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Noebels JL, Avoli M, Rogawski MA, et al., editors. Jasper's Basic Mechanisms of the Epilepsies. 5th edition. New York: Oxford University Press; 2024. doi: 10.1093/med/9780197549469.003.0027
Adenosine is an endogenous anticonvulsant, which is inversely linked to the expression levels of adenosine kinase (ADK), a ribokinase that metabolizes adenosine into AMP. ADK exists as two distinct isoforms, with unique cellular compartmentalization and functions. The short isoform (ADK-S) is localized primarily in the cytoplasm of astrocytes, where it controls, in conjunction with equilibrative adenosine transporters, the extracellular levels of adenosine and hence adenosine receptor-mediated mechanisms. On the other hand, the long isoform of ADK (ADK-L) is localized in the nucleus of astrocytes and a subset of neurons, where it regulates adenosine receptor independent epigenetic mechanisms. Overexpression of ADK-S was sufficient to generate spontaneous electrographic seizures, whereas increased ADK-L has been associated with epileptogenesis. Hence, targeting specific ADK isoforms is a rational approach for the suppression of seizures and the development of epilepsy. Systemic adenosine augmentation therapies are effective in seizure suppression, although accompanied by widespread cardiovascular side effects. The refinement of therapeutic administration strategies, the development of isoform-specific ADK inhibitors, or a combination of the two, will enable the formulation of more selective therapies for epilepsy and its prevention. Therapeutic tools to achieve focal adenosine augmentation, such as engineered stem cells and silk-based polymers, demonstrate robust protection from induced and spontaneous seizures. Metabolic therapies such as the ketogenic diet therapy have also been indicated to prevent disease progression via epigenetic mechanisms mediated by ADK. Small-molecule, ADK isoform-specific inhibitors are now in development with the goal to translate ADK-based therapies into clinical applications.
The ribonucleoside adenosine is a derivative of adenine, a purine base which was most likely present on the prebiotic primitive Earth (Miller and Urey, 1959). 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 the brain but also in the heart, skeletal muscle, kidney, and adipose tissue, in the sense of a “retaliatory metabolite” that protects the cell against excessive external stimulation (Newby et al., 1985).
Adenosine was first recognized as an endogenous modulator of neuronal excitability in 1980 (Dunwiddie, 1980). It is well recognized that adenosine exerts potent anticonvulsant (Dragunow, 1986) and neuroprotective (Dragunow and Faull, 1988) functions in the brain which 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 (Boison et al., 2010; Fredholm et al., 2005). Furthermore, a rise in endogenous adenosine has been documented during ongoing seizure activity in patients with epilepsy (During and Spencer, 1992), and adenosine has consequently been identified as an endogenous mediator of seizure arrest and postictal refractoriness (Dragunow, 1991; During and Spencer, 1992).
A tight control of adenosine levels is hence critical for normal brain function and is maintained by the enzymes adenosine kinase (ADK) and adenosine deaminase (ADA), in conjunction with equilibrative adenosine transporters. Although regulated by multiple enzymes and transporters, the high-affinity ADK-mediated metabolism of adenosine is the primary regulator of adenosine levels under physiological conditions (Arch and Newsholme, 1978). ADK is evolutionarily highly conserved and widely expressed in all forms of life, in tissues and organ systems (Park and Gupta, 2008). Alternative promoter use and splicing leads to the expression of two distinct ADK isoforms with varying exon lengths (Cui et al., 2009). These isoforms also vary in the subcellular localization, with the long form (ADK-L) found in the nucleus and the short form (ADK-S) in the cytoplasm (Cui et al., 2009). Further, it was identified that two independent promoters drive the expression of each isoform, raising the possibility that the isoforms may perform distinct physiological functions (Singh et al., 1996; Cui et al., 2011). In line with this, recent advances propose a role for ADK-L in nuclear transmethylation reactions and a role for ADK-S in adenosine receptor-dependent mechanisms and possibly cytosolic transmethylation functions (Boison, 2013). The distinct physiological and biochemical functions of the specific ADK isoforms and the ectopic changes to their functions during epileptic conditions are discussed in the next section.
According to the ADK hypothesis of epileptogenesis, maladaptive overexpression of ADK is intrinsically linked to epileptogenesis and disease progression (Boison, 2008a; Li et al., 2008). Our work demonstrates that focal disturbances in adenosine homeostasis is a first step in epileptogenesis (Li et al., 2012) and either direct focal augmentation of adenosine or indirect adenosine augmentation via ADK inhibition was sufficient to suppress seizures and delay disease progression (Fedele et al., 2004; Pritchard et al., 2010; Szybala et al., 2009; Wilz et al., 2008). Further, overexpression of ADK drives epileptogenesis through enhancing the rate of DNA-methylation (Williams-Karnesky et al., 2013a). Because ADK progressively increases in line with epilepsy development and progression (Li et al., 2007), and because epileptogenic activity originates from ADK-expressing brain areas (Li et al., 2008, 2012), ADK is a promising biomarker to identify epileptogenic brain areas and to monitor epileptogenesis longitudinally. Hence, the future development of positron-emission tomography (PET) or single photon emission computed tomography (SPECT) tracers for ADK in humans will enable the identification of the epileptogenic foci, monitor the development and progression of epilepsy, or to measure the effectiveness of antiepileptogenic therapeutic interventions (Li et al., 2012). At the same time, ADK is a rational target for therapeutic intervention. We highlight recent advances and our current understanding on targeting ADK-S and L isoforms for seizure suppression and prevention of epileptogenesis, respectively (see third section).
Targeting ADK as a therapeutic approach to manage epilepsy, pain, and inflammation has been underway since the late 1990s (Jarvis, 2019). These efforts generated multiple classes of highly potent inhibitors, which were, however, not selective for any of the isoforms (Boison and Jarvis, 2021). New insights regarding the role of the nuclear ADK-L isoform in the epigenetic modulation of maladaptive DNA methylation during epileptogenesis provide the rationale for identifying novel ADK-isoform selective inhibitors and new interventional strategies that are independent of adenosine receptor activation. Those renewed drug discovery efforts, encouraged by modern advances in medicinal chemistry, are geared toward the discovery of novel ADK isoform selective inhibitors (Toti et al., 2016) for the treatment and prevention of epilepsy.
Adenosine is omnipresent and is involved in a repertoire of fundamental biochemical pathways depending upon its cellular localization (Fig. 27–1). In addition to being a part of RNA, adenosine-based derivatives include energy metabolites (ATP, ADP, and AMP), secondary messenger systems such as cAMP, and a variety of biomolecules including S-adenosylhomocysteine (SAH), S-adenosylmethionine (SAM), and the adenine-containing co-enzymes NAD and FAD. Hence, any dysfunction in adenosine or its regulating enzyme ADK would in turn affect a wide range of biochemical and molecular pathways, which use adenosine as a substrate. In this section, we highlight the roles and regulation of extracellular (synaptic), intracellular, and intranuclear adenosine by the cytoplasmic (ADK-S) and nuclear (ADK-L) isoforms of the enzymes, and the associated biochemical pathways.
Biochemistry of adenosine: role in epilepsy. Adenosine is highly compartmentalized in extracellular, intracellular, and nuclear compartments. Extracellular adenosine levels are controlled by (i) rapid degradation of ATP by a cascade of ectonucleotidases (more...)
Synaptic levels of adenosine are largely controlled by an astrocyte-based adenosine cycle (Boison et al., 2008b, 2010). The major source of synaptic adenosine is its precursor ATP that can be released by astrocytes via regulated vesicular transport (Pascual et al., 2005) or via hemichannels (Kang et al., 2008). Once in the synaptic cleft, ATP is rapidly degraded into adenosine by a cascade of ectonucleotidases (Zimmermann, 2000). In contrast to classical neurotransmitters, which are removed from the extracellular space via specific energy-driven reuptake transporters, astrocyte membranes contain two types of equilibrative transporters for adenosine that rapidly equilibrate extra- and intracellular levels of adenosine (Baldwin et al., 2004). Due to the lack of a classical transporter-regulated reuptake system for adenosine, the intracellular astrocyte-specific enzyme ADK-S has likely adopted the role of a metabolic reuptake system for adenosine. Thus, by phosphorylation of adenosine into AMP, ADK drives the influx of adenosine into the astrocyte, thereby maintaining the physiological levels of synaptic adenosine (Boison, 2006, 2008b; Boison et al., 2010).
Extracellular adenosine affects neuronal excitability via the activation of G protein–coupled adenosine receptors (A1R, A2AR, A2BR, and A3R). A shift in the ratio of inhibitory A1R toward stimulatory A2ARs directly influences neuronal excitability. In general, the epileptic state is marked by decreased A1R signaling and/or enhanced A2AR signaling. The pharmacology and physiologic functions of the adenosine receptors have been extensively reviewed (Fredholm et al., 2000; Jacobson and Gao, 2006; Sebastiao and Ribeiro, 2009; Stone et al., 2009) and key roles in epilepsy are described (Weltha et al., 2019). It is currently unknown whether modifications in adenosine receptor expression are the cause or consequence of epilepsy.
Due to the abundance of ectonucleotidases in the extracellular compartment, it was originally assumed that adenosine was predominantly formed in the extracellular compartment (Meghji et al., 1985). However, a series of experiments using inhibition of ectonucleotidases to trap adenosine within the cell revealed that intracellular adenosine formed from the transmethylation pathway might be a substantial source of adenosine in addition to ATP catabolism via 5-AMP (Meghji et al., 1985; Headrick and Willis, 1989). The transmethylation reaction is a common biochemical reaction catalyzed by methyltransferase enzymes, in which a methyl group derived from the donor S-adenosylmethionine (SAM) is transferred onto an acceptor as varied as lipids, dopamine, or histamine. Depending upon the acceptor, the methyltransferase may be a phenylethanolamine N-methyltransferase (PNMT), histamine-N-methyltransferase (HNMT), or catechol-O-methyltransferase (COMT). S-adenosylhomocysteine (SAH), an end product of all transmethylation reactions, is subsequently cleaved by SAH hydrolase (SAHH) into adenosine and homocysteine. Indeed, it was noted that the intracellular transmethylation pathway contributes to 10 times higher adenosine levels in comparison to the extracellular adenosine levels found in heart tissue (Lloyd et al., 1988). Further, adenosine formed from the transmethylation pathway was found to be largely salvaged by ADK-S within the cell (Lloyd et al., 1988). Importantly, the thermodynamic equilibrium of the SAH hydrolysis reaction lies on the side of SAH formation. Thus, this reaction, which is critical for regulating the flux of methyl groups through the transmethylation pathway, can only proceed if adenosine levels are kept low (Hoffman et al., 1979) and adenosine is effectively removed through ADK (Boison et al., 2002). In line with this, coadministration of adenosine along with homocysteine in mice decreased the activities of COMT and HNMT found in whole-brain lysates (Schatz et al., 1981). Human patients with Adk mutations and demonstrated functional ADK deficiency were characterized by disrupted transmethyation, severe liver pathology, and encephalopathy (Bjursell et al., 2011). These findings suggest that elevated adenosine acts as a potent inhibitor of transmethylation reactions; hence, targeting ADK is a viable option for modulating methylation reactions.
The cytosolic methyltransferases PNMT and HNMT are involved in the biosynthesis of epinephrine and histamine, respectively, whereas COMT is crucial for the degradation of dopamine. Aberrant levels of methyltransferase activity, transmethylation levels, and resultant dysregulation of neurotransmitters such as histamine and dopamine have been reported in several models of epilepsy (Perry et al., 2008; McKenna et al., 2018; Schatz and Sellinger, 1975; Yawata et al., 2004; Schlesinger et al., 1975). In the EL mouse model for hereditary temporal lobe epilepsy, administration of either histidine, a substrate for histamine synthesis, or metoprine (2,4-diamino-5-(3,4-dichlorophnyl)-6-methyl-pyrimidine), an inhibitor of HNMT retarded the onset of seizure episodes in the mice (Yawata et al., 2004). In the DBA/2J mouse model of audiogenic seizures, the specific activity of cerebral HNMT and COMT seemed to determine seizure susceptibility, indicating that cerebral methylation is crucial for epileptogenesis (Doyle and Sellinger, 1980; Schlesinger et al., 1975). Unlike COMT and HNMT, which are more widely expressed in brain tissue, PNMT expression in the CNS is limited to a subset of epinephrine-producing neurons in the brain stem (Yang and Voogt, 2001; Ziegler et al., 2002). Following flurothyl-induced seizures, a marked increase in PNMT expression and a corresponding increase in c-Fos, a marker of neuronal activity was detected in the locus coeruleus and in the caudal ventrolateral medulla regions of the brainstem (Silveira et al., 2000). These findings indicate that flurothyl-induced generalized seizures correspond to changes in PNMT-mediated transmethylation reactions in the pons and medulla oblongata. Further studies are necessary to determine the involvement of PNMT in contributing to the autonomic manifestations that frequently accompany epileptic seizures. Taken together, these studies highlight the significance of transmethylation reactions in epilepsy. However, whether adenosine levels and ADK can regulate the activity of these cytosolic methyltransferases remains a major gap in our understanding that needs to be further investigated. The potential link in targeting ADK to regulate cytosolic transmethylation reactions and thereby the levels of neurotransmitters such as dopamine is novel and is of profound significance, particularly for the treatment of neuropsychiatric conditions such as addiction.
Independent lines of evidence from in vitro and in vivo studies show that ADK-L is specific for the nucleus, compared to its cytoplasmic ADK-S counterpart. Based on sequence homology studies that predict subcellular localization, it was initially speculated that both isoforms of ADK are located in the cytoplasm (Nakai and Horton, 1999; Sakowicz et al., 2001). However, distinct subcellular localizations of both isoforms of ADK were identified (Cui et al., 2009). Point mutations of the N-terminal sequence of ADK-L (PKPKKLKVE) prevented the nuclear localization of the isoform. These findings suggest that ADK-L contains a novel nuclear localization signal. The nuclear localization of ADK-L suggests a specific function for epigenetic regulation (Studer et al., 2006; Williams-Karnesky et al., 2013b). In the nucleus, ADK-L, via adenosine metabolism, regulates transmethylation reactions including DNA methylation and histone methylation, which are driven by DNA methyltransferases (DNMTs) and histone methyltransferases (HMT), respectively. Infusion of adenosine or homocysteine into the hippocampus of rats induced global DNA hypomethylation, whereas the infusion of SAM induced hypermethylation of DNA, demonstrating that the methylation status of DNA directly depended on the adenosine-sensitive transmethylation pathway. Importantly, blocking ADK with 5-iodotubercidin or genetic reduction of ADK expression resulted in hypomethylated DNA in the brain, whereas overexpression of either the cytoplasmic or the nuclear isoform of ADK resulted in increased DNA methylation in cultured cells, with the nuclear overexpression of ADK being more efficient in increasing the methylation status of the DNA (Williams-Karnesky et al., 2013b). These data suggest a novel adenosine receptor-independent role of ADK in regulating the methylation status of DNA and possibly histone, acting as an epigenetic regulator.
Altered patterns of DNA methylation have been reported in the epileptic hippocampus obtained from human temporal lobe epilepsy patients as well as in rodent models of epilepsy (Miller-Delaney et al., 2015; Williams-Karnesky et al., 2013b; Lusardi et al., 2015; Kobow et al., 2013). Although the role of histone methylation has not been directly investigated in epilepsy, epigenetic mechanisms (such as DNA and histone methylation) are thought to propagate epileptogenicity and thereby contribute to the epileptogenic memory in chronic and difficult-to-treat epilepsies (Henshall and Kobow, 2015). Indeed, the methylation hypothesis of epileptogenesis suggests that epigenetic chromatin modifications (DNA/histone methylation) may play a role in epileptogenesis (Kobow and Blumcke, 2011; see Chapter 35, this volume). Hence, targeting these reversible epigenetic mechanisms via ADK-L inhibition might help prevent epileptogenesis and is discussed in detail later.
In general, the effects of adenosine are largely inhibitory, capable of suppressing seizures, and overall neuroprotective. Although a short-lived adenosine surge is an acute consequence of seizures, chronic upregulated expression of ADK and a corresponding deficiency in adenosine levels is well-documented in several clinical conditions and in preclinical models of epilepsy (Aronica et al., 2011; Masino et al., 2011; Fedele et al., 2005; Gouder et al., 2003, 2004; Patodia et al., 2020). Hence ADK emerges both as a putative diagnostic marker to predict, as well as a prime therapeutic target to suppress seizures and possibly prevent epileptogenesis. Mounting evidence on ADK’s role in the development of epilepsy have led to the formulation of the “ADK hypothesis of epileptogenesis.” The evidence in support of this hypothesis includes the following: (1) Mouse models of epileptogenesis suggest a sequence of events leading from initial downregulation of ADK and elevation of ambient adenosine as an acute protective response, to changes in astrocytic adenosine receptor expression, to astrocyte proliferation and hypertrophy (i.e., astrogliosis), to consequential overexpression of ADK, reduced adenosine, and—finally—to spontaneous seizure activity restricted to regions of astrocytic overexpression of ADK (Fedele et al., 2005). (2) Transgenic mice overexpressing ADK (ADK-S and ADK-L) display increased sensitivity to brain injury and seizures (Gebril et al., 2020). (3) Inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy (Gouder et al., 2004). (4) Intrahippocampal implants of stem cells engineered to lack ADK prevent epileptogenesis (Fedele et al., 2004; Güttinger et al., 2005). (5) Transient treatment with an ADK inhibitor was able to prevent epilepsy in a mouse intrahippocampal kainate model of epilepsy (Sandau et al., 2019).
Targeting ADK is attractive from a drug discovery perspective and an ideal strategy for augmenting adenosine levels for the treatment and prevention of epilepsy. In tissue studies, the Km for adenosine is 1–2 orders of magnitude lower than that of adenosine deaminase. Thereby, a minor change in ADK activity can result in a major change in adenosine concentration (Boison, 2013; Arch and Newsholme, 1978). Another advantage of targeting ADK is the ability to use conventional drug delivery methods, compared to the need for specialized delivery systems for focal adenosine augmentation. The main evidence for this comes from an in vivo microdialysis study which demonstrated that systemic ADK inhibition selectively enhanced kainic acid-induced adenosine release without altering basal adenosine concentrations in rat striatum (Britton et al., 1999). This site- and event-specific potentiation of adenosine is particularly beneficial in the context of epilepsy, where focal effects of adenosine augmentation at the epileptogenic region can be achieved from systemic administration of ADK inhibitors (Britton et al., 1999). Hence, there is a strong neurochemical rationale for therapeutically targeting ADK, with strategies to target ADK-S and L for anti-ictogenic and antiepileptogenic effects, respectively.
Adenosine augmentation therapies capitalize on the anticonvulsant and neuroprotective properties of endogenous adenosine in the brain with the potential to suppress seizures (Boison, 2009b; Tescarollo et al., 2020). ADK inhibitors have been demonstrated to be powerful therapeutic agents to enhance the tissue tone of endogenous adenosine (Boison et al., 2013). Thus, augmentation of extracellular adenosine either directly via implantation of adenosine-releasing silk or indirectly by inhibiting ADK-S is beneficial in effecting seizure suppression and neuroprotection. In an intrahippocampal model of kainic acid-induced seizures, a mouse model of pharmacoresistant temporal lobe epilepsy, a biphasic regulation of ADK expression was noted in the epileptic hippocampus (Gouder et al., 2004). Further, astrogliosis was accompanied by overexpression of ADK (Gouder et al., 2004) and was later shown to be important for aggravation of the seizure phenotype (Fedele et al., 2005). Notably, the global ADK inhibitor 5-iodotubercidin (5-ITU) demonstrated robust efficacy in seizure suppression in a mouse model of pharmacoresistant epilepsy (Gouder et al., 2004). These findings confirm the therapeutic potential of targeting ADK to augment synaptic adenosine levels for seizure suppression.
To avoid side effects of systemic adenosine augmentation, focal adenosine delivery approaches have been developed with the goal to increase adenosine concentrations locally in the brain in the vicinity of a seizure producing focus (Boison, 2009a). Insertion of encapsulated Baby hamster kidney (BHK) cells engineered to release adenosine effectively suppressed seizures in response to electrical kindling. This strategy of focal adenosine augmentation demonstrated that the paracrine local release of adenosine was not only sufficient to suppress seizures but also to avoid sedative side effects associated with systemic ADK inhibition or A1R activation. In an independent study, the lentiviral expression of a microRNA (miRNA) directed against ADK was shown to lead to a 80% reduction of ADK expression in mesenchymal stem cells (Ren et al., 2007). Transplantation of these cells into mice before the injection of kainic acid significantly reduced acute brain injury and seizures in mice, confirming the seizure-suppressing effects of adenosine augmentation therapies (Ren and Boison, 2017).
In a more refined strategy for the focal delivery of adenosine to the epileptic brain, silk polymers were developed for the precise and controlled delivery of adenosine during a defined and limited time span of only 10 days with the goal to have a transient focal delivery system for adenosine (Pritchard et al., 2010; Szybala et al., 2009; Wilz et al., 2008). Using the rat electrical hippocampal kindling model in rats, the adenosine-releasing or control polymers were then implanted into the infrahippocampal fissure ipsilateral to the site of stimulation. It was demonstrated that only recipients of adenosine-releasing implants were completely protected from generalized seizures and seizure protection corresponded to the duration of sustained adenosine release (Szybala et al., 2009). Importantly, the injection of an A1R antagonist 30 minutes prior to a stimulation in protected animals allowed the development of a stage-5 seizure demonstrating the adenosine dependence of seizure suppression. These results suggest a powerful anti-ictogenic activity of focal implant-derived adenosine release in the range of 1000 ng per day (Szybala et al., 2009), as discussed in more detail below. The same adenosine-releasing silk implants were used to establish a novel antiepileptogenic role of adenosine.
The ketogenic diet is an alternative metabolic treatment option for epilepsy, and multiple retrospective and prospective studies confirm its clinical benefits (Kossoff and Rho, 2009; Neal et al., 2008). The high-fat, low-carbohydrate composition of the diet forces a ketone-based metabolism, which leads to anticonvulsant consequences. The proposed antiseizure and disease-modifying neuroprotective mechanisms of the diet are mediated by several converging mechanisms, which have been extensively reviewed (Murugan M, 2020; Masino et al., 2011; Youngson et al., 2017; D’Andrea Meira et al., 2019). Here, we highlight how the ketogenic diet augments adenosine availability and confers seizure suppression. It was noted that ketogenic diet administration suppressed seizures in a transgenic mouse with adenosine deficiency (Masino et al., 2011). Interestingly, the seizures suppression was reversed by administration of glucose (metabolic reversal) or an A1R antagonist (pharmacological reversal). Strikingly, in mice lacking A1Rs, spontaneous seizures were completely unaffected by the diet. Thus, genetic and pharmacological manipulations of the adenosine system provide compelling evidence that the diet’s anticonvulsant effect is mediated by A1R activation (Masino et al., 2011). In the Wistar Albino Glaxo/from Rijswijk (WAG/Rij) rat model of spontaneous seizures, the ketogenic diet–induced antiseizure effect was abolished by treatment with DPCPX, an A1R antagonist (Kovacs et al., 2017). These results demonstrate that the adenosine-dependent anticonvulsant effects of ketogenic treatments are consistent among different mechanistic models of epilepsy. Despite the proven neuroprotection yielded by the ketogenic diet, side effects and requisite strict compliance have limited widespread use, and a diet-based approach is often considered as a last resort.
Accumulating evidence suggests the antiepileptogenic effects of adenosine are mediated by its ability to regulate epigenetic mechanisms. Some of the early evidence comes from transgenic mice with a genetic reduction of forebrain ADK expression, which demonstrated resistance to the development of epilepsy, even when the epilepsy-triggering kainic acid was paired with a transient blockade of the A1R (Li et al., 2008). In addition, adenosine-releasing stem cells attenuated astrogliosis, restrained ADK expression, and mitigated the progression of spontaneous ictal activity after triggering epileptogenesis (Li et al., 2008; Güttinger et al., 2005). These studies for the first time suggested a hitherto unknown antiepileptogenic potential of adenosine releasing cellular brain implants.
Using a novel approach to deliver adenosine locally, the silk polymers described above were implanted into the brain ventricles of rats to deliver predefined target doses of adenosine with a limited duration of 10 days (Williams-Karnesky et al., 2013b). In order to assess the sustained antiepileptogenic role of adenosine, the silk polymers were implanted after the emergence of the first spontaneous recurrent seizures (9 weeks after kainic acid in rats) to release adenosine for 10 days. It was noted that DNMT activity was reduced and DNA methylation levels were restored in rat hippocampus treated with adenosine-releasing silk polymer compared to control-implant recipients (Williams-Karnesky et al., 2013b). Interestingly, those epigenetic benefits persisted for at least 3 weeks after depletion of adenosine release from the polymers, indicating a transient and localized dose of adenosine can exert long-lasting influence on maladaptive DNA methylation changes in epilepsy (Williams-Karnesky et al., 2013b). Importantly, epilepsy progression as measured by a gradual increase in weekly numbers of seizures in all control animals was completely prevented in recipients of adenosine-releasing brain implants for at least 3 months. In line with this, animals exposed to transient adenosine-releasing silk lacked progressive increases in mossy fiber sprouting, a hallmark feature of epilepsy severity, whereas the sham controls demonstrated a continuous increase in the sprouting of mossy fibers corresponding to their severe seizure phenotype.
Global inhibition of ADK using transient administration of the ADK inhibitor 5-ITU during the latent phase of epileptogenesis reduced seizures by at least 80% in 56% of mice at 6 weeks after intrahippocampal kainic acid (Sandau et al., 2019). This reduction in seizures was maintained in 40% of 5-ITU–treated mice at 9 weeks, suggesting an antiepileptogenic effect. 5-ITU also suppressed granule cell dispersion and prevented maladaptive ADK increases in these protected mice. These results demonstrate that transient inhibition of ADK is sufficient to prevent epileptogenesis.
The ability of ADK-L to regulate DNA methylation was confirmed in several in vitro studies, which systematically categorized the contribution of ADK-S and L to DNA methylation changes (Williams-Karnesky et al., 2013b; Wahba et al., 2021). BHK fibroblasts were engineered to either lack both forms of ADK or to selectively overexpress either ADK-S or ADK-L. It was noted that the methylation levels in cells overexpressing ADK-L were 400% higher, whereas global DNA methylation levels in the ADK-S-overexpressing cells were increased by only 50%, compared to ADK knockout cells (Williams-Karnesky et al., 2013b). A similar correlation was noted between ADK-L expression and DNA methylation levels in cancer cell lines with differential ADK isoform expression (Wahba et al., 2021). These findings further confirm that preferential ADK-L inhibitor activity would be desirable for epigenetic therapies aimed at blocking maladaptive methylation changes in a wide range of pathologies (Murugan et al., 2021).
A drug development platform for the synthesis of small molecules that selectively inhibit ADK-L is thus essential for the development of a new line of inhibitors capable of regulating DNA methylation (Toti et al., 2016; Iqbal et al., 2006). A recent study using a structure-based design approach and molecular dynamics simulation analysis of human ADK synthesized novel ADK inhibitors (MRS4380 and MRS4203) with higher potency for ADK inhibition over conventional nucleoside-based inhibitors for ADK (Toti et al., 2016). In another independent study, the lead compounds MRS4203 and MRS4380 were found to significantly reduce global DNA methylation in cancer cell lines in a concentration-dependent manner, confirming the ability to use ADK inhibitors as a novel platform to affect DNA methylation (Wahba et al., 2021). Isoform selective inhibitors are expected to overcome the limitations of conventional ADK inhibitors, which are indifferent to isoform selectivity. However, there are many challenges in developing inhibitors of nanomolar potency, given the considerations to structural features that allow entry into the nuclear compartment for targeting ADK-L. A structure-based design approach along with docking simulations is needed to help refine the pharmacological characteristics of the drug with efficient nuclear entry, improved ADK-L selectivity, and lasting epilepsy prevention.
In addition to seizure suppression, the ketogenic diet also offers long-lasting antiepileptogenic properties that prevail even after diet discontinuation (Murugan M, 2020; Kossoff and Rho, 2009; Masino et al., 2011). A preclinical study used two mechanistically different models of epilepsy to demonstrate that the diet, but not a conventional antiepileptic drug (valproic acid) prevented epileptogenesis (Lusardi et al., 2015). They showed that the seizure suppression after kindling-induced epileptogenesis was maintained even after the diet was terminated. A similar lasting reduction in seizures was noted in ketogenic diet–fed animals compared to control chow-fed animals in a pilocarpine model of epilepsy. Interestingly, diet treatment was associated with increased adenosine (among other metabolites) and decreased DNA methylation, the latter being maintained even after cessation of the diet (Lusardi et al., 2015; Williams-Karnesky et al., 2013b). Taken collectively, these studies indicate that the ketogenic diet–induced adenosine augmentation acts through dual mechanisms: (1) anti-ictogenic effects via adenosine receptor mediated mechanisms, and (2) antiepileptogenic effects via adenosine regulation of the DNA methylome. These findings highlight the significance of using the ketogenic diet for epilepsy prevention. However, further refinement in diet formulation and administration is needed to increase seizure suppression rates and epilepsy prevention rates, which are highly variable with the current diet strategies.
Data from acute and chronic models of epilepsy discussed above suggest that targeting adenosine kinase combines anticonvulsive, neuroprotective, and antiepileptogenic properties. Although initial drug discovery efforts to identify ADK inhibitors failed due to mechanistic and toxicity issues, novel insights into the highly compartmentalized, physiologically unique ADK isoforms have renewed the interest in targeting ADK and led to the launch of several new drug discovery programs (Toti et al., 2016; Iqbal et al., 2006) (Fig. 27–2).
Targeting ADK for epilepsy therapy. Schematic depicts mechanisms, strategies, therapeutic goals, and tools to target the ADK-S and L isoforms for anti-ictogenic and anti-epileptogenic effects, respectively. The bidirectional arrow between the anti-ictogenic (more...)
The conceptual rationale for targeting ADK for epilepsy treatment has an array of advantages over the conventional antiseizure drug (ASD) platform. (1) ASD development follows largely a neurocentric concept; hence, it is unlikely that the development of new ASDs acting on similar neuronal targets will lead to any significant improvement in antiepileptic therapy. In contrast, targeting ADK to augment 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. (2) Enhancing adenosinergic signaling via targeting ADK and metabolic therapies is effective in preclinical models of pharmacoresistant epilepsies (Boison, 2009b). (3) Metabolic strategies that increase adenosine levels not only demonstrate seizure suppression but also have antiepileptogenic properties, which has never been demonstrated with conventional ASDs. (4) ASDs are known to affect cognitive abilities and exacerbate comorbidities (Ortinski and Meador, 2004), whereas ADK-based therapies, in addition to seizure suppression, would ameliorate the affective, psychiatric, and cognitive deficits that often occur as comorbidities associated with epilepsy (Boison and Aronica, 2015).
The strategies for direct adenosine augmentation or selective targeting of ADK isoforms presented in this chapter have distinctive benefits and limitations that are summarized in Table 27–1. Several concerns need to be addressed before transitioning these therapeutic options to clinical practice. Some of these challenges include, but are not limited to (1) determination of ED50s and TD50s and the respective therapeutic index; (2) differentiation of antiepileptic efficacy in mechanistically different animal models and relevance in etiologically distinct human epilepsies; (3) determination of appropriate temporal window for therapeutic intervention; (4) demonstration of long-term efficacy; and (5) identification of any unintended or off-target effects.
Benefits and Limitations of Adenosine-Based Therapies.
Among the many modalities discussed, small-molecule ADK inhibitors are the most effective strategy to restore normal adenosine/DNA methylation levels. A transient low-dose therapy with the ability to selectively modulate nuclear ADK-L using either novel small-molecule ADK inhibitors or biological agents would be ideal as it would overcome the side effects of long-term global ADK inhibition. Moreover, the development of novel, selective inhibitors that capitalize on the epigenetic activity of ADK-L will be game-changing as it would be beneficial not only for the prevention of epilepsy, but in a repertoire of clinical conditions including brain injury, cancer diabetes, and vascular diseases (Murugan et al., 2021).
The work of the authors is supported by research grants from the National Institutes of Health (NIH—R01NS065957, R01NS103740) and the Citizens United for Research in Epilepsy (CURE—Catalyst Award).
The author declares no relevant conflicts.
This is an open access publication, available online and distributed under the terms of a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 International licence (CC BY-NC-ND 4.0), a copy of which is available at https://creativecommons.org/licenses/by-nc-nd/4.0/. Subject to this license, all rights are reserved.
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