameliorate memory deficits attributable to N-methyl-d-aspartate receptor (NMDAR) hypofunction. Disruption of glycine-reuptake near excitatory synapses is expected to enhance NMDAR function by increasing glycine-B site occupancy. Genetic models with conditional GlyT1 deletion restricted to forebrain neurons have yielded several promising promnesic effects, yet its impact on working memory function remains essentially unanswered because the previous attempt had yielded un-interpretable outcomes. The present study clarified this important outstanding lacuna using a within-subject multi-test approach. Here, a consistent lack of effects was convincingly demonstrated across three working memory tests - the radial arm maze, the cheeseboard maze, and the water maze. These null outcomes contrasted with the phenotype of enhanced working memory performance seen in mutant mice with GlyT1 deletion extended to cortical/hippocampal glial cells. It follows that glial-based GlyT1 might be more closely linked to the modulation of working memory function, and raises the possibility that neuronal and glial GlyT1 may regulate cognitive functions via dissociable mechanisms. Fil: Dubroqua, Sylvain. Laboratory Of Behavioural Neurobiology; Suiza Fil: Serrano, Lucas. Laboratory Of Behavioural Neurobiology; Suiza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Boison, Detlev. Dow Neurobiology Laboratories; Estados Unidos Fil: Feldon, Joram. Laboratory Of Behavioural Neurobiology; Suiza Fil: Gargiulo, Pascual Angel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina Fil: Yee, Benjamín A.. Laboratory Of Behavioural Neurobiology; Suiza

Extracellular levels of the brain's endogenous anticonvulsant and neuroprotectant adenosine largely depend on an astrocyte-based adenosine cycle, comprised of ATP release, rapid degradation of ATP into adenosine, and metabolic reuptake of adenosine through equilibrative nucleoside transporters and phosphorylation by adenosine kinase (ADK). Changes in ADK expression and activity therefore rapidly translate into changes of extracellular adenosine, which exerts its potent anticonvulsive and neuroprotective effects by activation of pre- and postsynaptic adenosine A(1) receptors. Increases in ADK increase neuronal excitability, whereas decreases in ADK render the brain resistant to seizures and injury. Importantly, ADK was found to be overexpressed and associated with astrogliosis and spontaneous seizures in rodent models of epilepsy, as well as in human specimen resected from patients with hippocampal sclerosis and temporal lobe epilepsy. Several lines of evidence indicate that overexpression of astroglial ADK and adenosine deficiency are pathological hallmarks of the epileptic brain. Consequently, adenosine augmentation therapies constitute a powerful approach for seizure prevention, which is effective in models of epilepsy that are resistant to conventional antiepileptic drugs. The adenosine kinase hypothesis of epileptogenesis suggests that adenosine dysfunction in epilepsy undergoes a biphasic response: an acute surge of adenosine that can be triggered by any type of injury might contribute to the development of astrogliosis via adenosine receptor-dependent and -independent mechanisms. Astrogliosis in turn is associated with overexpression of ADK, which was shown to be sufficient to trigger spontaneous recurrent electrographic seizures. Thus, ADK emerges as a promising target for the prediction and prevention of epilepsy.

Severe traumatic brain injury (TBI) is associated with a high incidence of acute mortality followed by chronic alteration of homeostatic network activity that includes the emergence of posttraumatic seizures. We hypothesized that acute and chronic outcome after severe TBI critically depends on disrupted bioenergetic network homeostasis, which is governed by the availability of the brain's endogenous neuroprotectant adenosine. We used a rat lateral fluid percussion injury (FPI) model of severe TBI with an acute mortality rate of 46.7%. A subset of rats was treated with 25 mg/kg caffeine intraperitoneally within 1 min of the injury. We assessed neuromotor function at 24 h and 4 weeks, and video-EEG activity and histology at 4 weeks following injury. We first demonstrate that acute mortality is related to prolonged apnea and that a single acute injection of the adenosine receptor antagonist caffeine can completely prevent TBI-induced mortality when given immediately following the TBI. Second, we demonstrate that neuromotor function is not affected by caffeine treatment at either 24 h or 4 weeks following injury. Third, we demonstrate development of epileptiform EEG bursts as early as 4 weeks post-injury that are significantly reduced in duration in the rats that received caffeine. Our data demonstrate that acute treatment with caffeine can prevent lethal apnea following fluid percussion injury, with no negative influence on motor function or histological outcome. Further, we show epileptiform bursting is reduced after caffeine treatment, suggesting a potential role in the modulation of epilepsy development after severe injury.

Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A(2A) receptors (A(2A)Rs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A(2A)Rs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A(2A)Rs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A(2A)Rs were focally removed from the NAc core or other A(2A)R-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain.

INTRODUCTION: Despite epilepsy being one of the most prevalent neurological disorders, one third of all patients with epilepsy cannot adequately be treated with available antiepileptic drugs. One of the significant causes for the failure of conventional pharmacotherapeutic treatment is the development of pharmacoresistance in many forms of epilepsy. The problem of pharmacoresistance has called for the development of new conceptual strategies that improve future drug development efforts. AREAS COVERED: A thorough review of the recent literature on pharmacoresistance in epilepsy was completed and select examples were chosen to highlight the mechanisms of pharmacoresistance in epilepsy and to demonstrate how those mechanistic findings might lead to improved treatment of pharmacoresistant epilepsy. The reader will gain a thorough understanding of pharmacoresistance in epilepsy and an appreciation of the limitations of conventional drug development strategies. EXPERT OPINION: Conventional drug development efforts aim to achieve specificity of symptom control by enhancing the selectivity of drugs acting on specific downstream targets; this conceptual strategy bears the undue risk of development of pharmacoresistance. Modulation of homeostatic bioenergetic network regulation is a novel conceptual strategy to affect whole neuronal networks synergistically by mobilizing multiple endogenous biochemical and receptor-dependent molecular pathways. In our expert opinion we conclude that homeostatic bioenergetic network regulation might thus be used as an innovative strategy for the control of pharmacoresistant seizures. Recent focal adenosine augmentation strategies support the feasibility of this strategy.

Behavioural characterisation of transgenic mice has been instrumental in search of therapeutic targets for the modulation of cognitive function. However, little effort has been devoted to phenotypic characterisation across environmental conditions and genomic differences such as sex and strain, which is essential to translational research. The present study is an effort in this direction. It scrutinised the stability and robustness of the phenotype of enhanced Pavlovian conditioning reported in mice with forebrain neuronal deletion of glycine transporter 1 by evaluating the possible presence of sex and circadian dependency, and its consistency across aversive and appetitive conditioning paradigms. The Pavlovian phenotype was essentially unaffected by the time of testing between the two circadian phases, but it was modified by sex in both conditioning paradigms. We observed that the effect size of the phenotype was strongest in female mice tested during the dark phase in the aversive paradigm. Critically, the presence of the phenotype in female mutants was accompanied by an increase in resistance to extinction. Similarly, enhanced conditioned responding once again emerged solely in female mutants in the appetitive conditioning experiment, which was again associated with an increased resistance to extinction across days, but male mutants exhibited an opposite trend towards facilitation of extinction. The present study has thus added hitherto unknown qualifications and specifications of a previously reported memory enhancing phenotype in this mouse line by identifying the determinants of the magnitude and direction of the expressed phenotype. This in-depth comparative approach is of value to the interpretation of behavioural findings in general.

Adenosine kinase (ADK) is the major negative metabolic regulator of the endogenous neuroprotectant and homeostatic bioenergetic network regulator adenosine. We used three independent experimental approaches to determine the role of ADK as a molecular target for predicting the brain's susceptibility to ischemic stroke. First, when subjected to a middle cerebral artery occlusion model of focal cerebral ischemia, transgenic fb-Adk-def mice, which have increased ADK expression in striatum (164%) and reduced ADK expression in cortical forebrain (65%), demonstrate increased striatal infarct volume (126%) but almost complete protection of cortex (27%) compared with wild-type (WT) controls, indicating that cerebral injury levels directly correlate to levels of ADK in the CNS. Second, we demonstrate abrogation of lipopolysaccharide (LPS)-induced ischemic preconditioning in transgenic mice with brain-wide ADK overexpression (Adk-tg), indicating that ADK activity negatively regulates LPS-induced tolerance to stroke. Third, using adeno-associated virus-based vectors that carry Adk-sense or -antisense constructs to overexpress or knockdown ADK in vivo, we demonstrate increased (126%) or decreased (51%) infarct volume, respectively, 4 weeks after injection into the striatum of WT mice. Together, our data define ADK as a possible therapeutic target for modulating the degree of stroke-induced brain injury.

Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral-mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures have received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain's endogenous anticonvulsant adenosine, as a potential therapeutic target for antisense-mediated seizure suppression.We developed adenoassociated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense or antisense orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors where injected into the CA3 of wild-type mice or spontaneously epileptic Adk-tg transgenic mice that overexpress ADK in brain. After virus injection, ADK expression was assessed histologically and biochemically. In addition, intracranial electroencephalography (EEG) recordings were obtained.We demonstrate in wild-type mice that viral overexpression of ADK within astrocytes is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event, whereas ADK downregulation via AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice.Our data demonstrate that modulation of astrocytic ADK expression can trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.

Schizophrenia is characterized by positive symptoms such as hallucinations, negative symptoms such as blunted affect, and symptoms of cognitive deficiency such as deficits in working memory and selective attention. N-methyl-d-aspartate receptor (NMDAR) hypofunction has been implicated in all three pathophysiological aspects of the disease. Due to the severe side effects of direct NMDAR agonists, targeting the modulatory co-agonist glycine-B site of the NMDAR is considered to be a promising strategy to ameliorate NMDAR hypofunction. To assess the antipsychotic and pro-cognitive potential of this approach, we examine the strategies designed to enhance glycine-B site occupancy through glycine transporter 1 (GlyT1) blockade. Among the existing transgenic mouse models with GlyT1 deficits, the one specifically targeting forebrain neuronal GlyT1 has yielded the most promising data on cognitive enhancement. Parallel advances in the pharmacology of GlyT1 inhibition point not only to an enhancement of attention, learning and memory but also include suggestions of mood enhancing effects that might be valuable for treating negative symptoms. Thus, interventions at GlyT1 are highly effective in modifying multiple brain functions, and dissection of their respective mechanisms is expected to further maximize their therapeutic potential for human mental diseases.

Sleep and sleep intensity are enhanced by adenosine and its receptor agonists, whereas adenosine receptor antagonists induce wakefulness. Adenosine kinase (ADK) is the primary enzyme metabolizing adenosine in adult brain. To investigate whether adenosine metabolism or clearance affects sleep, we recorded sleep in mice with engineered mutations inAdk. Adk-tg mice overexpress a transgene encoding the cytoplasmic isoform of ADK in the brain but lack the nuclear isoform of the enzyme. Wild-type mice andAdk+/−mice that have a 50% reduction of the cytoplasmic and the nuclear isoforms of ADK served as controls.Adk-tg mice showed a remarkable reduction of EEG power in low frequencies in all vigilance states and in theta activity (6.25–11 Hz) in rapid eye movement (REM) sleep and waking.Adk-tg mice were awake 58 min more per day than wild-type mice and spent significantly less time in REM sleep (102 ± 3 vs 128 ± 3 min in wild type). After sleep deprivation, slow-wave activity (0.75–4 Hz), the intensity component of non-rapid eye movement sleep, increased significantly less inAdk-tg mice and their slow-wave energy was reduced. In contrast, the vigilance states and EEG spectra ofAdk+/−and wild-type mice did not differ. Our data suggest that overexpression of the cytoplasmic isoform of ADK is sufficient to alter sleep physiology. ADK might orchestrate neurotransmitter pathways involved in the generation of EEG oscillations and regulation of sleep.

Adenosine signalling has an important role in cochlear protection from oxidative stress. In most tissues, intracellular adenosine kinase (ADK) is the primary route of adenosine metabolism and the key regulator of intracellular and extracellular adenosine levels. The present study provides the first evidence for ADK distribution in the adult and developing rat cochlea. In the adult cochlea, ADK was localized to the nuclear or perinuclear region of spiral ganglion neurons, lateral wall tissues, and epithelial cells lining scala media. In the developing cochlea, ADK was strongly expressed in multiple cell types at birth and reached its peak level of expression at postnatal day 21 (P21). Ontogenetic changes in ADK expression were evident in the spiral ganglion, organ of Corti, and stria vascularis. In the spiral ganglion, ADK showed a shift from predominantly satellite cell immunolabelling at P1 to neuronal expression from P14 onward. In contrast to the role of ADK in various aspects of cochlear development, the ADK contribution to the cochlear response to noise stress was less obvious. Transcript and protein levels of ADK were unaltered in the cochlea exposed to broadband noise (90-110 dBSPL, 24 hr), and the selective inhibition of ADK in the cochlea with ABT-702 failed to restore hearing thresholds after exposure to traumatic noise. This study indicates that ADK is involved in purine salvage pathways for nucleotide synthesis in the adult cochlea, but its role in the regulation of adenosine signalling under physiological and pathological conditions has yet to be established.

Traditionally, epilepsy has been considered to be a disorder of neuronal dysfunction. Based on this dogma, drug development efforts have largely focused on neurocentric model systems to screen for compounds that affect the function of neurons. Unfortunately, about 30% of all patients with epilepsy – or more than 20 million worldwide – are refractory to classical neurocentric pharmacotherapy. The failure of neurocentric pharmacotherapy in epilepsy requires radical rethinking and the search for novel therapeutic targets. Research from recent years suggests that epilepsy is a disorder of astrocyte dysfunction. Astrocytes are key regulators of the brain’s own anticonvulsant adenosine. Thus, any dysfunction in astrocyte metabolism will drastically affect the brain’s ability to control excitability via adenosinergic neuromodulation. This review will focus on the astrocyte-based enzyme adenosine kinase (ADK) as the key regulator of synaptic adenosine. Astrogliosis – a pathological hallmark of the epileptic brain – leads to overexpression of the adenosine-removing enzyme ADK and therefore to adenosine deficiency. Evidence from transgenic animals demonstrates that overexpression of ADK per se is sufficient to trigger seizures. Consequently, pharmacological inhibition of ADK is very effective in suppressing seizures that are refractory to classical antiepileptic drugs. The recent definition of ADK as rational target to predict and to prevent seizures in epilepsy has prompted the development of focal adenosine augmentation therapies (AATs) that have been designed to selectively reconstitute adenosinergic signalling within an area of astrogliosis-based adenosine-dysfunction. This therapeutic challenge has experimentally been met with polymeric or stem cell based brain implants to afford the focal delivery of adenosine.

Clinical evidence, in particular the wide use of theophylline as a bronchodilator, suggests that methylxanthines can cause seizures in patients without known underlying epilepsy. Theophylline is also known to be an added risk factor for seizure exacerbation in patients with epilepsy. The proconvulsant activity of methylxanthines can best be explained by their antagonizing the brain's own anticonvulsant adenosine. Recent evidence suggests that adenosine dysfunction is a pathological hallmark of epilepsy contributing to seizure generation and seizure spread. Conversely, adenosine augmentation therapies are effective in seizure suppression and prevention, whereas adenosine receptor antagonists such as methylxanthines generally exacerbate seizures. The impact of the methylxanthines caffeine and theophylline on seizures and excitotoxicity depends on timing, dose, and acute versus chronic use. New findings suggest a role of free radicals in theophylline-induced seizures, and adenosine-independent mechanisms for seizure generation have been proposed.

Due to its unique properties, silk fibroin was studied as a biodegradable polymer vehicle for sustained, local delivery of the anticonvulsant adenosine from encapsulated reservoirs. Silk is a biologically derived protein polymer that is biocompatible, mechanically strong and degrades to non-toxic products in vivo. To achieve local, sustained, controlled adenosine release from fully degradable implants, solid adenosine powder reservoirs were coated with silk fibroin. Material properties of the silk coating including thickness, crystallinity and morphology were investigated to assess the relationships between silk coating biomaterial features and adenosine release from silk encapsulated reservoirs. Reservoir coating thickness was varied through manipulation of the silk coating solution concentration and number of coatings applied. Release studies were also performed in proteinase type XIV to model the effects of degradation. Increasing the barrier to diffusion, either by increasing coating thickness or crystallinity was found to delay adenosine burst, decrease average daily release rate, and increase duration of release. In the case of encapsulated reservoirs coated with eight layers of 8% (w/v) silk, a linear release profile was observed and adenosine release was sustained for 14 days. The ability to achieve nearly constant release for two weeks for adenosine via control of the silk coating suggests these encapsulated reservoirs represent a novel system for delivering adenosine. We anticipate that this approach could also be extended to other implant needs and small molecule drugs to treat a range of clinical needs.

Local availability of glycine near N-methyl-D-aspartate receptors (NMDARs) is partly regulated by neuronal glycine transporter 1 (GlyT1), which can therefore modulate NMDAR function because binding to the glycine site of the NMDAR is necessary for channel activation. Disrupting GlyT1 in forebrain neurons has been shown to enhance Pavlovian conditioning and object recognition memory. Here, the authors report that the same genetic manipulation facilitated reversal learning in the water maze test of reference memory, but did not lead to any clear improvement in a working memory version of the water maze test. Facilitation in a nonspatial discrimination reversal task conducted on a T maze was also observed, supporting the conclusion that forebrain neuronal GlyT1 may modulate the flexibility in (new) learning and relevant mnemonic functions. One possibility is that these phenotypes may reflect reduced susceptibility to certain forms of proactive interference. This may be relevant for the suggested clinical application of GlyT1 inhibitors in the treatment of cognitive deficits, including schizophrenia, which is characterized by cognitive inflexibility in addition to the positive symptoms of the disease.

Despite major advances in a variety of neuroscientific research fields, the majority of neurodegenerative and neurological diseases are poorly controlled by currently available drugs, which are largely based on a neurocentric drug design. Research from the past five years has established a central role of glia to determine how neurons function and – consequently – glial dysfunction is implicated in almost every neurodegenerative and neurological disease. Glial cells are key regulators of the brain’s endogenous neuroprotectant and anticonvulsant adenosine. This review will summarize how glial cells contribute to adenosine homeostasis and how glial adenosine receptors affect glial function. We will then move on to discuss how glial cells interact with neurons and the vasculature and outline new methods to study glial function. We will discuss how glial control of adenosine-function affects neuronal cell death and its implications for epilepsy, traumatic brain injury, ischemia, and Parkinson’s disease. Eventually, glial adenosine-modulating drug targets might be an attractive alternative for the treatment of neurodegenerative diseases. There are, however, several major open questions that remain to be tackled.

Sudden unexpected death in epilepsy (SUDEP) is a significant cause of mortality in people with epilepsy. Two postulated causes for SUDEP, cardiac and respiratory depression, can both be explained by overstimulation of adenosine receptors. We hypothesized that SUDEP is a consequence of a surge in adenosine as a result of prolonged seizures combined with deficient adenosine clearance; consequently, blockade of adenosine receptors should prevent SUDEP. Here we induced impaired adenosine clearance in adult mice by pharmacologic inhibition of the adenosine-removing enzymes, adenosine kinase and deaminase. Combination of impaired adenosine clearance with kainic acid-induced seizures triggered sudden death in all animals. Most importantly, the adenosine receptor antagonist caffeine, when given after seizure onset, increased survival from 23.75 +/- 1.35 min to 54.86 +/- 6.59 min (p < 0.01). Our data indicate that SUDEP is due to overactivation of adenosine receptors and that caffeine treatment after seizure onset might be beneficial.

Various silk based drug delivery systems were designed to exploit silk's novel material properties in order to attain slow, sustained, controlled release. Using the small molecule model drug adenosine, release profiles from various silk delivery systems were characterized in vitro. The therapeutic potential of the implants was validated in a dose response study in the rat model of kindling epileptogenesis. Adenosine releasing silk implants engineered to release varied target release doses were implanted in rat brains and dose-dependent delays in epileptogenesis were observed over 14 days. We conclude that silk-based adenosine delivery systems represent a safe and efficient strategy to suppress seizures, and that these degradable, implantable biomaterials could potentially be applied to a range of therapeutics.

Converging evidence from pharmacological and molecular studies has led to the suggestion that inhibition of glycine transporter 1 (GlyT1) constitutes an effective means to boost N-methyl-d-aspartate receptor (NMDAR) activity by increasing the extra-cellular concentration of glycine in the vicinity of glutamatergic synapses. However, the precise extent and limitation of this approach to alter cognitive function, and therefore its potential as a treatment strategy against psychiatric conditions marked by cognitive impairments, remain to be fully examined. Here, we generated mutant mice lacking GlyT1 in the entire forebrain including neurons and glia. This conditional knockout system allows a more precise examination of GlyT1 downregulation in the brain on behavior and cognition. The mutation was highly effective in attenuating the motor-stimulating effect of acute NMDAR blockade by phencyclidine, although no appreciable elevation in NMDAR-mediated excitatory postsynaptic currents (EPSC) was observed in the hippocampus. Enhanced cognitive performance was observed in spatial working memory and object recognition memory while spatial reference memory and associative learning remained unaltered. These findings provide further credence for the potential cognitive enhancing effects of brain GlyT1 inhibition. At the same time, they indicated potential phenotypic differences when compared with other constitutive and conditional GlyT1 knockout lines, and highlighted the possibility of a functional divergence between the neuronal and glia subpopulations of GlyT1 in the regulation of learning and memory processes. The relevance of this distinction to the design of future GlyT1 blockers as therapeutic tools in the treatment of cognitive disorders remains to be further investigated.

Adenosine is a modulator of neuronal activity with anticonvulsant and neuroprotective properties. Conversely, focal deficiency in adenosine contributes to ictogenesis. Thus, focal reconstitution of adenosine within an epileptogenic brain region constitutes a rational therapeutic approach, whereas systemic augmentation of adenosine is precluded by side effects. To meet the therapeutic goal of focal adenosine augmentation, genetic disruption of the adenosine metabolizing enzyme, adenosine kinase (ADK) in rodent cells was used as a molecular strategy to induce adenosine release from cellular brain implants, which demonstrated antiepileptic and neuroprotective properties. Currently, the second generation of adenosine-releasing cells is under development based on the rationale to use human stem cell-derived brain implants to avoid xenotransplantation. To effectively engineer human stem cells to release adenosine, a lentiviral vector was constructed to express inhibitory micro-RNA directed against ADK. Lentiviral knockdown of ADK induced therapeutic adenosine release in human mesenchymal stem cells, which reduced acute injury and seizures, as well as chronic seizures, when grafted into the mouse hippocampus. The therapeutic potential of this approach suggests the feasibility to engineer autologous adenosine-releasing stem cells derived from a patient. Human embryonic stem cells (hESCs) have a high proliferative capacity and can be subjected to specific cellular differentiation pathways. hESCs, differentiated in vitro into neuroepithelial cells and grafted into the mouse brain, displayed intrahippocampal location and neuronal morphology. Using the same lentiviral micro-RNA vector, we demonstrated knockdown of ADK in hESCs. New developments and therapeutic challenges in using human mesenchymal stem cells and hESCs for epilepsy therapy will be critically evaluated.

Adenosine is an endogenous inhibitor of excitatory synaptic transmission with potent anticonvulsant properties in the mammalian brain. Given adenosine's important role in modulating synaptic transmission, several mechanisms exist to regulate its extracellular availability. One of these is the intracellular enzyme adenosine kinase (ADK), which phosphorylates adenosine to AMP. We have investigated the role that ADK plays in regulating the presence and effects of extracellular adenosine in area CA1 of rat hippocampal slices. Inhibition of ADK activity with 5'-iodotubercidin (IODO; 5 mu M) raised extracellular adenosine, as measured with adenosine biosensors, and potently inhibited field excitatory post-synaptic 2 potentials (fEPSPs) in an adenosine A(1)R-dependent manner. In nominally Mg2+-free aCSF, which facilitated the induction of electrically-evoked epileptiform activity, adenosine biosensor recordings revealed that seizures were accompanied by the transient release of adenosine. Under these conditions, IODO also inhibited the fEPSP and greatly suppressed epileptiform activity evoked by brief, high-frequency stimulation. During spontaneous seizures evoked by the AIR antagonist CPT, adenosine release was unaffected by IODO. This suggests that ADK activity does not limit activity-dependent adenosine release. On the basis of strong ADK immunoreactivity in GFAP-positive cells, astrocytes are likely to play a key role in regulating basal adenosine levels. It is this action of ADK on the basal adenosine tone that is permissive to seizure activity, and, by extension, other forms of activity-dependent neuronal activity such as synaptic plasticity. (c) 2008 Elsevier Ltd. All rights reserved.

Adenosine augmentation therapies (AAT) make rational use of the brain's own adenosine-based seizure control system and hold promise for the therapy of refractory epilepsy. In an effort to develop an AAT compatible with future clinical application, we developed a novel silk protein-based release system for adenosine. Adenosine releasing brain implants with target release doses of 0, 40, 200, and 1000ng adenosine per day were prepared by embedding adenosine containing microspheres into nanofilm-coated silk fibroin scaffolds. In vitro, the respective polymers released 0, 33.4, 170.5, and 819.0ng adenosine per day over 14 days. The therapeutic potential of the implants was validated in a dose-response study in the rat model of kindling epileptogenesis. Four days prior to the onset of kindling, adenosine releasing polymers were implanted into the infrahippocampal cleft and progressive acquisition of kindled seizures was monitored over a total of 48 stimulations. We document a dose-dependent retardation of seizure acquisition. In recipients of polymers releasing 819ng adenosine per day, kindling epileptogenesis was delayed by one week corresponding to 18 kindling stimulations. Histological analysis of brain samples confirmed the correct location of implants and electrodes. We conclude that silk-based delivery of around 1000ng adenosine per day is a safe and efficient strategy to suppress seizures.

Astrogliosis is a pathological hallmark of the epileptic brain. The identification of mechanisms that link astrogliosis to neuronal dysfunction in epilepsy may provide new avenues for therapeutic intervention. Here we show that astrocyte-expressed adenosine kinase (ADK), a key negative regulator of the brain inhibitory molecule adenosine, is a potential predictor and modulator of epileptogenesis. In a mouse model of focal epileptogenesis, in which astrogliosis is restricted to the CA3 region of the hippocampus, we demonstrate that upregulation of ADK and spontaneous focal electroencephalographic seizures were both restricted to the affected CA3. Furthermore, spontaneous seizures in CA3 were mimicked in transgenic mice by overexpression of ADK in this brain region, implying that overexpression of ADK without astrogliosis is sufficient to cause seizures. Conversely, after pharmacological induction of an otherwise epileptogenesis-precipitating acute brain injury, transgenic mice with reduced forebrain ADK were resistant to subsequent epileptogenesis. Likewise, ADK-deficient ES cell–derived brain implants suppressed astrogliosis, upregulation of ADK, and spontaneous seizures in WT mice when implanted after the epileptogenesis-precipitating brain injury. Our findings suggest that astrocyte-based ADK provides a critical link between astrogliosis and neuronal dysfunction in epilepsy.

The neuromodulator adenosine fulfills a unique role in the brain affecting glutamatergic neurotransmission and dopaminergic signaling via activation of adenosine A1 and A2A receptors, respectively. The adenosine system is thus ideally positioned to integrate glutamatergic and dopaminergic neurotransmission, which in turn could affect behavior and cognition. In the adult brain, adenosine levels are largely regulated by its key metabolic enzyme adenosine kinase (ADK), which may assume the role of an 'upstream regulator' of these two neurotransmitter pathways. To test this hypothesis, transgenic mice with an overexpression of ADK in brain (Adk-tg), and therefore reduced brain adenosine levels, were evaluated in a panel of behavioral and psychopharmacological assays to assess possible glutamatergic and dopaminergic dysfunction. In comparison to non-transgenic control mice, Adk-tg mice are characterized by severe learning deficits in the Morris water maze task and in Pavlovian conditioning. The Adk-tg mice also exhibited reduced locomotor reaction to systemic amphetamine, whereas their reaction to the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 was enhanced. Our results confirmed that ADK overexpression could lead to functional concomitant alterations in dopaminergic and glutamatergic functions, which is in keeping with the hypothesized role of ADK in the balance and integration between glutamatergic and dopaminergic neurotransmission. The present findings are of relevance to current pathophysiological hypotheses of schizophrenia and its pharmacotherapy.

Adenosine is a modulator of brain function uniquely positioned to integrate excitatory and inhibitory neurotransmission. The past few years brought a wealth of new data fostering our understanding of how the adenosine system is involved in the pathogenesis of neurological diseases. Thus, dysregulation of the adenosine system is implicated in epileptogenesis and cell therapies have been developed to locally augment adenosine in an approach to prevent seizures. While activation of inhibitory adenosine A(1) receptors is beneficial in epilepsy, chronic pain and cerebral ischemia, inhibition of facilitatory A(2A) receptors has profound neuroprotective effects, which are currently exploited in clinical trials in Parkinson's disease. A new era of adenosine-based therapies has begun, with the prospect to cover a wide range of neurological diseases.

Cell therapies based on focal delivery of the inhibitory neuromodulator adenosine were previously shown to provide potent seizure suppression in animal models of epilepsy. However, hitherto used therapeutic cells were derived from rodents and thus not suitable for clinical applications. Autologous patient-derived adenosine-releasing cell implants would constitute a major therapeutic advance to avoid both xenotransplantation and immunosuppression. Here we describe a novel approach based on lentiviral RNAi mediated downregulation of adenosine kinase (ADK), the major adenosine-removing enzyme, in human mesenchymal stem cells (hMSCs), which would be compatible with autologous cell grafting in patients. Following lentiviral transduction of hMSCs with anti-ADK miRNA expression cassettes we demonstrate up to 80% downregulation of ADK and a concentration of 8.5 ng adenosine per ml of medium after incubating 105 cells for 8 hours. hMSCs with a knockdown of ADK or cells expressing a scrambled control sequence were transplanted into hippocampi of mice 1 week prior to the intraamygdaloid injection of kainic acid (KA). While mice with control implants expressing a scrambled miRNA sequence or sham treated control animals were characterized by KA-induced status epilepticus and subsequent CA3 neuronal cell loss, animals with therapeutic ADK knockdown implants displayed a 35% reduction in seizure duration and 65% reduction in CA3 neuronal cell loss, when analyzed 24h after KA-injection. We conclude that lentiviral expression of anti-ADK miRNA constitutes a versatile tool to generate therapeutically effective adenosine releasing hMSCs, thus representing a model system to generate patient identical autologous adult stem cell grafts.

Protective mechanisms of the brain may reduce the extent of injury after focal cerebral ischemia. Here, we explored in a mouse model of focal cerebral ischemia potential synergistic neuroprotective effects of two mediators of neuroprotection: (i) neuronal or glial precursor cells and (ii) the inhibitory neuromodulator adenosine. Embryonic stem (ES) cells, engineered to release adenosine by biallelic disruption of the adenosine kinase gene, and respective wild-type cells were induced to differentiate into either neural or glial precursor cells and were injected into the striatum of mice 1 week before middle cerebral artery occlusion. All stem cell-derived graft recipients were characterized by a significant reduction in infarct volume, an effect that was augmented by the release of adenosine. Neuroprotection was strongest in adenosine-releasing glial precursor cell recipients, which were characterized by an 85% reduction of the infarct area. Graft-mediated neuroprotection correlated with a significant improvement of general and focal neurologic scores. Histologic analysis before and after ischemia revealed clusters of implanted cells within the striatum of all treated mice. We conclude that ES cell derived adenosine-releasing brain implants provide neuroprotection by synergism of endogenous precursor cell-mediated effects and paracrine adenosine release.

The amino acid glycine is the major inhibitory neurotransmitter of spinal cord and brain stem. The termination of the action of glycine is mediated by rapid reuptake into the presynaptic terminal or surrounding glial cells. Two glycine transporters, GLYT1 and GLYT2, are known. They display distinct distributions and are members of the sodium/chloride-dependent transporter family, which share 40-50% amino acid similarity and are characterized by 12 putative transmembrane regions. GLYT2 differs from GLYT1 in molecular structure, tissue specificity, and pharmacologic properties …

4-Aminobutyrate transaminase, a pyridoxal phosphate (PLP) dependent mitochondrial enzyme, is responsible for the degradation of gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, into succinic semialdehyde.

The incidence of drug-induced neutropenia in the United States is estimated at 1–3 million people per year. It is more common in females than in males. Neutropenia secondary to drug use can be due to direct toxicity or destruction of neutrophils via antibodies. It often resolves after stopping the offending drug but some times it can lead to severe infections. The overall mortality rate is approximately 15%. These infections warrant the use of broad-spectrum antibiotics and antifungals. In this module we review the different types of drugs leading to neutropenia, complications of neutropenia and its management.

The amino acid glycine acts as an inhibitory neurotransmitter and as a co-agonist of the NMDA-receptor in mammalian spinal cord and brain. The termination of the action of glycine is mediated by rapid reuptake into the presynaptic terminal or surrounding glial cells. Two glycine transporters, GLYT1 and GLYT2, are known. They display distinct distributions and are members of the sodium/chloride-dependent transporter family, which share 40-50% amino acid similarity and are characterized by 12 putative transmembrane regions. Three glycine transporter variants of GLYT1 (GLYT1a, b, and c) were identified with distinct localizations in the CNS and peripheral tissues …

Despite recent advances in the development of antiepileptic drugs, refractory epilepsy remains a major clinical problem affecting up to 35% of patients with partial epilepsy. Currently, there are few therapies that affect the underlying disease process. Therefore, novel therapeutic concepts are urgently needed. The recent development of experimental cell and gene therapies may offer several advantages compared to conventional systemic pharmacotherapy: (i) Specificity to underlying pathogenetic mechanisms by rational design; (ii) specificity to epileptogenic networks by focal delivery; and (iii) avoidance of side effects. A number of naturally occurring brain substances, such as GABA, adenosine, and the neuropeptides galanin and neuropeptide Y, may function as endogenous anticonvulsants and, in addition, may interact with the process of epileptogenesis. Unfortunately, the systemic application of these compounds is compromised by limited bioavailability, poor penetration of the blood-brain barrier, or the widespread systemic distribution of their respective receptors. Therefore, in recent years a new field of cell and gene-based neuropharmacology has emerged, aimed at either delivering endogenous anticonvulsant compounds by focal intracerebral transplantation of bioengineered cells (ex vivo gene therapy), or by inducing epileptogenic brain areas to produce these compounds in situ (in vivo gene therapy). In this review, recent efforts to develop GABA-, adenosine-, galanin-, and neuropeptide Y- based cell and gene therapies are discussed. The neurochemical rationales for using these compounds are discussed, the advantages of focal applications are highlighted and preclinical cell transplantation and gene therapy studies are critically evaluated. Although many promising data have been generated recently, potential problems, such as long-term therapeutic efficacy, long-term safety, and efficacy in clinically relevant animal models, need to be addressed before clinical applications can be contemplated.

Adenosine is an endogenous neuromodulator with anticonvulsive and neuroprotective activity. Adenosine levels are normally kept in the range of 20 to 200 nmol/L by low basal expression of its main metabolic enzyme, adenosine kinase (ADK). Dysfunction of the adenosinergic system has been demonstrated to contribute to epileptogenesis. To investigate whether upregulation of ADK may render the brain more susceptible to ischemic cell death, mutant mice overexpressing an Adk transgene in brain were subjected to middle cerebral artery occlusion (MCAO). One day after either 15 or 60 mins of MCAO, wild-type (WT) animals had infarct areas encompassing about 5% and 50% of their ischemic hemisphere, respectively. In marked contrast, the volume of the infarcts increased three-fold in Adk transgenic mutants after 15 mins of MCAO, and after 60 mins of MCAO all mutants died within 24 h. Pretreatment of the mutants with the ADK inhibitor 5-iodotubercidin led to lesions similar to those in WT mice. Thus, low levels of ADK are essential to maintain adenosine-mediated neuroprotection. We conclude that pathologic overexpression of ADK as in epilepsy may also render the brain more susceptible to injury from ischemia. Consequently, ADK emerges as a rational therapeutic target to enhance neuroprotection.

Adenosine is a potent modulator of excitatory neurotransmission, especially in seizure-prone regions such as the hippocampal formation. In adult brain ambient levels of adenosine are controlled by adenosine kinase (ADK), the major adenosine-metabolizing enzyme, expressed most strongly in astrocytes. Since ontogeny of the adenosine system is largely unknown, we investigated ADK expression and cellular localization during postnatal development of the mouse brain, using immunofluorescence staining with cell-type specific markers. At early postnatal stages ADK immunoreactivity was prominent in neurons, notably in cerebral cortex and hippocampus. Thereafter, as seen best in hippocampus, ADK gradually disappeared from neurons and appeared in newly developed nestin- and glial fibrillary acidic protein (GFAP)-positive astrocytes. Furthermore, the region-specific downregulation of neuronal ADK coincided with the onset of myelination, as visualized by myelin basic protein staining. After postnatal day 14 (P14), the transition from neuronal to astrocytic ADK expression was complete, except in a subset of neurons that retained ADK until adulthood in specific regions, such as striatum. Moreover, neuronal progenitors in the adult dentate gyrus lacked ADK. Finally, recordings of excitatory field potentials in acute slice preparations revealed a reduced adenosinergic inhibition in P14 hippocampus compared with adult. These findings suggest distinct roles for adenosine in the developing and adult brain. First, ADK expression in young neurons may provide a salvage pathway to utilize adenosine in nucleic acid synthesis, thus supporting differentiation and plasticity and influencing myelination; and second, adult ADK expression in astrocytes may offer a mechanism to regulate adenosine levels as a function of metabolic needs and synaptic activity, thus contributing to the differential resistance of young and adult animals to seizures.

Adenosine kinase deficient (Adk−/−) embryonic stem cells (ESCs) encapsulated in synthetic polymers have previously been shown to provide therapeutic adenosine release and transient seizure suppression in epileptic rats. Here we explored the utility of biopolymer-substrates to promote long-term adenosine release from Adk−/− ESCs. Three different substrates were studied: (1) type I collagen (Col-1), (2) silk-fibroin (SF), and (3) poly( l -ornithine) (PO) coated tissue culture plastic. Adk−/− or wild type (wt) ESC-derived glial precursor cells were seeded on the substrates and cultured either in proliferation medium containing growth factors or in differentiation medium devoid of growth factors. In proliferation medium cell proliferation was higher and metabolic activity lower on Col-1 and PO substrates as compared to SF. Cells from both genotypes readily differentiated into astrocytes after growth factor removal on all substrates. Adk−/− cells cultured on biopolymers released significantly more adenosine than their wt counterparts at all developmental stages. Adenosine release was similar on SF and PO substrates and the amounts released from Adk−/− cells (>20 ng/ml) were considered to be of therapeutic relevance. Taken together, these results suggest that silk matrices are particularly suitable biomaterials for ESC encapsulation and for the design of adenosine releasing bioincubators for the treatment of epilepsy.

Summary: Purpose: Stem cells and their derivatives have emerged as a promising tool for cell-based drug delivery because of (a) their unique ability to differentiate into various somatic cell types, (b) the virtually unlimited donor source for transplantation, and (c) the advantage of being amenable to a wide spectrum of genetic manipulations. Previously, adenosine-releasing embryonic stem (ES) cells have been generated by disruption of both alleles of adenosine kinase (Adk−/-). Lack of ADK did not compromise the cells' differentiation potential into embryoid bodies or glial precursor cells. The aim of the present study was to investigate the potential of differentiated Adk−/- ES cell progeny for seizure suppression by paracrine adenosine release. Methods: To isolate paracrine effects of stem cell–derived implants from effects caused by network integration, ES cell–derived embryoid bodies and glial precursor cells were encapsulated into semipermeable polymer membranes and grafted into the lateral brain ventricles of kindled rats. Results: While seizure activity in kindled rats with wild-type Adk+/+ implants remained unaltered, rats with adenosine-releasing Adk−/- ES cell–derived implants displayed transient protection from convulsive seizures and a profound reduction of afterdischarge activity in EEG recordings. Long-term seizure suppression was precluded by limited viability of the encapsulated cells. Conclusions: We thereby provide a proof-of-principle that Adk−/- ES cell–derived brain implants can suppress seizure activity by a paracrine mode of action. Adk-deficient stem cells therefore represent a potential tool for the treatment of epileptic disorders.

Endogenous adenosine in the brain is thought to prevent the development and spread of seizures via a tonic anticonvulsant effect. Brain levels of adenosine are primarily regulated by the activity of adenosine kinase. To establish a link between adenosine kinase expression and seizure activity, we analyzed the expression of adenosine kinase in the brain of control mice and in a kainic acid-induced mouse model of mesial temporal lobe epilepsy. Immunohistochemical analysis of brain sections of control mice revealed intense staining for adenosine kinase, mainly in astrocytes, which were more or less evenly distributed throughout the brain, as well as in some neurons, particularly in olfactory bulb, striatum, and brainstem. In contrast, hippocampi lesioned by a unilateral kainic acid injection displayed profound astrogliosis and therefore a significant increase in adenosine kinase immunoreactivity accompanied by a corresponding increase of enzyme activity, which paralleled chronic recurrent seizure activity in this brain region. Accordingly, seizures and interictal spikes were suppressed by the injection of a low dose of the adenosine kinase inhibitor 5-iodotubercidin. We conclude that overexpression of adenosine kinase in discrete parts of the epileptic hippocampus may contribute to the development and progression of seizure activity.

entla (ent) is a novel recessive phenotype of mice. The underlying mutation was mapped to chromosome 9 (60.1 centimorgans) and identified as an allele of the Cacna2d2 gene encoding the alpha2delta-2 subunit of voltage-gated calcium channels. The Cacna2d2entla allele harbors a 38-kb duplication comprising the 117 nucleotides of exon 3. The predicted duplication of 39 amino acid residues near the subunit's N terminus results in the expression of a full-length, membrane-associated protein. Western blot data were consistent with correct cleavage of the alpha2delta-2entla precursor into alpha2entla and delta2 proteins but indicated loss of the disulfide linkage between the two proteins. ent/ent mice develop ataxia by postnatal day 13-15, followed by paroxysmal dyskinesia a few days later. Two distinct types of cortical and hippocampal epileptic activity at 2 and 4 Hz were recorded, indicative of absence epilepsy. Homozygotes display reduced size and weight, increased mortality before weaning, and female infertility. No overt neuroanatomical abnormalities were detected. Ca2+ current densities recorded from acutely dissociated Purkinje cells of homozygous entla animals were reduced by 50% compared with wild type. Ligand binding assays using the antiepileptic drug [3H]gabapentin, a specific ligand of the alpha2delta-1 and alpha2delta-2 subunits, revealed a60% reduced maximum binding to cerebellar membranes of ent/ent compared with unaffected littermates. entla is allelic to ducky and ducky2J, representing the third murine Cacna2d2 allele identified and so far the only one encoding an untruncated protein that is incorporated into membranes.

A recent study describing two epileptic patients with brain cysts has suggested that elevated concentrations of excitatory amino acids in cysts may play a role in induction and maintenance of epileptogenesis [Epilepsy Res. 28 (1997) 245]. Here, we report that only in 3 out of 22 patients with brain cysts undergoing brain surgery cyst fluids displayed highly increased amounts of the excitatory amino acids aspartate and/or glutamate. Two of these patients experienced epileptic seizures prior to neurosurgical intervention. Thus, highly increased excitatory amino acid levels are present only in a subset of patients with brain cysts. Our observation that one patient with highly increased glutamate and aspartate concentrations in the cyst did not display seizures or typical epileptiform potentials in the EEG questions that these excitatory amino acids in the cyst fluid are directly involved in epileptogenicity. This patient displayed an increased level of adenosine in the cyst fluid, which is known to have anticonvulsant properties and might provide protection from seizures. In summary, there is no evidence for a close correlation between excitatory amino acids in brain cysts and the occurrence of seizures.