1.Introduction
Epilepsy is a common neurological disorder in which excessive and abnormal neuronal discharges can be observed and is characterized by recurrent seizures(Chen et al., 2020). At present, antiepileptic drugs (AEDs) are still the major methods of treatment for epilepsy in clinic and about 1/3rd of patients with epilepsy are drug-resistant (DRE)(Moshé et al., 2015). The epileptogenesis is usually involved in neuropathological processes such as ion channel dysfunction, neuronal injury, inflammatory response, synaptic plasticity, glial cell proliferation and mossin fibrosis, whereby affecting neuronal function in the brain(Gan et al., 2015). Owing to the neuropathological process of epilepsy is complex and changeable, the pathogenesis of epilepsy is not completely clear, which brings difficulties to the prevention and treatment of epilepsy. In addition, an increasing number of epileptic patients with potential genetic causes fully proves that the regulation of genes may play a pathophysiological role in the epileptogenesis or progression of epilepsy.
In gene regulation, epigenetics mainly regulates gene expression through DNA methylation, histone modification and RNA methylation, so as to change the genetic information of organisms, but does not change the sequence of DNA nucleotides(Huang et al., 2021). At present, epigenetic mechanisms have been proved to be necessary to maintain neuronal function in the human brain(Hauser et al., 2018). A growing number of studies have shown that epigenetic regulation including histone modification, DNA methylation, ncRNAs and REST/NRSF, is also involved in epilepsy(Henshall and Kobow, 2015; Citraro et al., 2017; Butler-Ryan and Wood, 2021). In cultured hippocampal neurons of rats, it has been found that the cellular memory of epileptogenesis may be related to epigenetic regulation of epileptic target genes(Kiese et al., 2017). Importantly, epigenetic regulation can influence susceptibility and severity of SE, and in turn SE can also drive the changes of epigenetic markers that influence the expression of epileptogenesis-associated genes(Henshall, 2018). In the CNS, DNA methylation has been demonstrated to be involved in the expression of nerve cell-specific genes and is significantly altered in the animal models of epilepsy and human epileptic tissues(Zhu et al., 2012; Dębski et al., 2016). Meanwhile, improved cognitive function and hyperexcitability phenotypes after status epilepticus (SE) is also associated with altered DNA methylation(Henshall, 2018). In addition, in animal models of epilepsy and epileptic patients, altered histone acetylation have been thought to be involved in epileptogenesis and prolonged seizures also modify chromatin compaction by histone acetylation (Citraro et al., 2017; Henshall, 2018). Different brain-specific microRNAs have been observed to be abnormally expressed in animal models of epilepsy and epileptic patients, particularly in temporal lobe epilepsy (TLE)(Cattani et al., 2016). In addition, ncRNAs including microRNAs and lncRNAs have been studied in epileptogenesis and treatment of epilepsy and may play an antiepileptic role as a new therapeutic strategy since they have highly selective targeting(Xiao et al., 2018). MicroRNAs, as potential molecular biomarkers, can affect SE by targeting epilepsy-related gene networks through post-transcriptional mechanisms. Meanwhile, lncRNAs can also influence expression of some genes which are involved in electrophysiological functions of neurons by targeting microRNAs in epilepsy(Henshall, 2018). Thus, discovering new therapeutic targets related to epigenetics and exploring the correlation between epigenetics and epileptogenesis are crucial for the prevention and treatment of epilepsy. Here, we discussed the role of epigenetic mechanisms in the occurrence and treatment of epilepsy.
2. Epigenetic modification inepilepsy
2.1DNA methylation
DNA methylation, as an epigenetic modification, has been shown to be involved in a variety of CNS disorders, including epilepsy(Portales-Casamar et al., 2016). DNA methylation refers to the formation of 5-methylcytosine (5mC) from cytosine under the catalytic action of DNA methyltransferase, which occurs mainly in cytosine-guanine dinucleotides (CpGs). In this process, DNA methyltransferases mainly include DNMT1, DNMT3a and DNMT3b(Figure1 )(Heikkinen et al., 2022). In the genome of normal cells, most CpG sequences are methylated and the hypomethylated regions of DNA function are considered as elements to regulate gene expression, such as promoters and enhancers(Nishiyama and Nakanishi, 2021). Meanwhile, DNA methylation can also promote the binding of various transcription factors(Nishiyama and Nakanishi, 2021). Currently, two major regulatory mechanisms of DNA methylation in modifying gene activity have been proposed. On the one hand, DNA methylation can directly block the binding of transcription factors, resulting in gene silencing. On the other hand, DNA methylation can attract methyl-binding proteins, such as MBD1, MBD2, MBD3, MBD4 and MeCP2, which recognize methylated cytosine, thereby indirectly leading to the changes of gene expression(Heikkinen et al., 2022; Xie et al., 2022). In addition, the methyl groups required for DNA methylation depend mainly on the transmethylation of S-adenosine methionine (SAM) in the methionine cycle of organisms, resulting in the formation of S-adenosine homocysteine (SAH). Then, SAH is cleaved into adenosine and homocysteine. Adenosine kinase (ADK) is a cytoplasmic enzyme that catalyzes the conversion of adenosine to AMP. ADK activation represents the main pathway of adenosine clearance, which can increase the DNA methylation status of epigenome through the transmethylation pathway, whereas experimental or therapeutic adenosine augmentation prevents the reactions of DNA methylation(Williams-Karnesky et al., 2013). In epilepsy, ADK also regulates intracellular adenosine to modulate epileptogenesis by the epigenetic mechanism(Williams-Karnesky et al., 2013; Xu et al., 2017). In addition, the ketogenic diet (KD), as an important treatment for epilepsy, can enhance the production of adenosine which is a metabolic feedback inhibitors of DNA methylation(Lusardi et al., 2015; Longo et al., 2019). Relevant studies have shown that DNA methylation contributes to neuron cell-specific gene expression, which is significantly changed in the animal models of epilepsy and epileptic patients(Zhu et al., 2012; Dębski et al., 2016). In a word, nutrient metabolism and DNA methyltransferases may serve as a potentially modifiable upstream mechanism regulating DNA methylation in epileptogenesis.
In the KA-induced and pilocarpine-induced SE models, the similar patterns of DNA hypermethylation have been demonstrated in the epileptic hippocampal neurons (Murugan and Boison, 2020). Meanwhile, in the three models of chronic epilepsy (pilocarpine injection, focal amygdala stimulation and post-TBI), genome-wide changes in global DNA methylation were also investigated (Dębski et al., 2016). This study has concluded that changes in genomic DNA methylation provide the general pathological mechanism of epileptogenesis(Dębski et al., 2016). It have been reported that upregulated DNMT activity and associated changes of DNA methylation in patients with TLE, including focal cortical dysplasia (FCD)(Dixit et al., 2018). Moreover, FCD subtypes including FCDIa, FCDIIa and FCDIIb may also be distinguished by DNA methylation profiles, which suggesting that DNA methylation may serve as a biomarker for FCD(Kobow et al., 2019). Recently, a study about human epilepsy has shown 224 genes with differential DNA methylation persons in epilepsy patients and healthy people(Wang et al., 2016b). In the epileptic samples, three genes (TUBB2B, ATPGD1, HTR6 ) exhibited relative transcriptional regulation by DNA methylation. TUBB2B and ATPGD1 showed hypermethylation and reduced mRNA levels, while HTR6 showed hypomethylation and increased mRNA levels(Wang et al., 2016b). These findings suggest that some genes are differentially regulated by DNA methylation in human epilepsy. Previous studies have shown that 27 hypomethylated genes and 119 hypermethylated genes are present in hippocampal tissue from patients with DR-TLE compared with healthy people(Miller-Delaney et al., 2015). Meanwhile, DNMT1 and DNMT3a are highly expressed in the temporal neocortex of DR-TLE patients, which are involved in DNA methylation(Zhu et al., 2012). The lower levels of global DNA methylation and the lower expression of DNMT3a2 were found in the hippocampus of TLE. Interestingly, compared with control and TLE groups, the expression of DNMT3a1 and DNMT3a2 was more significant reduced in the hippocampus of TLE with febrile seizures (FS) history(de Nijs et al., 2019). Mesial temporal lobe epilepsy with hippocampal sclerosis (mTLE-HS) is the most common focal epilepsy, which characterized by strong drug resistance. In the intracortical KA-induced mouse models of mTLE-HS, contralateral hippocampus (CLH) shows substantial changes in gene expression and DNA methylation in glia and neurons, in which some genes and pathways associated with antiepileptic effects were upregulated(Berger et al., 2020). Although changes of gene expression in CLH and ILH overlap to a large extent, changes of DNA methylation were not overlapped(Berger et al., 2020). As the seizures last longer, DNA methylation of many genomic sites is significantly changed, and progression of epilepsy is also relevant to changes in DNA methylation of inflammatory-related genes(Martins-Ferreira et al., 2021). Therefore, seizures may be responsible for altered DNA methylation of certain genes(Caramaschi et al., 2020). And changes of DNA methylation may also contribute to the development of epilepsy.
At present, the regulation of synaptic function in epilepsy may also have light with DNA methylation of related genes. Relevant studies have shown that DNA methylation-dependent endocytosis may be involved in the regulation of synaptic function in inhibitory cortical interneurons of mice, which have potential significance in TLE(Pensold et al., 2020). Moreover, synaptic NMDAR expression is also regulated by DNA methylation. Related study has found that activated NMDAR-containing GluN2A subunits control DNMT3a1 levels in neurons and drive degradation of DNMT3a1 in a ubiquitin-like dependent manner, which may be associated with synaptic plasticity and memory formation(Bayraktar et al., 2020). At the same time, the upregulation of DNMT3b expression is also regulated by NO produced by NMDAR activation in the hippocampus(de Sousa Maciel et al., 2020). Moreover, glutamate dysfunction and cognitive decline are also closely associated with changes in GRIN2Bpromoter methylation(Fachim et al., 2019). Overexpression of DNMT1 in ES cells leads to epigenetic changes, which results in abnormal neuronal differentiation with upregulated functional NMDARs(D’Aiuto et al., 2011). SE can trigger early and late changes of BDEF andGRIN2B DNA methylation levels in the hippocampus(Ryley Parrish et al., 2013). In the epileptic hippocampus, increased levels ofGRIN2B DNA methylation lead to decreased expression of GluN2B and decreased levels of BDNF DNA methylation also result in increased expression of BDNF. Meanwhile, inhibition of DNMT can decreaseGRIN2B mRNA expression and increase excitatory postsynaptic potential in hippocampus of epileptic rats(Ryley Parrish et al., 2013).
The blocking of DNA methylation may play an important role in epileptogenesis and treatment of epilepsy. Abnormal DNA methylation ofRASgrf1 is closely associated with epilepsy. RG108 as a DNMT inhibitor, can block hypermethylation of the RASgrf1 promoter and inhibited acute epileptic activity in the KA-induced epilepsy models(Chen et al., 2017). The high-dose 5-Aza-2dC as a DNMT inhibitor, significantly increase seizure threshold and attenuate seizures in PTZ-kindled model of rats, which suggesting inhibited DNMT activity can reduce epilepsy acquisition and seizure susceptibility(Williams-Karnesky et al., 2013). Adenosine intervention can reverse DNA hypermethylation in the epileptic brain, thereby inhibiting sprouting of mossy fibers in the hippocampus and preventing the progression of epilepsy in a rat model of TLE(Williams-Karnesky et al., 2013). Studies have shown that methylation level of EPHX1 promoter is significantly correlated with epilepsy that is resistant to carbamazepine (CBZ). EPHX1methylation may be a potential target of CBZ treatment and a potential marker of DREs for CBZ (Lv et al., 2019). Elevated levels of matrix metalloproteinase-9 (MMP-9) have been implicated in epileptogenesis of humans and animals. Upregulated MMP-9 expression is primarily regulated by deletion of MMP-9 gene proximal promoter including interweaved potent silencing mechanisms-DNA methylation and polycomb repressive complex 2 (PRC2)-related repression(Zybura-Broda et al., 2016). In addition, DNA demethylation has also been reported to depend on the gradual dissociation of DNMT3a and DNMT3b, as well as the progressive binding of DNA demethylation promoter Gadd45β to the MMP-9 proximal gene promoter in vivo(Zybura-Broda et al., 2016). These studies identify MMP-9 expression is regulated by DNA methylation in human epilepsy.
In conclusion, DNA methylation is involved in the occurrence and treatment of epilepsy. However, the regulatory mechanism of DNA methylation in epilepsy is still not completely clear and needs further exploration.