Adenosine kinase is a new therapeutic target to prevent ischemic neuronal death

Abstract

The brain has evolved several endogenous mechanisms to protect itself from the deleterious consequences of stroke. One of those endogenous neuroprotective systems is centered on the purine ribonucleoside adenosine, which exerts potent neuroprotective functions within the brain. One major goal in therapeutic stroke research is to explore and utilize such endogenous neuroprotective mechanisms therapeutically. This review illustrates molecular approaches to study the role of the adenosine system within the context of stroke and highlights innovative therapeutic approaches aimed at increasing adenosinergic function. New research data suggest that the major adenosine regulating enzyme adenosine kinase (ADK) plays a prominent role in determining the brain’s susceptibility to ischemic injury. Thus, endogenous ADK is rapidly downregulated following a stroke, possibly an endogenous neuroprotective mechanism aimed at raising ambient levels of adenosine in brain. Conversely, transgenic overexpression of ADK in brain renders the brain more susceptible to stroke-induced neuronal cell loss. In the present review we will first summarize the physiological role of adenosine metabolism within the context of ischemic brain injury. Next, we will highlight the key role of ADK in determining the brain’s susceptibility to ischemic injury, and finally we will discuss potential therapeutic applications of adenosine augmentation to provide neuroprotection in stroke.

1. Introduction

Ischemic stroke has a mortality rate of around 30% and is the third leading cause of death in major industrialized countries. In addition, it represents a major cause for long-lasting disabilities. Ischemic stroke can result from the occlusion of a major brain artery, either by an embolus or by local thrombosis, which leads to transient or permanent reduction in cerebral blood flow and extensive neuronal cell death [1, 2]. Despite intensive research and the development of neuroprotective drugs, to date, no adequate therapy exists to prevent neuronal injury after stroke. Previous strategies to prevent neuronal cell loss have mostly focused on the N-methyl-D-aspartate (NMDA) receptor, which is considered to be the main target responsible for excitotoxic Ca²⁺ overload in the ischemic brain that is linked to apoptotic signal transduction pathways [3, 4]. Based on this dogma, most experimental strategies to prevent ischemic neuronal cell death include caspase inhibitors [2] and blockade of calcium-permeable acid sensitive ion channels to prevent glutamate receptor-independent Ca²⁺ toxicity [3]. Although several neuroprotective drugs have been developed with demonstrated positive results in animal models of stroke, none of those has proven efficacy in human clinical trials [4–7].

2. Molecular basis of adenosine system

The brain has evolved several endogenous mechanisms to protect itself from the deleterious consequences of ischemic brain injury. The purine ribonucleoside adenosine is one of those endogenous neuroprotectant mediators.

2.1. Adenosine receptors

Adenosine – present already in the pre-biotic world – has evolved as a potent homeostatic regulator in all known forms of life and in all mammalian organ systems. In brain, adenosine is an important modulator of neuronal function and regulates transmitter release and neuronal excitability [8, 9]. Adenosine exerts potent neuroprotective functions through activation of four different subtypes of G-protein coupled adenosine receptors (Fig. 1) which also influence a wide range of other brain functions in both physiological and pathophysiological situations. For instance, adenosine is critically involved in the regulation of sleep, locomotion, anxiety, cognition and memory [8–10], as well as in the pathophysiology of stroke [11], and epilepsy [12].

Figure 1.

Adenosine exerts its effects through its four subtypes of receptors, A₁R, A_2AR, A_2BR and A₃R. Adenosine activation of G_i/o-coupled A₁Rs and A₃Rs inhibits activity of cAMP cyclase. In contrast, adenosine-activation of G_s/olf-coupled A_2AR and G_q-coupled A_2BR enhances the activity of cAMP cyclase. Therefore, four subtypes of adenosine receptors manipulate cellular cAMP levels, and thus consequently affect downstream protein kinase pathways, i.e. PKA, PKC and PKB.

At the molecular level, adenosine modulates the release of many neurotransmitters, i.e. dopamine, glutamate, GABA, serotonin, noradrenaline and acetylcholine, while the inhibition of excitatory neurotransmitter release (e.g. glutamate) is most pronounced [10, 13, 14]. These modulations are mediated by the interplay of A₁ and A_2ARs that have opposing actions mediated by different sets of G-proteins (Fig. 1), which explains the high degree of complexity in the overall effects of adenosine [15–18]. For example, the adenosine-dependent inhibitory modulation of neurotransmitter release is largely mediated through activation of G_i/o - coupled A₁Rs, which reduce neurotransmitter release at pre-synaptic nerve terminals and depresses neuronal firing at postsynaptic sites [13, 19, 20]. In contrast, adenosine activates G_s/olf - coupled A_2ARs to exert largely an excitatory activity based on stimulating the release of glutamate, acetylcholine, but also of GABA in striatum and hippocampus [21–25].

2.2. Adenosine regulation and ADK

The metabolism of adenosine in brain has extensively been reviewed elsewhere [8, 9, 14, 26]. Briefly, intra- and extra-cellular adenosine levels are in a dynamic exchange by equilibrative and concentrative nucleoside transporters [27, 28]. Intra- and extra-cellular enzymes of adenosine metabolism include adenosine kinase (ADK), adenosine deaminase (ADA), S-adenosylhomocysteine- hydrolase (SAH-hydrolase), and ecto- and 5′-nucleotidases (NT) (Fig. 2).

Figure 2.

Adenosine metabolic pathways and major enzymes involved: ecto - nucleotidase (ecto-NT), 5′-nucleotidase (5′-NT), adenosine kinase (ADK), S-adenosylhomocysteine-hydrolase (SAH-hydrolase), S-adenosylmethionine (SAM), equilibrative nucleoside transporter (ent).

Physiologically, adenosine is generated extracellularly as a product of the breakdown of adenine nucleotides, such as ATP, by a variety of ecto-nucleotidases (ecto-NT). Adenosine can also be formed intracellularly by hydrolysis of S-adenosylhomocysteine (SAH) via SAH-hydrolase [29]; thereby, adenosine is an obligatory end product of all transmethylation reactions [30]). A second intracellular source for adenosine is dephosphorylation of AMP by cytoplasmic 5′-nucleotidase (5′-NT) [31]. Adenosine levels are largely regulated by an astrocyte-based adenosine cycle (Fig. 2) [32, 33] Due to the existence of 2 types of equilibrative adenosine transporters in the astrocyte membrane, the intracellular astrocyte-specific enzyme ADK fulfills the role of a metabolic re-uptake system for adenosine [32, 34]. The removal of adenosine is thus predominantly regulated by ADK via conversion of adenosine into AMP, whereas the degradation of adenosine into inosine by adenosine deaminase (ADA) plays only a minor role in regulating adenosinergic function [35, 36]. It is important to note that extracellular and intracellular levels of adenosine are usually maintained in the same range because most cells possess highly efficient equilibratory facilitated diffusion transporters for nucleosides [37].

3. Adenosine and stroke

3.1. Adenosine responses to stroke

Under physiological conditions, extracellular levels of adenosine (25–250 nM) remain within the affinity range of the A₁ and A_2ARs. However, adenosine levels can rapidly rise to micromolar levels in response to metabolic stress situations, such as excessive energy consumption or lack of oxygen. Under those conditions, when an imbalance of energy supply and demand occurs, adenosine fulfils the role of a “retaliatory metabolite” to reset the energy balance [38].

In vitro, it has been demonstrated that adenosine levels rise as an acute consequence of ischemia and increased neuronal activity [39], as well as under external stimuli such as high K⁺, electrical stimulation, glutamate receptor agonists, hypoxia, and hypoglycemia [26]. Sustained enhancement of the tone of adenosine was found in hippocampal slices after the induction of ischemia [40] or seizures [41]. In line with those findings, adenosine levels were found to rise after cerebral ischemia in vivo [42]. The following mechanisms have been suggested to account for the increase of extracellular adenosine levels following ischemic events: (i) increased ATP release from cells and accelerated extracellular breakdown of ATP by ecto-nucleotidases [43]; (ii) inhibition of intracellular adenosine removal, via decreased activity of ADK [44]; and (iii) inhibition of equilibrative nucleoside transporters between cell membranes [10].

3.2. Adenosine receptors and stroke

Within the context of stroke, the adenosine receptor-mediated net effect of adenosine can best be summarized by the following findings, which have recently been reviewed [34, 45–47]: Activation of the adenosine A₁ receptor (A₁R) provides presynaptic inhibition, neuronal hyperpolarization and is involved in ischemic preconditioning. Consequently, mice with a genetic deletion of the A₁R exhibit decreased tolerance to hypoxia and loss of preconditioning in several tissues [48, 49]. However, in A₁R knockout mice the damage resultant from global ischemia was not different from WT mice, although pretreatment with A₁R antagonist aggravated damage in WT mice [50].

The role of the A_2AR in stroke appears to be more complex; depending on the context, activation of this receptor can either have beneficial or detrimental effects [51, 52]. Thus, both activation and inactivation of the A_2AR can provide protection against ischemic damage [53]. (1) Beneficial neuroprotective effects of A_2AR activation can be attributed to peripheral effects such as inhibition of platelet aggregation, vasodilatation, and anti-inflammatory actions [54, 55]. Meanwhile, it has been demonstrated that ischemic brain injury was aggravated in neonatal mice with genetic A_2AR deletion compared to WT [51, 56]. (2) Detrimental effects of A_2AR activation can be attributed to increased glutamate outflow [57–59], mitogen activated protein kinase (MAPK) activation [60], as well as activation of pro-inflammatory cytokines via activation of A_2ARs on bone marrow cells [61]. Genetic evidence thus demonstrated that a knockout of the A_2AR led to increased tolerance to ischemia [51, 56] and significantly attenuated infarct volumes and improved neurological behavioral deficit scores against ischemia when compared to WT mice [62]; this effect was further demonstrated to be largely dependent on A_2ARs present on bone marrow-derived (BMDC) cells [61]. Based on these findings, the inhibition of central A_2ARs is generally thought to be neuroprotective in their net effects.

The effects of A_2BR during ischemia are not as well characterized as other adenosine receptors, which is perhaps the least studied member of the adenosine receptor family. The A_2BR seems to be involved in the regulation of inflammatory processes that play an important role in the long-term outcome parameters after stroke [63], whereas the A₃R is involved in preconditioning in some species [64]. In addition, genetic deletion of the A₃R demonstrated an increase of ischemic injury after brain stroke, indicating A₃R-dependent endogeneous neuroprotection against ischemia [65].

3.3. Adenosine’s role in ischemic preconditioning

3.3.1. Ischemic preconditioning

In the treatment of cerebral stroke two basic principles are used: (i) Limitation of the acute injury-induced damage, and (ii) prophylactic approaches to afford tolerance or resistance to injurious processes that follow a cerebral stroke [66]. Preconditioning of the brain to afford tolerance to the effects of stroke has emerged as an attractive therapeutic strategy.

Ischemic preconditioning, tolerance, and endogenous neuroprotection of the brain have been reviewed extensively [1, 67–69]. It is generally known that certain antecedent treatments or events can protect individuals from injury due to an ischemic episode. For the induction of ischemic preconditioning in brain, a variety of stimuli, from short periods of ischemia or hypoxia, cortical spreading depression, brief seizures, exposure to inhaled anesthetic, or low dose of bacterial endotoxin [1, 70, 71], may produce functional protection of the brain through preconditioning. In view of the protective time window, ischemic preconditioning in the brain is either based on rapid (classical) preconditioning, induced almost immediately after stimulation; or delayed preconditioning, induced one to three days after injury, a process that requires protein synthesis[72, 73].

3.3.2. Involvement of the adenosine system in ischemic preconditioning

It has been suggested that the local rapid increase of adenosine is one of the mechanisms involved in acute neuronal ischemic preconditioning [11, 74]. In fact, the increase of adenosine during stroke is not limited to the CNS; Adenosine increases have also been documented in serum following a transient ischemic attack (TIA), or after stroke in humans [75]. Moreover, the involvement of adenosine in ischemic preconditioning has also been confirmed in the heart [76, 77], indicating that adenosine-based preconditioning might be a general protective mechanism. In addition, adenosine-based mechanisms might also contribute in delayed preconditioning. Long-term effects of adenosine include modulation of the brain’s immune response, induction of microglial proliferation, and phagocytosis of dead cells and debris in the affected tissue [78]. Adenosine may also be involved in tissue remodeling after injury by promoting angiogenesis and, thus, facilitating the replacement of dysfunctional blood vessels [79].

Apart from the involvement of adenosine as such in ischemic preconditioning, adenosine receptors are also implicated. Thus, the neuroprotective A₁R was shown to be upregulated rapidly in the brain as a consequence of an ischemic episode [80]. The involvement of the A₁R in ischemic preconditioning is also supported by pharmacological studies showing that DPCPX, a selective A₁ receptor antagonist, attenuated the protective effects of preconditioning, when administrated during a brief conditioning treatment [81].

4. ADK as therapeutic target against ischemic brain injury

Adenosine kinase (ADK), the major adenosine regulating enzyme, has recently been identified as a novel therapeutic target to prevent ischemic neuronal cell death [35, 44], seizures and epileptogenesis[32, 82], as well as other neurological conditions such as chronic pain [83]. In the following overview, we will first describe the pathological responses of ADK within the context of stroke and then highlight the key regulatory role of ADK to determine the susceptibility to ischemic brain injury and possible mechanisms involved. Finally, we will discuss potential therapeutic applications of focal adenosine augmentation therapies against stroke.

4.1. ADK-based genetic tools to study preconditioning

Given the crucial role of ADK as key regulator for adenosine and the demonstration that pharmacological inhibition of ADK potentiates adenosine-based neuroprotection in ischemic cerebral stroke [83, 84], several ADK-based molecular tools have been developed during the past several years to facilitate research on adenosine-based mechanisms in stroke and ischemic preconditioning:

First, Adk cDNA has successfully been cloned and part of the Adk gene has been incorporated into gene expression and targeting vectors [30, 85, 86]. Second, genetically engineered mouse lines have been generated and characterized, which include a systemic knockout of the Adk-gene that is lethal [30], a line with brain-wide overexpression of ADK (Adk-tg) [86], or that have reduced ADK expression in cortex and hippocampus (fb-Adk-def) [82]. Third, an antibody against ADK was generated and validated in ADK knockout mice [30]. Fourth, therapeutic cell lines, including embryonic stem cells, have been engineered to release adenosine based on genetic disruption of ADK [87–89]. Fifth, micro-RNAs directed against ADK have been expressed in a lentiviral expression system and shown to effectively downregulate ADK [90]. These molecular tools are now available and are expected to move the adenosine field forward in our attempts to understand and to exploit adenosinergic signalling therapeutically within the context of stroke therapy and prevention.

4.2. Genetic upregulation of ADK increases vulnerability to ischemic injury

Whereas pharmacological inhibition of ADK is not able to separate systemic from brain-specific effects, transgenic mice with regionally altered brain expression levels of ADK afford the unique possibility to study the roles of increased or decreased endogenous ADK on the brain’s susceptibility to stroke-induced neuronal cell loss [91]. First, we demonstrated that brain-wide transgenic overexpression of ADK (Adk-tg mice; 141% ADK of control) renders the brain more susceptible to ischemic cell death [91]. Remarkably, Adk-tg mice showed a 3-fold increase in infarct volume after 15 min of MCAO and 24 hour of reperfusion, compared to the infarct volume observed in wild-type mice with normal ADK expression. More strikingly, 60 min of MCAO was lethal in all Adk-tg mice within 24 hours of reperfusion, whereas all wild-type mice survived this procedure with 50% infarct volumes. In an attempt to reproduce an injury in Adk-tg mice that reproduces the injury in wild-type mice observed after 60 min of MCAO, we successively decreased the occlusion time of the middle cerebral artery in Adk-tg mice. Our findings demonstrate that 30 min of MCAO in Adk-tg mice leads to an infarct volume that is comparable to the infarct volume obtained after 60 min of MCAO in wild-type mice (Fig. 3).

Figure 3.

Triphenyltetrazolium chloride (TTC) staining of two brain slices from Adk-tg (left) and wild-type (WT) mice (right) subjected to MCAO and 24 h of reperfusion. 30 min of MCAO in Adk-tg mice leads to an infarct volume that is comparable to the infarct volume obtained after 60 min of MCAO in wild-type mice.

Conversely, injury levels corresponding to wild-type mice were recreated by pre-treatment of Adk-tg mice with the ADK inhibitor 5-ITU prior to MCAO. These experiments demonstrate that low levels of ADK are essential to maintain adenosine-mediated neuroprotection [91]. In line with the above findings, a subsequent study suggested that the expression levels of endogenous ADK might be critically involved in the brain’s susceptibility to neuronal cell loss after MCAO [44]. In this study it was demonstrated that the hippocampus of wild-type mice was consistently spared from ischemic injury after 60 min of MCAO followed by 24 hours of reperfusion, a time span during which the endogenous hippocampal ADK was significantly reduced, leading to a 2.2-fold increase of adenosine with a maximum after 3 hours of reperfusion. In contrast, hippocampal injury in Adk-tg mice became evident after only 15 mins of MCAO. The high susceptibility to stroke-induced hippocampal cell loss in Adk-tg mice can best be explained by constitutive transgenic overexpression of ADK and resulting inability to raise adenosine levels by sufficient downregulation of the enzyme. Therefore, increased activity of ADK and consequent dysregulation of the adenosine system promotes cell death in stroke.

4.3. Downregulation of ADK as endogenous protective mechanism

The key adenosine-regulating enzyme ADK is highly conserved in evolution [92] and features ubiquitous species and tissue distribution in all eukaryotes [93]. It catalyzes the reaction: adenosine + ATP → AMP + ADP. ADK is a remarkable enzyme as it is regulated by all of its four substrates and metabolites (adenosine, AMP, ADP, and ATP). It is therefore ideally positioned to function both as a sensor for the energy state of a cell as well as a switch to adjust adenosine levels according to metabolic needs. While ADK shows a dominant neuronal expression pattern during neonatal brain development [94], in adult brain the enzyme is highly specific for astrocytes and has been demonstrated to play a crucial role in maintaining a protective tone of adenosine [36, 85].

Endogenous astrocytic ADK is rapidly downregulated as an acute response to stroke or other brain insults that are associated with lack of oxygen and/or glucose, or that are associated with high levels of oxygen consumption and resulting pH changes [32, 44, 85]. Downregulation of ADK as an acute response to stroke appears to be an efficient endogenous neuroprotective mechanism aimed at raising ambient levels of adenosine in brain. Following stroke, the extracellular amounts of adenosine can increase rapidly and excessively to micromolar and up to milimolar levels [26] and are thus critically positioned to act as an endogenous neuroprotectant during cerebral ischemia and other neuronal insults [8, 9]. In vitro, oxygen-glucose deprivation in cultured cortical neurons led to downregulation of ADK and a rapid rise in extracellular adenosine, that was maintained for at least one hour [95]. Importantly, the rapid elevation of levels of ambient adenosine was demonstrated to be due to the release of adenosine as such – as a consequence of changes in intracellular ADK-dependent metabolism – rather than by extracellular cleavage of ATP [96]. The notion of downregulation of ADK as a general protective response to brain injury was corroborated in pharmacological studies using an ADK inhibitor in the rat MCAO model of transient focal cerebral ischemia [84]. In a recent in vivo study, ADK was shown to be transiently downregulated in ischemic mouse brain suggesting that acute downregulation of ADK following stroke causally participates in the endogenous adenosine-dependent protective mechanism of the brain [44]. Similarly, protective downregulation of ADK has also been observed within 2 hours after acute seizures in mice [85].

Due to the existence of a highly active substrate cycle between adenosine and AMP that is catalyzed by ADK and 5′-nucleotidase, minor changes in ADK activity can rapidly translate into major changes of adenosine [97, 98]. ADK is encoded by one of the largest known genes with a size of about 500,000 base pairs in rodents and human; it is not known yet, whether ADK is regulated at the genomic or transcriptional level. Protein phosphorylation as a mechanism for the regulation of ADK activity has been excluded [99]. In order to be effective and efficient as a rapid modulator of ambient levels of adenosine, a more rapid mechanism to regulate ADK activity is needed. Pioneering work from Datta and colleagues performed on Leishmania ADK suggests the following mechanism for ADK regulation [100–102]: ADK is active as a monomer that is stabilized by a cyclophilin. Dissociation from the cyclophilin and aggregation leads to rapid enzyme inactivation. Interestingly, the aggregate is stabilized by ADP. Thus, when energy reserves run low, the inactive form of ADK is stabilized by an increase in ambient ADP. Inhibition of ADK leads to increases in adenosine, which due to its inhibitory role on metabolism resets the energy balance of the cell. This is a feasible and highly likely mechanism how ADK could fulfill the role of a rapid “neuroprotective switch” under ischemic conditions. The neuroprotective effects of ADK inactivation in stroke can further be potentiated by reduced clearance of endogenously released adenosine. In sum, direct increases in adenosine in combination with reduced clearance combine to exert sustained suppression of excitatory neurotransmission. Thus, under conditions of ischemic or oxidative stress increased activation of A₁Rs leads to the suppression of overstimulation of glutamate receptors and thereby to suppression of neuronal excitation and excitotoxicity [103–105].

4.4. Stem cell-based focal adenosine augmentation prevents stroke-induced injury

Focal stem cell therapies can provide regeneration and repair combined with the sustained delivery of therapeutic compounds. Consequently, several cell therapies are currently under development for the treatment of neurological disorders. We have recently used focal stem cell-based adenosine delivery to demonstrate that providing adenosine to the brain by stem cell-derived brain implants can decrease ischemic neuronal damage [106]. In this study, the intrastriatal transplantation of embryonic stem cell-derived neural precursor (NP) or glial precursor (GP) cells (transplanted 7 days prior to ischemia) demonstrated significant protection against ischemic brain injury. This implies that stem cell derived implants have endogenous mechanisms to protect the brain from ischemic insults.

However, when the same cells were engineered to release adenosine by bi-allelic disruption of the Adk-gene, their therapeutic efficacy was potentiated about two-fold. Thus, Adk-deficient GPs transplanted into mouse brain (again 7 days prior to MCAO) protected almost the entire brain from injury related to 60 min of MCAO followed by 24 hours of reperfusion. These findings clearly demonstrate that focal augmentation of the adenosine function is sufficient to provide extensive protection in cortex and striatum.

4.5. ADK: a rational therapeutic target

The neurochemical rationale outlined above clearly defines ADK as a potential therapeutic target for ischemic brain injury. ADK inhibition is expected to exert powerful neuroprotection through the following mechanisms:

Activation of A₁Rs can (i) inhibit the presynaptic release of excitatory neurotransmitters, such as dopamine and glutamate [107–109]; (ii) reduce Ca²⁺ influx and enhance the K⁺ and Cl⁻ conductance postsynaptically in neurons leading to membrane hyperpolarization [110]; (iii) hyperpolarize astrocyte cell membranes and improving the astrocytic uptake of excessive extracellular K⁺ and glutamate [111]; (iv) attenuate basal and NMDA - induced production of nitric oxide [112]. In addition, increased A_2AR activation by increased levels of adenosine (caused by ADK-inhibition), has neuroprotective properties that are context dependent. For instance, the activation of A_2ARs may (i) inhibit platelet aggregation, thus reducing the potential of vessel obstruction [55]; (ii) exert anti-inflammatory actions during the post-stroke period [52, 80, 113]. Finally, stimulation of the adenosine A₃ receptor may also be beneficial in brain ischemia, although the mechanisms underlying this effect are less clear [114].

5. Adenosine-based pharmacological approaches

5.1. Neuro-protection via acute A₁R agonism or chronic A₁R antagonism

Since A₁R activation is known to mediate the neuroprotective activity of adenosine, the majority of pharmacological studies has focused on acute A₁R activation. Thus, the majority of A₁R agonists exert potent neuroprotective properties in a wide range of animal models of either global or focal ischemia. For instance, either acute systemic or intracerebroventricular administration of the A₁R agonist, N6-cyclohexyl-adenosine (CHA) attenuated hippocampal neuronal cell loss and improved neurological deficits in gerbils and rats subjected to global forebrain ischemia [115–117]. In line with these findings, the local administration of an adenosine analogue, 2-chloroadenosine (CADO), attenuated hippocampal cell loss in rats subjected to transient global forebrain ischemia [118]. Similarly, other A₁R agonists, N6-cyclopentyladenosine (CPA) and 2-chloro-N6-cyclopentyladenosine (CCPA), reduced neuronal loss and mortality in gerbils after global forebrain ischemia [119, 120]. Moreover, the A₁R agonist adenosine amine congener (ADAC) increased survival in the gerbil when administrated after the ischemic event [121]. All above confirm that stimulation of A₁Rs exert neuroprotective effects both in vitro and in animal models of hypoxia/ischemia. However, the therapeutic use of selective A₁R agonists is severely hampered by unwanted peripheral side effects, i.e. sedation, bradycardia and hypotension [122].

On the other hand, A₁R antagonists have protective effects after chronic administration (effect inversion after chronic dosage). For example, pretreatment with both 2-chloro-N6-cyclopentyladenosine (DPCPX) and caffeine given chronically for 2 to 3 weeks before an ischemic insult reduced neuronal injury in rats and gerbils [120, 123]. Similar neuroprotective effects were also observed in gerbils subjected to global forebrain ischemia after caffeine was given for 4 weeks [119, 124]. Above findings suggest the beneficial effects of chronic administration of A₁R antagonists, most likely mediated by up-regulation of A₁R.

5.2. Neuroprotection via A_2AR antagonists

The neuroprotective properties of A_2AR antagonists have also been increasingly demonstrated recently [125–128]. For instance, pharmacological data showed that both the selective A_2AR antagonists, ZM241385 and 8-(3-chlorostyryl) caffeine (CSC), as well as the less selective ones, CGS15943 and CP66,713, were able to attenuate global forebrain ischemia-induced hippocampal neuronal cell loss in gerbils or rats [53, 125, 128, 129]. Similarly, the selective A_2AR antagonist SCH58261 was shown to reduce cortical infarct volume in the focal cerebral ischemia model of permanent middle cerebral artery occlusion [127]. In addition, SCH58261 reduced brain damage in neonatal rats with unilateral carotid artery occlusion [130]. In contrast to the central effects of A₁R agonists, neuroprotection mediated by A_2AR antagonism is mainly due to peripheral mechanisms including vasodilation, inhibition of platelet aggregation and the suppression of neutrophil superoxide generation [51, 131, 132]. However, it needs to be mentioned that in several studies, A_2AR agonists exhibit neuroprotective activity, for example in the global ischemia model in the gerbil [53]

5.3. Neuroprotection by A₃R manipulation

In addition to the pharmacological manipulation of A₁ and A_2ARs, the protective effects of A₃R agonists have also been evaluated recently. For instance, the selective A₃R agonist, chloro-N(6)- (3-iodo-benzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA) when given intracerebroventricularly or intravenously by repeated administration not only protected primary cortical cultures against hypoxia-mediated decrease in cell viability in vitro, but also, protected MCAO-treated ischemic mice by decreasing cerebral infarction [133]. However, paradoxical effects of A₃R agonists in the brain have also been demonstrated: High concentration of Cl-IB-MECA have lethal effects, whereas low, nanomolar concentrations of A₃R agonists protect against apoptosis or ischemic damage [134].

5.4. ADK inhibition protects against ischemia

As a result of efforts in medicinal chemistry, adenosine-potentiating agents have been developed that elevate endogenous adenosine levels by inhibition of adenosine metabolism. Thus, inhibitors of ADK, ADA, or adenosine transporters have been developed and shown to protect from ischemic brain injury in different in vivo models [83, 84, 135–137]. Based on these findings, the pharmacological inhibition of ADK has been proposed to be the most effective strategy to provide neuroprotection in stroke.

Since Miller et al (1996) first presented a dose-dependent neuroprotection in the rat middle cerebral artery occlusion (MCAO) model afforded by the ADK inhibitor 5′-deoxyiodotubercidin (5-lTU) [136], it was realized as an attractive therapeutic possibility that ADK inhibition can provide neuroprotection when administrated during the pre-and peri-stroke period. Thus, adenosine augmentation via ADK-inhibition was seen as a viable strategy to overcome the limited therapeutic time-window of systemic adenosine augmentation. Later on, a study from Tatlisumak et al (1998) demonstrated that a different ADK inhibitor - GP683 [4-(N-Phenylamino) - 5-phenyl-7- (5′-deoxy ß-D-ribofurasonyl) pyrrolo [2,3-d] pyrimidine] - was effective in reducing the infarction volume in a rat model of focal cerebral ischemia. Similar to 5-ITU, GP683 was also demonstrated to provide effective neuroprotection when given 30 min after the induction of ischemia [137].

These pharmacological studies using ADK-inhibition suggested the following: (i) In contrast to A₁R agonists, ADK inhibition, either by 5-ITU or by GP683, did not induce hypothermia, suggesting that its anti-ischemic effects are mediated by mechanisms other than brain hypothermia. Importantly, ADK inhibition avoided systematic side-effects of hypothermia. (ii) The delayed administration of ADK inhibitors, both 5-ITU (0.33 mg/kg, i.v.) and GP683 (1.0 mg/kg, i.p.), at 30 min after the induction of ischemia resulted in a significant, 32% and 44% respectively, reduction in infarct volume. These unique features of an ADK-based therapeutic approach suggest a wider therapeutic index of ADK inhibitors in stroke. Based on the concept that ADK inhibition may potentiate an event-specific surge in adenosine and thereby provide event-specific benefits, thereby avoiding systemic side effects [83, 138–140], ADK inhibitors were further developed for the potential treatment of stroke, epilepsy, and chronic pain [141]. However, due to liver toxicity of ADK inhibition [30], and due to brain hemorrhage in preclinical models in dogs [139, 140]. ADK-inhibitors might not be a therapeutic option and drug development efforts have largely been stalled.

6. Concluding remarks and outlook

Therapeutic strategies aimed at enhancing adenosinergic function have widely been explored for their capability to protect the brain from ischemic injury. However, although the use of adenosine as a neuroprotective agent against stroke has been evaluated extensively in previous studies [142, 143], currently no adenosine-based therapeutics exist for the treatment of cerebral ischemia and reperfusion-induced brain damage.

Two major limitations have accounted for the delayed development of adenosine-based therapeutic strategies:

• Potent peripheral and central side effects of adenosine [144], which include suppression of cardiac functions, sedation, and renal impairment, preclude the systemic use of adenosine or its receptor agonists [12].

• The short physiological half-life of adenosine limits drug delivery and sustained therapeutic activity. Therefore, a novel generation of adenosine-based therapeutics is needed. The development of therapeutics for stroke therapy requires (i) local application/action combined with sustained delivery and (ii) the identification of novel therapeutic targets.

The identification of ADK as key regulator for ambient levels of adenosine and thus as a control point for adenosine-mediated downstream effects unravels a promising therapeutic target. Thus, inhibition of ADK as an effective strategy to increase adenosine levels represents a logical neurochemical rationale for therapeutic intervention. Importantly, ADK-inhibition was demonstrated to have several key advantages in terms of site and event selectivity compared to alternative adenosine-based approaches. The rationale for ADK-based therapeutics is based on the following arguments:

• ADK inhibitors can selectively enhance adenosine availability at traumatized tissue sites [145]; therefore ADK inhibitors may selectively amplify local endogenous adenosine responses.

• Local ADK inhibition may be of prophylactic value in stroke.

• Compared to direct-acting adenosine agonists, ADK inhibition offers the possibility to provide sustained augmentation of protective adenosine, which may extend the therapeutic window available for stroke therapy [83] (Figure 4).

Figure 4.

Model for adenosine-based neuroprotection after stroke. The endogenous surge of adenosine (ADO, blue) initiated after ischemia onset reaches a peak at 3 h post-stroke and is partially responsible for delayed neuronal cell loss (red) after stroke. Adenosine levels reach baseline values within 24 hours and might explain concurrence of lack of adenosine-based neuroprotection with neuronal cell loss. Therapeutic inhibition of ADK (green) may extend the duration of the endogenous adenosine-surge and therefore might provide extended neuroprotection.

In conclusion, the development of ADK-based pharmacotherapeutics still needs to overcome limitations on in vivo half-life, cellular penetrability and oral bioavailability. Limitations of systemic ADK inhibition may also include liver toxicity and the risk of brain hemorrhage. Eventually, local cell or gene-based approaches might become a future therapeutic option.