Abstract

Despite the availability of numerous antiseizure drugs (ASDs), treatment of epilepsy remains a clinical challenge, with over 30% of patients not responding to any pharmacological intervention. In addition, ASDs are mainly symptomatic without significantly impacting on disease progression. Thus, there is a strong focus on the identification of drug targets with a novel mechanism of action that are independent on GABAergic and glutamatergic neurotransmission and that possess a disease-modifying potential. That extracellular released purines such as adenosine triphosphate (ATP) and adenosine act as signaling molecules in the brain has been known for decades. While the anticonvulsant potential of adenosine is well established, increasing evidence suggests also an important role for ATP during the generation of seizures and the development of epilepsy. Usually present at low extracellular concentrations, ATP is released in response to several pathological stimuli including increased neuronal activity acting as danger signal and activating specific purinergic P2 receptors divided into the metabotropic P2Y and ionotropic P2X receptors. Suggesting a role during seizures and epilepsy, data from experimental models and patients show widespread expression changes of both P2Y and P2X receptor family members during epilepsy and, critically, drugs targeting both receptor subtypes, in particular, the P2Y1 and P2X7 receptors, confer both anticonvulsive and antiepileptic effects. This chapter provides a detailed summary of the current evidence suggesting ATP-gated receptors as novel drug targets for epilepsy, discusses the possible mechanisms for how these receptors contribute to increased hyperexcitability, and briefly outlines how the purinergic system can be used as novel tool for the diagnosis of epilepsy.

Purinergic Signaling in the Brain

Compelling and exciting evidence gathered over the past decades has demonstrated a role for purinergic signaling in the generation of seizures and the development of epilepsy. These discoveries provide new promising therapeutic avenues for the treatment of drug-refractory seizures and epilepsy. As the field of purinergic signaling is fast moving and many readers may not be familiar with it, we start our review by considering the mechanisms of purinergic signaling that are likely to be relevant to the development and regulation of seizures.

The purinergic signaling system is similar in some ways to other neurotransmitter systems in the brain. However, it is marked out from other possibly more familiar systems by a complexity that encompasses the multiplicity of possible ligands, release pathways, receptor systems, and the fact that the inactivation products are signaling agents in their own right (Fig. 73–1). Adenosine triphosphate (ATP) and adenosine comprise the canonical purinergic ligands. However, there are other compounds present in the central nervous system (CNS) that can be released or generated in a regulated fashion and can activate some of the purinergic receptors: Adenosine diphosphate (ADP), diadenosine polyphosphates (Pintor et al., 1992; Miras-Portugal et al., 1999), uridine triphosphate (UTP), uridine diphosphate (UDP), and UDP-glucose (Moore et al., 2003; Lazarowski, 2006).

Figure 73–1.

Scheme that shows the major elements of purinergic signaling. There are two major mechanisms of release: via gap junction hemichannels (HC); and exocytosis. ATP is sequestered in vesicles via the vesicular nucleotide transporter. ATP can also be released (more...)

Targeting Purinergic Receptors for Seizure Control and the Treatment of Epilepsy

P2 Receptors

P2 Receptor Expression Following Seizures and during Epilepsy

P2Rs are widely distributed throughout the brain where they are expressed and functional on both neurons and glial cells (Burnstock, 2013; Guzman and Gerevich, 2016). While a detailed description of potential expression changes during epilepsy is still missing for most of the P2Rs, substantial progress has been made for the P2Y1R and P2X7R subtype with data stemming from both experimental models and patients (Table 73–1). Expression analysis of brain tissue was, however, mainly limited to the hippocampus and the cortex.

Table 73–1

P2 Expression Changes and Impact of P2 Receptor-Targeting during Acute Seizures, Epileptogenesis, and Epilepsy.

Changes in the expression for P2XRs following seizures (i.e., status epilepticus) or during epilepsy have been reported for the P2X2, P2X4, and P2X7R, while no changes have been found so far in the expression for the remaining P2XRs (i.e., P2X1R, P2X3R, P2X5R, and P2X6R). Regarding the P2X2R and P2X4R, whereas hippocampal P2X2R expression has been shown to be downregulated following intra-amygdala KA (Engel et al., 2012), P2X4R expression increases in the hippocampus following systemic KA injections (Avignone et al., 2008; Ulmann et al., 2013), but not intra-amygdala KA- or pilocarpine-induced status epilepticus (Dona et al., 2009; Engel et al., 2012). In contrast, P2X4R expression was found decreased in the hippocampus of pilocarpine-treated epileptic rats (Dona et al., 2009).

Among the P2XR family, most data are available on the P2X7R subtype. P2X7R expression has consistently been reported to be increased following status epilepticus including intraperitoneal KA in mice (hippocampus) (Avignone et al., 2008), intra-amygdala KA-induced status epilepticus in mice (hippocampus, cortex, striatum, thalamus, cerebellum) (Engel et al., 2012; Jimenez-Pacheco et al., 2013; Morgan et al., 2020) and pilocarpine-induced status epilepticus in rats (hippocampus) (Dona et al., 2009). While increases in P2X7R expression are well established, the cell-type-specific expression pattern of P2X7R in the setting of seizures and epilepsy remains controversial, in line with a wider ongoing debate surrounding its expression in the CNS (Illes et al., 2017; Miras-Portugal et al., 2017). Using immunohistochemistry approaches, P2X7R expression has been detected at glutamatergic nerve terminals (Dona et al., 2009). Neuronal P2X7R expression has also been detected using a soluble enhanced green fluorescent protein (EGFP) bacterial artificial chromosome (BAC) transgenic P2X7R reporter mouse model (Engel et al., 2012; Jimenez-Pacheco et al., 2013). In this model EGFP is under the transcriptional control of the P2rx7 promoter. These results have been challenged by the use of a P2X7R reporter mice in which P2X7R is fused to EGFP. This approach detected P2X7R expression within the CNS mainly in microglia and oligodendrocytes (Kaczmarek-Hajek et al., 2018; Morgan et al., 2020). A recent study comparing both transgenic P2X7R reporter mouse lines has shown abnormal P2X7R expression and increased P2X4R expression in the soluble EGFP BAC transgenic P2X7R mouse model, questioning the validity of this reporter mouse (Ramirez-Fernandez et al., 2020). P2X7R expression has not been detected on astrocytes (Engel et al., 2012; Morgan et al., 2020). Similarly to status epilepticus, there is wide consensus that increased P2X7R expression occurs during epilepsy in the brain (hippocampus and cortex) with evidence from both rodent models and patients with TLE (Vianna et al., 2002; Dona et al., 2009; Barros-Barbosa et al., 2016b; Jimenez-Pacheco et al., 2013, 2016). Again, as in status epilepticus, P2X7R expression increases in microglia, neurons, and oligodendrocytes, but was not detected in astrocytes. Vianna et al. found P2X7R expression increased on mossy fibers and glutamatergic nerve terminals of epileptic pilocarpine-treated rats (Vianna et al., 2002; Dona et al., 2009). A neuronal P2X7R expression during epilepsy was confirmed later using the soluble EGFP BAC transgenic P2X7R mouse model (Jimenez-Pacheco et al., 2013, 2016). However, as for status epilepticus, neuronal expression of P2X7R during epilepsy could not be confirmed in a more recent study using the fused P2X7R-EGFP reporter mice (Morgan et al., 2020). Thus, while compelling evidence suggests P2X7R to be increased in microglia following status epilepticus and during epilepsy, it is still a matter of debate as to whether P2X7R expression increases in neurons.

Although less investigated, changes in the expression of the different P2YR subtypes have also been reported in both rodent models and patients (Alves et al., 2018). P2ry6, P2ry12, and P2ry13 mRNA transcripts were found increased in the hippocampus post-status epilepticus using the intraperitoneal KA mouse model (Avignone et al., 2008). A later study carried out by Alves et al. using the intra-amygdala KA mouse model reported that while mRNA levels of adenine nucleotide-sensitive receptors were decreased (P2ry1, P2ry12, and P2ry13), transcript levels of uracil nucleotide-sensitive receptors were increased (P2ry2, P2ry4, and P2ry6) in the hippocampus. When looking at the protein level, P2YRs coupled to Gq were increased (P2Y₁R, P2Y₂R, P2Y4R, and P2Y6R), and P2YRs coupled to Gi were downregulated or not changed (P2Y12R, P2Y13R, P2Y14R) (Alves et al., 2017), suggesting that receptor expression is dependent on substrate (mRNA) and receptor-downstream signaling (protein). In contrast to the hippocampus, however, P2YRs expression in the cortex was mainly upregulated post status epilepticus (Alves et al., 2019b). An upregulation of P2YRs was also the predominant response during epilepsy in both epileptic mice and patients (Sukigara et al., 2014; Alves et al., 2017). Very little is known regarding the cell-type-specific expression. Whereas P2Y1Rs have been shown to be increased on microglia post-intra-amygdala KA-induced status epilepticus (Alves et al., 2019a), P2Y2Rs and P2Y4Rs have been detected on astrocytes in brain tissue from patients with intractable epilepsy (Sukigara et al., 2014).

Targeting P2 Receptors during Epilepsy

P2X Receptors

Among the P2XRs, the P2X7R subtype has attracted by far the most attention as a potential therapeutic target for seizure suppression and treatment of epilepsy (Beamer et al., 2017) (Table 73–1). The only other two P2XRs for which functional data are available are P2X3R and P2X4R.

The first study suggesting anticonvulsive potential of targeting the P2X7R was published by Engel et al. in 2012. Here the authors showed that P2X7R deletion or blocking of the P2X7R (antagonists and P2X7R-targeting antibodies) reduced both seizure severity during status epilepticus and resulting neurodegeneration in the intra-amygdala KA mouse model (Engel et al., 2012; Jimenez-Pacheco et al., 2013). Suggesting the potential of P2X7R antagonists as adjunctive treatment for pharmaco-resistant status epilepticus, P2X7R antagonists potentiated effects of the anticonvulsant lorazepam (Engel et al., 2012). An anticonvulsive effect of P2X7R antagonism was confirmed in a more recent study using a rat model of coriaria lactone-induced status epilepticus (Huang et al., 2017). This anticonvulsive potential of targeting P2X7R, seems, however, heavily dependent on the model used. Fischer et al. showed no anticonvulsant effects provided by P2X7R antagonism using the maximal electroshock seizure threshold test and the PTZ seizure threshold test in mice. In the same study, the authors, however, observed that P2X7R antagonists potentiated anticonvulsant effects of carbamazepine in the maximal electroshock seizure test, supporting the potential of P2X7R-based treatment as adjunctive therapy (Fischer et al., 2016). Similar to these results, in a study published one year later, Nieoczym et al. (2017) only found a weak anticonvulsant effect of targeting the P2X7R in the intravenous pentylenetetrazole (PTZ) seizure threshold, maximal electroshock seizure threshold, and 6 Hz psychomotor seizure threshold tests. More recently, Dogan et al. showed no effect of P2X7R antagonism on seizures in WAG/Rij rats, a model of genetic absence epilepsy (Dogan et al., 2020). On the other hand, a pro-convulsive function of P2X7R is suggested by Kim et al., who reported in 2012 that seizure severity was exacerbated in P2X7R knockout mice subjected to intraperitoneal pilocarpine. This may be unique to the pilocarpine model, as P2X7 knockout mice treated with intraperitoneal KA showed no increase in seizure severity (Kim and Kang, 2011). In subsequent studies the same group showed, using a pilocarpine rat model, that P2X7R antagonism protected from astroglial cell death, reduced the infiltration of neutrophils into the frontoparietal cortex and increased status epilepticus–induced hippocampal neurodegeneration (Kim et al., 2009, 2010, 2011).

In addition to anticonvulsive effects during acute seizures, an impact of targeting the P2X7R has also been investigated during the development of epilepsy and on established epilepsy. In 2015, Soni et al. showed that P2X7R antagonism decreased the mean kindling score and restored cognitive deficits and motor coordination in the PTZ kindling model in rats, suggesting antiepileptogenic potential (Soni et al., 2015). These antiepileptogenic effects were confirmed in two more recent studies by Fischer et al. using the same model (Fischer et al., 2016) and by Amorim et al. using P2X7R-targeting siRNA post status epilepticus in the pilocarpine rat model (Amorim et al., 2017). Jamali-Raeuly et al., meanwhile, showed that treatment with a P2X7R antagonist and linagliptin, which blocks the enzyme dipeptidyl peptidase-4 and is used for the treatment of diabetes, reduced more efficiently seizure severity and neuronal cell death when given in combination in a KA rat model (Jamali-Raeufy et al., 2020). Similar, however, to status epilepticus, treatment with a P2X7R antagonist post pilocarpine-induced status epilepticus in mice resulted in the development of a more severe epileptic phenotype (Rozmer et al., 2017).

Less conflicting results have been obtained during epilepsy with two studies, published both in 2016, demonstrating antiepileptic effects of P2X7R antagonism. In the study carried out by Amhaoul et al., P2X7R antagonism during epilepsy reduced seizure severity, without, however, affecting the total number of seizures (Amhaoul et al., 2016). Jimenez-Pacheco et al. meanwhile showed that mice subjected to intra-amygdala KA and treated with a P2X7R antagonists starting 10 days post status epilepticus, when mice usually have experienced their first epileptic seizure, experienced fewer seizures during treatment and, remarkably, during a 1-week drug-washout period, suggesting disease-modifying potential (Jimenez-Pacheco et al., 2016).

As mentioned before, the only other P2XRs for which a functional role has been investigated in the setting of seizures and epilepsy include the P2X3R and P2X4R subtype. Here, Xia et al. showed, using a PTZ-induced kindling rat model, that treatment with a P2X3R antagonist reduced the mean kindling score and improved other pathological parameters such as memory deficits, motor activity, neuronal damage, and hippocampal inflammation (Xia et al., 2018). Using P2X4R knockout mice subjected to intraperitoneal KA-induced status epilepticus, Ulmann et al. showed that, despite experiencing no changes in seizure severity during status epilepticus, P2X4R deficiency partially protects from seizure-induced neurodegeneration (Ulmann et al., 2013).

P2Y Receptors

While less investigated, increasing evidence also suggests a role of the metabotropic P2YRs during epilepsy (Engel et al., 2016; Alves et al., 2018) with the P2Y1R and P2Y12R being the most studied (Alves et al., 2018; Table 73–1). In one of the first studies published in 2014, Eyo et al. demonstrated increased seizure severity in P2Y12R knockout mice subjected to intraperitoneal KA which was accompanied by a reduction in microglia process extension in the hippocampus (Eyo et al., 2014). Meanwhile, more recently, Milior et al. showed, using resected hippocampal tissue from patients with TLE, that while low doses of ADP induce microglial process extension, which was suppressed by treatment with P2Y12R antagonists, high doses of ADP cause microglia process retraction and membrane ruffling, blocked by the co-application of antagonists against the P2Y1R and P2Y13R (Milior et al., 2020).

One of the first investigations suggesting a role for the P2Y1R during seizures stems from a study carried out by Simoes et al. in 2018. Here, the authors showed that blocking of the P2Y1R reduced neuronal cell death following intraperitoneal KA-induced status epilepticus in rats. Seizure severity was, however, not affected by treatment (Simoes et al., 2018). A more complex role of P2Y1R was, however, suggested by a more recent study published by Alves et al. in 2019 in which mice deficient for P2Y1R experienced more severe seizures during intra-amygdala KA-induced status epilepticus with an associated increase in seizure-induced neurodegeneration (Alves et al., 2019a). These results were confirmed via pharmacological approaches where specific P2Y1R antagonists prior to intra-amygdala KA led to a more severe seizure phenotype. Conversely pretreatment with a P2Y1R agonist reduced seizure severity during status epilepticus (Alves et al., 2019a). However, and in contrast to their pretreatment regime, treatment of mice with P2Y1R agonists and antagonists post intra-amygdala KA injection, after the occurrence of the first seizure burst, showed the opposite result. In this case, P2Y1R antagonists reduced seizure severity and neurodegeneration, while treatment with a P2Y1R agonist increased seizure severity. Further confirming the anticonvulsant potential of P2Y1R antagonists once pathological changes are ongoing, data from the same study showed that treatment with a P2Y1R antagonist post-status epilepticus delayed the onset of epilepsy, and, when applied during epilepsy, suppressed epileptic seizures. However, in contrast to targeting the P2X7R, this effect did not persist following treatment withdrawal (Alves et al., 2019a). P2Y1R-based treatment is therefore highly dependent on the time of intervention relative to the onset of epilepsy. Whether this context-specific role during seizures is also true for other purinergic receptors remains to be established.

P2 Receptor–Mediated Molecular Mechanisms Contributing to Seizures and Epilepsy

P2 receptors have been implicated in multiple cellular processes pertinent to seizures and epilepsy, including synaptic reorganization, the regulation of the blood–brain barrier (BBB), cellular survival, circadian rhythms, neurogenesis, and inflammation (Burnstock and Ralevic, 2014; Pitkanen et al., 2015; Khan et al., 2018; Klein et al., 2018; Ali et al., 2020). While the exact mechanism(s) of how P2 receptors contribute to increased hyperexcitability in the brain remain to be established, inflammation has attracted particular attention. In this section we will briefly summarize evidence suggesting that P2 receptors impact on inflammatory processes during epilepsy. As mentioned before, it is, however, important to keep in mind that purinergic signaling is not restricted to driving inflammatory processes and that its impact on seizures is most likely the combination of several altered cellular pathways rather than a single one.

A role for inflammation on neuronal excitability is well established. Cytokines, such as the P2X7R downstream target Interleukin (IL)-1β, have been shown to be upregulated during seizures and epilepsy and to modulate GABAergic and NMDA receptor-dependent synaptic transmission (e.g., Wang et al., 2000a; Yang et al., 2005; Roseti et al., 2015; Stellwagen et al., 2005; Vezzani et al., 2019). Critically, drugs targeting inflammatory signaling pathways have shown promising effects during acute seizures and epilepsy (Vezzani et al., 2019).

One of the P2 receptors which has been most closely associated with inflammatory processes is the P2X7R. The P2X7R is highly expressed on microglia under both physiological conditions and during epilepsy where it contributes to microglial activation and proliferation (Monif et al., 2009; Kaczmarek-Hajek et al., 2018; Morgan et al., 2020). Evidence suggesting regulation of inflammatory processes during seizures and epilepsy by P2X7Rs stems from studies showing that blockade of the P2X7R leads to decreased hippocampal IL-1β levels post intra-amygdala KA-induced status epilepticus (Engel et al., 2012) and reduced microgliosis and astrogliosis during epilepsy (Jimenez-Pacheco et al., 2016). Although P2X7Rs are not detected on astrocytes during epilepsy, P2X7R antagonism protects against astrocyte death following pilocarpine-induced status epilepticus (Kim et al., 2009). Astrocytes can reduce seizure threshold via various mechanisms (e.g., dysregulation of the extracellular ionic balance, impaired neurotransmitter reuptake, release of pro-inflammatory cytokines and purines [e.g., ATP], removal of extracellular adenosine via ADK) (Bedner et al., 2015; Robel et al., 2015; Illes et al., 2019; Boison, 2008). Supporting this indirect role for P2X7Rs in astrocyte function, P2X7R-deficient mice subjected to intraperitoneal KA showed decreased astroglial autophagy through the regulation of FAK- and PHLPP1/2-mediated AKT-S473 phosphorylation (Lee and Kim, 2020).

The P2X4R subtype, primarily expressed on activated microglia where it drives microglia motility, process extension, activation, and recruitment to the site of damage (Raouf et al., 2007, Ohsawa et al., 2007, Montilla et al., 2020), represents another receptor subtype possibly contributing to inflammatory processes during seizures. Its contribution to seizure-induced inflammation is supported by Ullman et al., who found that P2X4Rs localize to activated microglia during intraperitoneal KA-induced status epilepticus and that mice deficient in P2X4R showed an impairment in several characteristics of microglia activation, including cell recruitment and upregulation of voltage-dependent potassium channels (Ulmann et al., 2013).

In contrast to the P2X7R and P2X4R, the P2Y12R seems to perform a protective function during seizures. In the brain, P2Y12R expression is mainly restricted to microglia (Haynes et al., 2006), where it has been involved in several processes, including microglial migration, the regulation of microglial-neuron interactions, and microglia injury detection (Gomez Morillas et al., 2021). While P2Y12R immunoreactivity has been widely accepted as a marker of resting microglia (Mildner et al., 2017), that is, microglia in nonpathological conditions, the primary role of P2Y12R activation is to induce reactivity of microglia and to stimulate their migratory movement via PI3k/PLC signaling to sites of inflammation (Ohsawa et al., 2007; Irino et al., 2008). In this line, Eyo et al. and, more recently, Milior et al. showed by using mouse models of epilepsy and resected brain tissue from TLE patients that both the deletion of P2Y12 (Eyo et al., 2014) and treatment with P2Y12R antagonists (Milior et al., 2020) reduced microglia process extension thereby preventing possible seizure-suppressive microglia-neuron interactions. Avignone et al. observed an increase in microglia motility following treatment with the P2Y12R agonist 2-Me-ADP following intraperitoneal KA-induced status epilepticus when compared to control (Avignone et al., 2015). This further supports a role for P2Y12Rs in regulating microglia during seizures.

In the case of the P2Y1R, findings are more complex with cell-specific expression changes and anti- and pro-convulsive functions that depend on the disease stage and model used. Whereas Alves et al. suggested that the proconvulsant effects of P2Y1R are mediated via P2Y1R acting on microglia, others have suggested these effects are mediated via astrocytes (Alvarez-Ferradas et al., 2015; Martorell et al., 2020). In support of a pro-convulsive role of P2Y1Rs via activation of microglia, Alves et al. showed that P2Y1Rs become rapidly up-regulated in microglia following intra-amygdala KA and that P2Y1R antagonism suppressed seizures. Thus, a likely explanation is that P2Y1Rs contribute to increased hyperexcitability via driving inflammatory processes (Alves et al., 2019a). In the same study, P2Y1R was mainly found to be expressed on neurons during physiological conditions. Because P2Y1R antagonism applied before intra-amygdala KA injection increased seizure severity, the authors concluded that, in contrast to P2Y1R on microglia, neuronal P2Y1R expression may have anticonvulsive effects. By contrast, and supporting the hypothesis that P2Y1R acts via astrocytes, P2Y1R has been suggested to contribute to seizures and epilepsy via the mediation of astrocytic-Ca²⁺ oscillations (Alvarez-Ferradas et al., 2015). More recently, and again in agreement with P2Y1R acting on astrocytes, Martorell et al. showed that P2Y1R inhibition rescues both the abnormal pattern of astroglial Ca²⁺-activity and plastic properties of CA3-CA1 synapses in the epileptic hippocampus (Martorell et al., 2020).

In a nutshell, while P2X4, P2X7, and P2Y1Rs seem to activate microglia, thereby lowering seizure threshold and contributing to the development of epilepsy, P2Y12Rs seem to exert an anticonvulsive function maintaining microglia in a nonactivated state. In addition, P2Y1Rs may also contribute to seizures via the activation of astrocytes (Fig. 73–2). It is, however, important to keep in mind that both cell types, astrocytes and microglia, maintain a tight relationship during seizures and epilepsy, thereby impacting on each other’s activation status and function (Sano et al., 2021). Microglial inflammatory action may precede astrocytic action, with microglia known to stimulate the transition of astrocytes to a neuroprotective phenotype (Shinozaki et al., 2017) and to tightly regulate their proliferation (Quintas et al., 2011). Moreover, while microglia were previously believed to have a mainly pro-convulsant function via the secretion of pro-inflammatory cytokines (e.g., IL-1β) (Vezzani et al., 1999), more recent research now has suggested also a protective function of microglia. In this line, the depletion of microglia leads to an amplification and synchronization of neuronal activity culminating in the generation of epileptic seizures. Interestingly, this effect was highly dependent on microglia’s ability to sense and catabolize extracellular ATP, which is released by neurons and astrocytes following neuronal activation (Badimon et al., 2020). This detrimental effect was further confirmed by another two recent studies where microglia were depleted in a similar fashion (Zhao et al., 2020; Liu et al., 2020).

Figure 73–2.

Positive feedforward loop of inflammation and P2 signaling underlying epileptogenesis. ATP is released from cells via exocytotic mechanisms, through leakage across damaged membranes and through purinergic channels such as the P2X7R and Pannexin-1 following (more...)

In summary, increasing evidence suggests P2 receptors as important contributors to inflammatory processes during epilepsy. Whether P2 receptor-driven changes in inflammation are the main signaling pathway of how these receptors impact on seizures and epilepsy and exactly which cell types are involved remain to be fully established. This will, however, most likely require the use of cell-specific knockout mice (e.g., Cre-LoxP system) or cell-type-specific targeting of different receptors (e.g., via viral vectors).

Purines as Diagnostics for Epilepsy

Together with the lack of effective pharmacological approaches in over 30% of patients and the absence of disease-modifying treatments, epilepsy diagnosis remains a clinical challenge, adding significantly to the disease burden (Moshe et al., 2015; Thijs et al., 2019). A correct diagnosis of epilepsy is critical to inform treatment. To date, epilepsy diagnosis relies, however, heavily on clinical examination and history and, patient monitoring via long-term video-EEG recording at hospitals remains the gold standard for the diagnosis of epilepsy. This is, however, time-consuming, costly, low-throughput, and requires a high level of specialist expertise (Engel et al., 2013; Moshe et al., 2015). Misdiagnosis rates are high and clinical signs can easily be confused with disorders, which present in a similar way, such as psychogenic nonepileptic attacks (Benbadis, 2009). Accordingly, there is significant interest in the discovery and validation of novel molecular biomarkers to identify patients with epilepsy. Ideally, these biomarkers should be noninvasive, be measured via a reproducible, easy-to-use, and economically feasible analysis platform and associated with cellular and molecular changes occurring during seizures and epilepsy (Engel et al., 2013).

Our laboratories have pioneered the development and use of biosensors for purines, and biosensors for adenosine and the downstream purines inosine and hypoxanthine have been developed to allow rapid and convenient measurement of purines in whole blood (Beamer et al., 2021; Martin et al., 2019; Fisher et al., 2019; Dale et al., 2019; Tian et al., 2007, 2017). As we have discussed previously, purines are produced in the brain during seizure activity. They also readily cross the BBB and can thus be detected in whole blood (Tian et al., 2017). This make purines a potentially valuable and convenient biomarker that could be elevated in the blood of epilepsy patients.

We have explored this concept in both mouse models of epilepsy and human patients. In a recent study, we found that induction of status epilepticus in mice rapidly elevated whole blood purine levels by nearly 4-fold (Beamer et al., 2021). Importantly, the extent of elevation correlated with the intensity of seizures and the degree of neurodegeneration in the hippocampus that followed induction of seizures, suggesting that the magnitude of the purine signal could indicate the severity of the epileptic insult. The sensitivity and specificity of these measurements had excellent performance in the mouse model with an area under the receiver operating characteristic (ROC) curve of 0.95.

Based on these results, we performed an initial study on 26 epileptic patients and 13 controls. Despite the patients not having experienced a seizure within 24 h of the measurement, we found that the baseline level of whole blood purines of epileptic patients was elevated by 2-fold relative to comparable healthy controls (Beamer et al., 2021). Overall, the diagnostic performance (area under the ROC curve of 0.79) in this study was highly promising and suggests that whole blood purine measurements could provide a rapid biomarker test suitable for triaging suspected epilepsy patients for more detailed clinical assessment (Beamer et al., 2021). Further trials are needed to explore this further and to study the extent to which, like in the mouse model, the level of whole-blood purines in human patients could indicate the severity of the condition.

Conclusion

We now have a substantial body of evidence demonstrating the therapeutic potential of targeting the purinergic system during epilepsy. This includes the well-known anticonvulsive and antiepileptogenic effects of targeting adenosine-dependent pathways, and more recently also ATP-dependent receptors (Fig. 73–3). While there are still important questions to be answered, such as possible cell-type-specific effects of certain receptors (e.g., P2Y1R) or the confirmation of results in animal models mimicking closer the human condition (e.g., TBI-induced epilepsy, genetic models), we have come a long way and it is now clear that the purinergic signaling system represents numerous promising targets for seizure control and treatment of epilepsy and, as shown more recently, as a diagnostic tool. Of particular promise are the disease-modifying effects observed via targeting of the ATP-gated P2X7R or ADK (Jimenez-Pacheco et al., 2016; Sandau et al., 2019). With drugs targeting the purinergic signaling system quickly moving from preclinical to clinical trials stage for CNS diseases (Timmers et al., 2018; Bhattacharya, 2018), the first clinical trials targeting this system in epilepsy can be expected in the near future.

Figure 73–3.

Targeting purinergic signaling to treat seizures and epilepsy. Schematic illustrating possible therapeutic intervention points targeting the purinergic system to suppress seizures and to treat epilepsy. (1) Purine release: Purines (e.g., ATP and adenosine) (more...)

Acknowledgments

Disclosure Statement

References

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