The epilepsies are a group of debilitating and progressive neurologic conditions affecting about 1% of the population (Hauser et al., 1993), and are characterized by spontaneous recurrent seizures. All current medical therapies for epilepsy symptomatically suppress seizure activity, but they are not disease-modifying, having no effect on the underlying propensity of the brain to generate seizures. Current treatments also have no significant effects on the psychiatric comorbidities associated with epilepsy, such as anxiety, depression, and psychosis, which are common in patients and significantly add to the disability burden (Tellez-Zenteno et al., 2007; Hermann et al., 2008; Vega et al., 2011). A major goal of translational epilepsy research is to identify treatments that are not merely symptomatic, but truly disease-modifying (Galanopoulou et al., 2012). There have been several recent reports of experimental successes using therapies that interfere with epileptogenesis (Blumenfeld et al., 2008; Russo et al., 2010; Wong, 2010; McClelland et al., 2011), suggesting that this goal is achievable.
Ethosuximide (ESX) is a first-line clinical symptomatic treatment for absence seizures. Recent research has demonstrated that chronic treatment with ESX, when initiated prior to the onset of the epilepsy, has antiepileptogenic effects in the WAG/Rij rat model of genetic generalized epilepsy (GGE) with absence seizures (Blumenfeld et al., 2008; Russo et al., 2010; Sarkisova et al., 2010; Russo et al., 2011). This treatment regimen also reduces the depression-like behaviors that are present in WAG/Rij rats (Sarkisova et al., 2010). Without corroboration of the antiepileptogenic potential of ESX in other models, it is unclear whether this property is limited to an isolated cause of GGE (thereby limiting the translatability of this finding), or whether it is effective in other GGE models, thereby establishing a broad applicability of this drug across animal models, and potentially extending to human cases. In this study, we investigated whether chronic treatment with ESX has disease-modifying effects against epilepsy and behavioral comorbidities in a different model of GGE with absence seizures, Genetic absence epilepsy rats from Strasbourg (GAERS).
GAERS are a well-validated polygenic model of GGE (Danober et al., 1998), with a genetic causation that is different from that of WAG/Rij rats (Gauguier et al., 2004; Rudolf et al., 2004; Powell et al., 2009). The epilepsy develops in GAERS at around 8–9 weeks of age in our colony (Jones et al., 2008), manifesting as bilateral spike-and-wave discharges on electroencephalography (EEG), which are responsive to drugs used clinically to treated absence seizures, including ESX (Tringham et al., 2012). In addition, we have shown previously that GAERS from our colonies in Melbourne exhibit an anxiety-like behavioral phenotype using the elevated plus maze and open field tests of anxiety (Jones et al., 2008). Because anxiety disorders are prevalent in pediatric generalized epilepsy patients (Caplan et al., 1998; Ott et al., 2001, 2003; Caplan et al., 2005; Jones et al., 2007; Caplan et al., 2008; Vega et al., 2011), this high-anxiety phenotype makes GAERS an appropriate model to study the influence of disease-modifying drugs on behavioral outcomes (Jones & O'Brien, 2012).
We also investigated whether ESX treatment alters expression of key components of the epigenetic molecular machinery, which may implicate an epigenetic mechanism in any long-term effects on the epilepsy and behavioral phenotype. Specifically we examined the expression levels of the DNA methyltransferase (DNMT) family of enzymes in the somatosensory cortex—a region shown previously to play a key role in spike-wave seizure expression in rodent models (Meeren et al., 2002; Polack et al., 2007; David et al., 2008; Zheng et al., 2012) following both chronic and acute ESX treatment. DNMTs catalyze the covalent attachment of methyl (CH3) groups to specific cytosine residues in the DNA sequence—an epigenetic modification termed DNA methylation, which is strongly associated with transcriptional repression of genes (Attwood et al., 2002). Any change in DNMT enzyme expression would be expected to induce enduring alterations of the expression patterns of key genes, an effect that may influence epileptogenesis.
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Here we demonstrate that chronic treatment with ESX induces a disease-modifying effect in the GAERS rat model of GGE. This extends upon previous research demonstrating an antiepileptogenic action of chronic ESX treatment in WAG/Rij rats (Blumenfeld et al., 2008; Russo et al., 2010, 2011; Sarkisova et al., 2010), a model of GGE with different genetic causation (Gauguier et al., 2004; Rudolf et al., 2004; Powell et al., 2009), suggesting that this effect is broadly applicable across absence epilepsy models. In addition, we showed that chronic suppression of seizures mitigates the anxiety-like behavioral phenotype in GAERS, an effect that presented itself only after the onset of seizures.
GAERS from our Melbourne colonies exhibit a highly anxious phenotype, which is present prior to the onset of the epilepsy (Jones et al., 2008). Our previous studies characterizing this phenotype employed GAERS/NEC rats originally obtained from Hull, United Kingdom, in 2002 (Jones et al., 2008, 2010; Bouilleret et al., 2009), and contrasted from a much earlier report on the original Strasbourg colony, which did not find an hyperanxious phenotype (Vergnes et al., 1991). It is important to note that we now replicate this phenotype using GAERS/NEC rats from our inbred colonies sourced from the founder colony from Strasbourg in 2007, providing broad validation of the association of this behavioral trait with different GAERS colonies. Exposure to a novel open field is a widely used measure of anxiety-like behavior that demonstrates predictive validity (Prut & Belzung, 2003), and this apparatus gives strong and reproducible data when testing GAERS. However, caution must be taken when using only a single test for behavioral outcomes, and future studies should use batteries of assays to comprehensively characterize anxiety-like phenotypes in this and other rat strains.
An intriguing aspect of the disease-modifying effects of ESX treatment observed here was the mitigation of the anxiety-like behavioral comorbidity. Because the GAERS strain was originally bred for the epilepsy phenotype, the emergence of the behavioral abnormality is likely to be linked in some way to the epilepsy, even though it appears to manifest before seizures begin. Clinically, this ontogeny has also been reported, with pediatric patients with GGE frequently experiencing anxiety disorders prior to the first recognized seizure (Jones et al., 2007). Here we show that the behavioral deficit is significantly attenuated by ESX treatment, but only in chronically epileptic animals, suggesting an indirect effect of the treatment on behavior that is dependent on the presence of seizures. This finding also agrees with a previous report describing amelioration of depression-like behavior during chronic ESX treatment in the WAG/Rij model (Sarkisova et al., 2010), and further enhances the postulate of bidirectionality between epilepsy and psychiatric comorbidities (Kanner, 2011). Here, we did not examine any measure of depression-like behaviors, so we are unable to comment on whether depression phenotypes are similarly improved by ESX in GAERS.
An intriguing question posed by the effects of ESX treatment relates to the mechanism by which it induces disease-modifying effects. ESX is widely believed to act as an antagonist at low threshold T-type calcium channels, and there is evidence from pilocarpine-induced status epilepticus studies that T-type calcium channels play a role in limbic-acquired epileptogenesis (Becker et al., 2008), suggesting involvement of these ion channels. However, recent studies suggest that ESX may not be working as a pure antagonist at T-type channels (Goren & Onat, 2007), so the role of T-type channels in these effects is not clear. It is also feasible that the effect of ESX represents the inhibition of a “kindling-like” phenomenon, whereby a cycle of seizures begetting seizures is interrupted by the ESX treatment. Supporting evidence comes from observations that chronic treatment with other antiabsence drugs (e.g., levetiracetam and zonisamide) induce similar sustained effects on epilepsy development in WAG/Rij rats (Russo et al., 2011). However, chronic treatment with carbamazepine, which is recognized to acutely increase seizures in models of absence epilepsy (Liu et al., 2006), did not induce the anticipated sustained increase in seizure activity (Russo et al., 2011).
It is recognized that many changes in gene expression occur in GAERS over the course of disease development (Jones et al., 2011), potentially representing causal or contributory drivers of the epileptic and behavioral phenotypes. Epigenetic mechanisms represent methods of controlling gene expression, and they are typically mediated by changes in the structure of chromatin and other DNA binding proteins that modify the accessibility of transcription factors to their binding domains (Borrelli et al., 2008). Here we demonstrate that chronic ESX treatment in GAERS results in alterations in the expression levels of the DNMT enzymes that catalyze DNA methylation, the most widely studied epigenetic modification. Indeed, such alterations in DNA methylation enzyme expression may also be relevant to other types of epilepsy, since a recent report identified increases in DNMT1 and DNMT3a in brain samples from human patients with temporal lobe epilepsy (Zhu et al., 2012). The expression changes identified here would be expected to modify the DNA methylation landscape and influence expression of many downstream genes, some of which may be relevant to the long-term disease-modifying effects of ESX. The attraction of this explanation lies in the prevailing assumption that DNA methylation is a stable and enduring mark, and therefore able to continue to mediate an ongoing gene expression profile long after the intervention has ceased (in this case, ESX treatment). DNMT1 acts as the maintenance methyltransferase during DNA replication, adding methyl groups onto the new DNA strand in appropriate positions (Leonhardt et al., 1992). In addition to its role in cell division, it is highly expressed in postmitotic cells (Veldic et al., 2004), suggesting it also plays a role in regulating DNA methylation patterns in mature neurons, which may be relevant to neurologic function and dysfunction. DNMT3A and DNMT3B are de novo methyltransferases, acting during development to dramatically alter the DNA methylation landscape (Okano et al., 1998), and DNMT3A has also been shown to affect neuronal function in adulthood (LaPlant et al., 2010). We found a significant gene versus drug interaction for DNMT3A expression, such that this enzyme was upregulated in GAERS only if the animals received ESX treatment. A similar trend, but without a statistical interaction, was observed for DNMT1. These effects did not appear to be acute effects of ESX, since a single drug injection did not affect DNMT expression. This is intriguing, and it suggests that these changes may act to alter DNA methylation patterns to mediate the effects of ESX on disease development and severity. It may also explain why the ESX had no effect on anxiety-like behavior in the NEC rats. Elevated levels of DNMT3B in both treated and untreated GAERS compared to NEC rats indicates that, although this enzyme may play roles in any DNA methylation differences between the strains, it is unlikely to be a mediator of any long-term beneficial effects of ESX. Further studies are required to demonstrate how ESX treatment might influence DNMT expression in GAERS, whether the changes in DNMT levels are relevant to the effects of ESX, and by extension whether this biologic process can be targeted to induce disease-modifying effects in epilepsy (Qureshi & Mehler, 2010).
Another point of interest raised from this study is whether there is a critical developmental window where treatment must be initiated for effects on disease progression to be realized. The ESX treatment in this study, and the previous studies in WAG/Rij rats, was started in young rats (3 weeks of age) prior to the onset of spontaneous recurrent seizures. This is relevant to any potential translation of this research to humans, where in most circumstances patients present to the clinic after their first seizure. An important next step will be to examine whether chronic ESX treatment initiated after the onset of epilepsy also produces disease-modifying and anxiolytic effects observed in this study. Overall, the current work supports an increasing literature base suggesting that targeting epileptogenic pathways is a feasible therapeutic strategy, and perhaps brings closer the prospect of identifying the holy grail of epilepsy research: a cure.