Adenosine is an endogenous anticonvulsant that is controlled by an astrocyte-based adenosine cycle and expression levels of its key negative regulator adenosine kinase (ADK). Astrogliosis, a pathological hallmark of the epileptic brain, is universally accompanied by overexpression of ADK and resulting adenosine deficiency. Thereby deregulated ADK provides a molecular link between astrogliosis and neuronal dysfunction in epilepsy. Since overexpression of ADK and resulting adenosine deficiency are sufficient to initiate spontaneous recurrent seizures, focal adenosine augmentation therapies (AATs) constitute a rational approach for the suppression and prevention of seizures. Systemic pharmacological AATs using either ADK inhibitors or adenosine A1 receptor agonists are effective in seizure suppression, however accompanied by wide-spread, largely cardiovascular, side-effects that can only be avoided via focal delivery approaches. As therapeutic tools to achieve focal AAT, stem cells have been engineered to release therapeutic amounts of adenosine using RNA interference or gene targeting technology. In rodent models, stem cell-derived brain-implants provide robust protection from induced and spontaneous seizures through the paracrine delivery of adenosine. Most recently, silk-based polymers have been bioengineered to release adenosine with the goal to translate focal AATs into clinical applications.
The ribonucleoside adenosine is based on the purine base adenine, which was most likely already present on the pre-biotic primitive Earth.1 Being the core molecule of the energy metabolite adenosine-5′-triphosphate (ATP) as well as being an integral component of both DNA and RNA, adenosine likely played an important role in early evolution as ideally positioned negative feedback regulator to adjust cellular activity (DNA, RNA) to available energy supplies (ATP). Adenosine has therefore evolved as an important modulator of function in brain, but also in heart, skeletal muscle, kidney, and adipose tissue, in the sense of a “retaliatory metabolite” that protects the cell against excessive external stimulation.2
Adenosine was first recognized as an endogenous modulator of neuronal excitability in 1980.3 Today it is well recognized that adenosine exerts potent anticonvulsant4 and neuroprotective5 functions in brain that are largely mediated via the activation of pre- and postsynaptic G protein-coupled adenosine A1 receptors (A1Rs) providing presynaptic inhibition and stabilization of the postsynaptic membrane potential, respectively.6, 7 Furthermore, a rise in endogenous adenosine has been documented during ongoing seizure activity in patients with epilepsy,8 and adenosine has consequently been identified as an endogenous mediator of seizure arrest and postictal refractoriness.8, 9 In addition, A1Rs, which are the dominant receptor subtype in the limbic system, are crucial in preventing the spread of seizures10 and lack of the receptor is associated with spontaneous electrographic seizures,11 and with increased susceptibility to seizure-11 or trauma-12 induced mortality. Apart from the direct role of A1Rs in regulating the susceptibility to seizures, stimulatory A2ARs, which in brain have their highest expression levels in striatum, appear to be involved in the regulation of several pathways thought to be implicated in epileptogenesis. Notably, A2ARs directly interact with various transmitter systems to modulate both inflammatory, as well as cognitive processes.13, 14
Clinical evidence, in particular the widespread use of the adenosine receptor antagonist theophylline as bronchodilator, suggests that methylxanthines can cause seizures in patients without known underlying epilepsy.15 Conversely, pharmacological activation of A1Rs provides effective seizure control in animal models,16 albeit accompanied by significant, largely cardiovascular, side effects that are common after systemic use of adenosinergic drugs.17 Importantly, pharmacological activation of A1Rs effectively suppressed seizures in mice that were resistant to treatment with conventional antiepileptic drugs.18 Despite 30 years of research on adenosine and epilepsy, only the recent advent of novel molecular tools has led to the discovery of adenosine-related pathogenetic mechanisms in epilepsy and the translation of those findings into novel therapeutic approaches.
ADENOSINE DYSFUNCTION IN EPILEPSY
Although astrogliosis is a pathological hallmark of the epileptic brain, the important role of astrocytes in the control of neuronal excitability has only recently been appreciated.19, 20 Via the formation of tripartite synapses, astrocytes control synaptic transmission and neurovascular coupling.21 Importantly, both purinergic22, as well as glutamatergic23 signalling from astrocytes was shown to influence neuronal networks and an astrocytic basis of epilepsy has been proposed.23
Astrocytes and Adenosine Kinase are Key Regulators of Synaptic Adenosine
Synaptic levels of adenosine are largely controlled by an astrocyte-based adenosine cycle.6, 24 The major source of synaptic adenosine is its precursor ATP that can be released by astrocytes via regulated vesicular transport22 or via hemichannels25. Once in the synaptic cleft, ATP is rapidly degraded into adenosine by a cascade of ectonucleotidases.26 Proof that the astrocytic release of ATP plays a crucial role in regulating neuronal excitability via adenosine was provided using inducible transgenic mice that express a dominant-negative SNARE domain selectively in astrocytes to block the astrocytic release of ATP. Using these mice it was shown that by releasing ATP, which accumulated as adenosine, astrocytes tonically suppressed synaptic transmission.22 In contrast to classical neurotransmitters, which are removed from the extracellular space via specific energy-driven re-uptake transporters, astrocyte membranes contain two types of equilibrative transporters for adenosine that rapidly equilibrate extra- and intracellular levels of adenosine.27 Due to the lack of a classical transporter-regulated reuptake system for adenosine, the intracellular astrocyte-specific enzyme adenosine kinase (ADK) has likely adopted the role of a metabolic reuptake system for adenosine. Thus, by phosphorylation of adenosine into 5′-adenosine monophosphate (AMP), ADK drives the influx of adenosine into the astrocyte thereby reducing the concentration of synaptic adenosine.6, 24, 28 The notion that ADK is the key enzyme for the regulation of ambient adenosine in brain is further supported by the following evidence: (i) In adult brain, ADK expression is restricted to astrocytes.29(ii) Inhibition of ADK in hippocampal slices increases endogenous adenosine and depresses neuronal firing, whereas inhibition of ADA has no effect.30
(iii) Pharmacological inhibition of ADK suppresses seizures in models of epilepsy.31(iv) Genetic disruption of ADK induces an increase in adenosine.32–35(v) Overexpression of ADK triggers seizures by reduction of ambient adenosine.36(vi) A substrate cycle between AMP and adenosine, which involves ADK and 5′-nucleotidase, enables minor changes in ADK activity to rapidly translate into major changes in adenosine.37
Adenosine Kinase is a Target for Seizure Prediction and Prevention
The crucial role of ADK in regulating the synaptic availability of adenosine and thus neuronal excitability has led to the proposal of the “adenosine kinase hypothesis of epileptogenesis”.24 In an acute response to injury to the brain, a surge in micromolar levels of adenosine is triggered38 that is further potentiated by acute downregulation of ADK during the first hours after experimental stroke39 or status epilepticus.40
The acute injury-induced surge of adenosine is increasing the neuroprotective tone during the first hours following an insult to the brain, and seems to be a ubiquitous endogenous protective mechanism.24 However, this acute surge in adenosine is also able to trigger several downstream pathways that can all contribute to the initiation of epileptogenesis. Several downstream mechanisms can be influenced by an acute surge of adenosine: Adenosine is an important modulator of inflammation.41 Both, activation of A1 as well as of A2A receptors triggers the proliferation of microglial cells.42 In addition, adenosine A2A receptor activation promotes inflammatory responses that are specific to the CNS.43 Furthermore, increased levels of adenosine can induce downregulation of A1Rs but upregulation of A2ARs on astrocytes.44 Increased occupancy of astrocytic A2ARs was shown to increase astrocyte proliferation and activation.45,46 Therefore, any injury-induced surge in adenosine can be a direct trigger for subsequent astrogliosis.
Based on the mechanisms discussed above, it is not surprising that astrogliosis is a common pathological consequence of different types of brain injury, such as status epilepticus, trauma, or stroke, but is also a common pathological hallmark of a variety of neurodegenerative and neuropsychiatric disorders including Alzheimer’s Disease and autism (). As will be discussed in more detail below, astrogliosis is accompanied by overexpression of ADK, thereby inducing focal adenosine deficiency and spontaneous electrographic seizures. Adenosine deficiency can also explain a wide spectrum of co-morbidities that are common in epilepsy or after brain injury (). Evidence that ADK, expressed by astrocytes, is a key molecular link between astrogliosis and neuronal dysfunction in epilepsy has been obtained in seizure models as well as through genetic and pharmacological approaches.
Astrogliosis and adenosine deficiency link diverse pathologies with co-morbidities. Astrogliosis – a common pathological hallmark of conditions as diverse as epilepsy, traumatic brain injury (TBI), Alzheimer’s disease and autism – (more...)
Evidence from Seizure Models
Temporal lobe epilepsy is characterized by a complex pathophysiology including mossy fiber sprouting, granule cell dispersion, neuronal cell loss, ectopic neurons, and astrogliosis. Given this complex situation it is almost impossible to attribute specific cause-effect relationships between those pathological changes and the development of seizures. To dissect out the specific contribution of astrogliosis to seizure generation a mouse model was created that isolates astrogliosis from other components of the epileptogenic cascade.47 In this model, a single unilateral injection of the excitotoxin kainic acid (KA) into the basolateral amygdala triggers status epilepticus that is terminated after 30 minutes with an intravenous infusion of lorazepam. This manipulation results in neuronal cell loss that is restricted to the ipsilateral CA3 area of the hippocampal formation.47 The induced acute injury constitutes a trigger for subsequent epileptogenesis and three weeks after KA the ipsilateral CA3 is characterized by prominent astrogliosis and overexpression of ADK.47 Remarkably frequent spontaneous electrographic seizures (around 4 seizures per hour, with a duration of 20 seconds per seizure) result that are restricted to the ipsilateral CA3.47 These findings demonstrate spatial colocalization of astrogliosis, overexpressed ADK and spontaneous seizures, and document that astrogliosis and/or overexpression of ADK per se and in the absence of any other component of the epileptogenic cascade is sufficient to trigger seizures. Remarkably, during epileptogenesis in this model, astrogliosis, overexpression of ADK, and first emergence of spontaneous seizures also coincide temporally around day 12 following the KA injection, further highlighting the possible causal relationship between those events.11
Evidence from Genetic Approaches
The seizure model described above does not allow distinguishing whether astrogliosis or overexpression of ADK are required for seizure generation. To address this question, transgenic mice were engineered with a global brain-wide overexpression of an Adk-transgene on top of a deletion of the endogenous Adk gene (Adk-tg mice). These animals are characterized by spontaneous hippocampal seizures in the absence of astrogliosis or any other histopathological alterations.11 These data demonstrate that overexpression of ADK in the absence of any histopathological alteration usually associated with an epileptic brain is sufficient to trigger seizures. More recently, an injection of an adeno associated virus (AAV) overexpressing a cDNA of Adk selectively in astrocytes was shown to trigger the same type of spontaneous seizures in otherwise healthy wild-type mice, whereas the injection of an AAV expressing an Adk antisense construct into the CA3 of Adk-tg mice was able to almost completely abolish spontaneous seizures in those animals.48 Together these data define ADK as rational target for therapeutic intervention.
Evidence from Pharmacological Approaches
The identification of overexpressed ADK and resulting adenosine deficiency as major inducer of seizures implies that adenosine augmentation therapies (AATs) should be highly effective in seizure suppression. Indeed, adenosine A1R agonists effectively inhibit neuronal activity, suppress seizures49 and have been the subject of intense drug development efforts.50, 51 Although A1R agonists are effective in a variety of models including one of pharmacoresistant epilepsy,18 the systemic application of those drugs leads to profound cardiovascular and sedative side effects.52 Sedative side-effects of systemic adenosine augmentation can best be explained by the sleep-promoting effects of A1R activation.53 Since any type of injury or stress to the brain leads to an increase in endogenous adenosine,8, 38, 54 agents (e.g. the ADK inhibitor ABT-70255, 56) that amplify this site- and event-specific increase in adenosine could provide antiseizure activity comparable to adenosine receptor agonists.31, 57 Consequently, pharmacological inhibition of ADK is effective in inhibiting epileptic seizures31, 40 with an improved therapeutic window compared to A1R agonists58. However, systemic ADK inhibitors might not be a longterm therapeutic option for epilepsy due to interference with methionine metabolism in liver59, 60 and the risk of brain hemorrhage61, 62.
FOCAL ADENOSINE AUGMENTATION
Rationale for Focal Drug Delivery Approaches in Epilepsy
Side effects from systemic adenosine augmentation can most effectively be avoided by focal treatment approaches. In general, spatially restricted therapies are considered to be safe and feasible alternatives for systemic drug use and given the focal nature of many epilepsies, focal interventions might be preferable.63 The identification of neurochemical deficits which are specific for an epileptogenic focus provide a direct rationale for focal intervention (). Endogenous antiepileptic “drugs” such as GABA,64–66 adenosine,67–69 galanin,70–72 or NPY73, 74 are therefore logical candidates for therapeutic intervention. Tools for focal drug delivery have been reviewed recently75–78 and include polymeric brain implants,79, 80 cell therapy,81–84 or gene therapy.85–87 In contrast to conventional systemically used antiepileptic drugs, the rational focal use of endogenous anticonvulsants is uniquely posed to reconstitute normal signalling within an epileptogenic focus and thereby not only to suppress seizures but also to affect disease progression in the absence of systemic side effects (). As outlined above, manipulation of ADK and focal adenosine augmentation are considered to be an effective and rational strategy for epilepsy therapy.
Rationale for local drug delivery in epilepsy.
Molecular Approaches to Induce Adenosine Augmentation
The most effective strategy to increase levels of ambient adenosine is disruption of metabolic adenosine clearance. Pharmacologically, it has been demonstrated that inhibition of ADK is more effective in raising adenosine and in enhancing presynaptic inhibition, than blockade of adenosine deaminase (ADA).30 Likewise, engineered fibroblasts with a lack of ADK released 2.3 times more adenosine compared to fibroblasts lacking ADA.35 Consequently, ADK is an effective target to induce cellular adenosine release. Two molecular strategies have been used to augment the adenosine system: (i) Gene targeting to disrupt the endogenous Adk gene,34 and (ii) RNA interference (RNAi) to knockdown ADK expression.88
Using a gene targeting construct to disrupt the endogenous Adk gene by homologous recombination in murine embryonic stem cells (ESCs) and subsequent biochemical selection for ADK-deficiency, ESCs were isolated with a biallelic disruption of their endogenous Adk gene.34 Upon directed differentiation in vitro into either neuronal or glial cell populations, the ADK-deficient cells released up to 40 ng adenosine per hour per 105 cells, an amount considered to be of therapeutic relevance. To induce therapeutic adenosine release in adult stem cells, an RNAi approach was used to induce a knockdown of ADK in human mesenchymal stem cells (hMSCs). This was achieved by constructing a lentivirus expressing an artificial micro-RNA directed against ADK. Transduction of hMSCs with the virus yielded cell populations in which ADK expression was reduced by up to 80%; this manipulation resulted in a release of about 1 ng adenosine per hour per 105 cells.32 Control cells, transduced with a lentivirus expressing a scrambled control sequence did not release detectable amounts of adenosine. These results demonstrate that although a complete genetic disruption of the Adk gene is more effective in inducing adenosine release, RNAi based strategies can effectively be used to engineer stem cells to release adenosine.
Seizure Suppression and Prevention by Focal Adenosine Augmentation
Proof of Principle
The first proof of principle that focal adenosine augmentation might be effective for seizure control was demonstrated in the rat kindling model. Synthetic ethylene vinyl acetate copolymers were engineered to release an amount of around 20 to 50 ng adenosine per day.89 Individual polymers were implanted into the lateral brain ventricles of rats that had been kindled in the hippocampus. Recipients of adenosine-releasing polymers were characterized by a strong reduction of stage 5 seizures for at least 7 days and by reduction of epileptiform electrical afterdischarges up to 3 days. In line with a transient delivery of adenosine, the antiepileptic effects gradually decreased and were no longer evident two weeks after polymer implantation. Control implants loaded with BSA failed to display any therapeutic effects. This was the first published demonstration that a focal release of adenosine in doses of 20 to 50 ng adenosine per day can suppress epileptic seizures.89 Subsequently, robust but transient seizure suppression in kindled rats was demonstrated using intraventricular implants of encapsulated fibroblasts engineered to release adenosine.35 The proof of principle that focal adenosine delivery can be of therapeutic benefit was further validated by an independent research group using intracranial adenosine injections in a rat seizure model.69
Stem Cell Based Adenosine Delivery
In the adult brainthe subgranular zone of the hippocampal formation, as well as the subventricular zone contain primitive cell sources and stem cells capable of repairing the injured brain.90 Therefore, stem cell therapies are a logical choice for regeneration and repair of the epileptic hippocampal formation. While stem cell-derived brain implants may indeed repair the injured hippocampus in epilepsy and may be of therapeutic value by modifying circuitry through synaptic interactions,91 stem cell-based brain implants may also exert direct anticonvulsant activity via the paracrine release of adenosine.92 Based on the therapeutic efficacy of paracrine adenosine delivery,35, 92 stem cell-based adenosine delivery can effectively be achieved by injecting the cells into the infrahippocampal fissure of rodents thereby avoiding interference with hippocampal circuitry.93 Infrahippocampal implants of neural precursor cells derived from murine or human ESCs, as well as hMSCs survive within the infrahippocampal fissure for at least several weeks.32, 47, 93–95
Infrahippocampal implants of adenosine-releasing stem cells provided potent seizure control in several experimental paradigms: (i) ESC-derived neural precursor cells injected into rats prior to the onset of kindling provided robust suppression of kindling epileptogenesis.93(ii) The same cells injected after intraamygdaloid kainic acid induced status epilepticus in mice, prevented the development of spontaneous seizures three weeks after the injury, a time point when recipients of control cells had developed recurrent electrographic CA3-seizures at a rate of about 4 seizures per hour;47 remarkably, those animals with the adenosine releasing implants were characterized by significantly reduced astrogliosis and almost normal expression levels of ADK. Together, these data suggest a novel disease-modifying and possibly antiepileptogenic effect of focal adenosine augmentation.47(iii) Adenosine-releasing hMSC-implants were demonstrated to provide both neuroprotective as well as antiepileptic effects in the mouse intraamygdaloid KA model of CA3-restricted epileptogenesis;32, 95 however, due to 40-times lower amounts of released adenosine, those effects were less profound compared to those observed in ESC-recipients.
Silk Based Adenosine Delivery
Although stem cell-based adenosine delivery yielded robust antiepileptic and possibly antiepileptogenic effects in two different models of epilepsy, those approaches still seem far from future clinical implementation. To provide a platform for rapid clinical implementation of focal adenosine augmentation, the natural biopolymer silk was recently evaluated for the therapeutic delivery of adenosine.79, 96 Purified silk fibroin presents a unique option for therapeutic adenosine delivery as it is biocompatible and biodegrades slowly.97 Degradation kinetics can be regulated to allow control of release from weeks to years.97 Both silk as well as adenosine are FDA-approved and the frequent use of silk sutures in brain confirms the feasibility of implanting silk biomaterials into brain.
In the first therapeutic silk-based adenosine delivery approach, a hierarchically structured silk-based implant was engineered with target release rates of 0, 40, 200, and 1000 ng adenosine per day.79 The devices were implanted into the infrahippocampal fissure of rats prior to the onset of electrical kindling. It was demonstrated that focal adenosine release from silk-based polymers dose-dependently retarded kindling epileptogenesis. Importantly, recipients of polymers releasing a target dose of 1000 ng adenosine per day did not display any behavioral seizures during the time window of active adenosine release, whereas control rats experienced convulsions during the same period. As soon as adenosine-release from the polymers began to wear off, seizures gradually re-appeared with progressive intensity.79
Likewise, when implanted after completion of kindling into fully kindled rats (reproducible stage 4 to 5 seizures), implants engineered to release 1000ng adenosine for a defined time frame of ten days, effectively suppressed seizures for ten days in fully kindled rats. After expiration of adenosine release from the polymers, stage 4 to 5 seizures recurred.98 To assess a potential antiepileptogenic effect of focal adenosine delivery, unilateral (ipsilateral to side of kindling) implantations with the same adenosine-releasing or control polymers were performed prior to the onset of kindling. All animals received a total of 30 kindling stimulations between day 4 and 8 after polymer implantation. This treatment resulted in stable stage 5 seizure expression in control animals, whereas seizures stages in adenosine-treated animals did not progress beyond stage 1. The lack of convulsive seizures in the adenosine group can either be attributed to antiepileptogenic effects of focal adenosine delivery or merely to seizure suppression by adenosine (masking potential antiepileptogenic effects). To distinguish between the two possibilities, kindling was discontinued to allow expiration of adenosine release from the polymers. At day 18 kindling was resumed. Whereas control animals continued with generalized stage 5 seizures, animals from the adenosine group resumed kindling at stage 0 to 1, indicating that epileptogenesis was indeed suppressed during the time window of active adenosine delivery.98 Pharmacological control experiments with the adenosine A1 receptor antagonist DPCPX further corroborated disease-modifying effects of focal adenosine delivery: Whereas a dose of 1 mg/kg DPCPX has no effects in non-kindled control animals, DPCPX restores stage 5 seizures in fully kindled rats that are protected from transient adenosine delivery.35 Importantly, DPCPX did not trigger seizures in kindled animals that received adenosine-releasing implants prior to the onset of kindling, a result that further substantiated a possible antiepileptogenic effect of focal adenosine delivery.98
Together these studies document the therapeutic potential of silk-based adenosine delivery, capitalizing on a biopolymer that fulfills crucial requirements for future clinical application, such as: biocompatibility, delivery of defined doses of adenosine, safety, and therapeutic efficacy in a widely used preclinical model. Although the polymer design used in this study 79 precluded long-term applications due to adenosine depletion over a period of two weeks, the system would nevertheless be suitable for initial short-term clinical safety and feasibility trials.
CHALLENGES AND IMPACT
Data from the acute and chronic models of epilepsy discussed above suggest that focal AAT’s may combine anticonvulsive, neuroprotective, and possibly antiepileptogenic properties. The conceptual rationale for focal AAT development differs from classical AED development. Since current AED development follows largely a neurocentric concept, it is unlikely that the development of new AEDs acting on similar neuronal targets will lead to any significant improvement in antiepileptic therapy. In contrast, augmentation of adenosine as an upstream modulator of several downstream pathways is uniquely suited to affect neuronal excitability on the network level and therefore constitutes a new pharmacological principle that has not yet been exploited in clinical epilepsy therapy. In addition, adenosine is an endogenous anticonvulsant and therefore subject to physiological clearance. Rather than leading to toxic accumulations of adenosine, adenosine augmentation is likely to restore the adenosinergic equilibrium thereby avoiding undue side effects.
Stem cell-based and silk-based focal AAT approaches discussed here have distinctive benefits and limitations that are summarized in . Before focal AATs can move into the clinical realm several issues need to be resolved. These include, but are not limited to: (i) determination of ED50s and TD50s and the respective therapeutic index; (ii) differentiation of antiepileptic efficacy in mechanistically different animal models; (iii) determination of appropriate time points for therapeutic intervention; (iv) determination of appropriate therapeutic target populations (e.g. TLE vs. cortical dysplasia), and (v) demonstration of long-term efficacy.
Benefits and limitations of stem cell-based or silk polymer-based AATs.
AATs rationally utilize the brain’s endogenous adenosine-based seizure-control system, thereby presenting significant therapeutic potential for epilepsy. Adenosine is already FDA-approved for the treatment of supraventricular tachycardia and has been used in intrathecal infusions in phase I clinical trials for the treatment of chronic pain.99 Based on its relative safety profile, focal AATs, in particular those involving silk-based adenosine delivery, could rapidly be translated from animal studies to clinical trials. One possibility for first safety and feasibility studies could be the infusion of adenosine into an epileptic temporal lobe during its surgical removal. When coupled to synchronous EEG recordings, a proof-of-principle could be established that adenosine is effective in pharmacoresistant human epilepsy.
Given the potential disease modifying effects of focal AAT, eventually the implantation of a bio-resorbable adenosine-releasing silk polymer into an epileptogenic brain region could provide lasting benefit even after expiration of adenosine delivery. To make best possible use of those disease-modifying effects, it is of benefit to use a carrier system that can degrade with time without having any long-lasting residual impacts.
ACKNOWLEDGMENT
The work of the author is supported by grants R01NS058780, R01NS061844, R01NS065957 and R01MH083973 from the National Institutes of Health (NIH).
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