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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/9780197549469.003.0035
Epigenetics refers to those mechanisms that allow a transient stimulus to induce long-lived programs of gene expression changes without altering the underlying DNA sequence itself. Epigenetic regulators function by catalyzing the modification of DNA or histone protein tails, which, in turn, up- or down-regulate the transcription of target genes. Originally thought to be static in terminally differentiated cells, it is now known that epigenetic processes are active and dynamic in mature neurons. Their function or perturbation may be sufficient to develop common neurological disorders such as epilepsy. This chapter provides a critical overview of key epigenetic changes detected in human patients and experimental epilepsy models. The chapter reflects on how these data impact epileptogenesis, what role epigenetic mechanisms may play in facilitating disease pathogenesis, and how it influences the chronic epileptic state. In the end, the chapter briefly discusses the potential for targeting epigenetic modulators as a new source of therapeutic intervention in epilepsy.
The origin of epigenetics began with the study of evolution and an attempt to understand how a fertilized zygote develops into a complex, mature organism (i.e., development; Felsenfeld, 2014). Given that all cells in an organism carry the same blueprint for life, Conrad Waddington in 1942 defined epigenetics as a complex set of developmental processes that guide the genotype-to- phenotype transition (Deichmann, 2016). Since then, the field of epigenomics has grown to encompass efforts that study how the genome responds to transient stimuli or environmental changes; what proteins and molecular drivers are responsible for epigenetic modification; what conditions render the genome to acquire epigenetic variation; and how perturbations in these processes may lead to human disease. The modern definition of epigenetics is information heritable during cell division other than the DNA sequence itself (Feinberg, 2007). However, the same regulatory mechanisms that transmit information through cell division are also present and active in postmitotic neurons in the adult brain. In this chapter, they will be referred to as “epigenetic” as they maintain and accumulate information over time. All primary epigenetic mechanisms, including DNA methylation, histone modifications, noncoding RNAs, and chromatin remodeling, will be discussed with their potential contribution to seizure development and participation in a cellular memory of epileptogenesis.
Every cell in the human body contains approximately 2 m of DNA with the blueprint for constructing nearly 23,000 protein-encoding genes. To fit these instructions inside a cell, 3 × 109 bases of DNA must be packaged into the nucleus such that the genome is accessible for transcription by nuclear machinery but also maintains local heterochromatic (tightly packed) structure for long-term silencing of genomic regions such as telomeres. In eukaryotes, the packaging and compaction of DNA is facilitated by wrapping 146 bp of DNA around histone octamers (i.e., nucleosomes) consisting of two H3 histones, two H4 histones, two H2A histones, and two H2B histone proteins (Luger et al., 1997), and further hierarchical folding into higher-order chromatin fibers (Dekker and Misteli, 2015) (Fig. 35–1). Each chromosome and many genes have a characteristic distribution of positioning within the cell nucleus, adhering to specific chromosome territories (Jercovic and Cavalli, 2021) and specific active or repressive microenvironments (Wang et al., 2016). The nonrandom positioning of genes in the nucleus allows functionally related genes to be spatially clustered in 3D space and share overlapping activating or repressing protein complexes. Also, long-range interactions with distant regulatory features such as enhancers are possible through chromatin looping (Dekker and Misteli, 2015). In summary, local and global spatial chromatin organization holds a central role in controlling gene expression under physiological and pathological conditions.
A simplified overview of epigenetics and organization of genetic material starting from densely packed nucleosomes, which are twice-looped DNA wrapped around an octamer of histone proteins. Energy-dependent chromatin remodeling complexes can slide, eject, (more...)
The nucleosome not only serves as an inert structure for wrapping DNA and facilitating compaction into the cell. The nucleosome can also be a source of important gene regulatory instructions given by DNA inside every cell. One of the several ways gene expression is regulated after a developmental, environmental, or biochemical stimulus is through modifications added by specific enzymes known as epigenetic “writers.” Writers will add a covalent chemical modification to DNA or accessible N-terminal histone tails, which can subsequently be recognized and interpreted by a class of proteins known as “readers.” These modifications on top of DNA or histone tails are reversible, meaning they can be removed via another type of enzymes known as “erasers” (Nicholson et al., 2015). Through constant incorporation and removal of novel information or stimuli in our environment, epigenetic writers, readers, and erasers are in a consistent state of complex interplay and ultimately shape the level of gene transcription. Deficiencies in the epigenetic machinery have been demonstrated to contribute to many neurological diseases and cancers in both humans and rodents. Because epigenetic chromatin marks are reversible, this opens multiple avenues for appropriate therapeutic intervention.
The genome contains regulatory information beyond nucleotide sequences. This information can either be dynamic and transitory or relatively stable and can be passed onto daughter cells (Wang et al., 2020). In addition, epigenetic regulatory information can be passed onto offspring via germline, as occurs with parentally imprinted genes (Almouzni and Cedar, 2016; Staubli and Peters, 2021). Studies of human populations and animal models suggest that a mother’s or father’s experiences, such as diet or environmental stress, can influence the health and development of their descendants (Kaelin and McKnight, 2013; Sun et al., 2018; DiTroia et al., 2019). In so-called cis memory, epigenetic information is proposed to be physically stored in local chromatin states that are associated, for example, with DNA methylation or histone modifications (Almouzni and Cedar, 2016). By contrast, in “trans memory,” epigenetic information is stored in the concentration of a diffusible factor such as a transcriptional repressor (Dean, 2017). The full details on how these effects are transmitted across generations still need to be untangled.
DNA methylation occurs predominantly at palindromic CpG dinucleotides by adding a methyl group to the 5ʹ position of the cytosine pyrimidine ring, thereby generating 5-methylcytosine (5mC). It may also occur in non-CpG sequences. Three enzymes, DNA methyltransferase 1 (DNMT1), DNMT3A, and DNMT3B, either methylate DNA de novo or maintain genomic methylation patterns during cell division. Active DNA demethylation in mammals involves the ten-eleven translocation (TET) dioxygenases, which oxidize the 5-methyl group of 5mC to produce 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxylcytosine (5caC). All of these intermediates have been proposed to exert regulatory function, for example, through precluding or enhancing the binding of several (“reader”) MBD proteins (Dawson and Kouzarides, 2012: Kriaucionis and Tahiliani, 2014).
DNA methylation is widespread in mammalian genomes, with around 70% of CpG dinucleotides being methylated (Li and Zhang, 2014). The exception to this pattern is CpG-rich regions (i.e., CpG islands) within the promoters of active genes, which are characteristically unmethylated. DNA methylation is primarily noted within centromeres, telomeres, inactive X-chromosomes, and repeat sequences (Ichikawa et al., 2017; Smith and Meissner, 2013; Greenberg and Bourc’his, 2019). It is fundamental to stably suppress gene expression during development and aging, for gene dosage compensation and allelic imprinting, and maintenance of genome stability (Iguchi-Ariga and Schaffner, 1989; Reik et al., 2001; Reik and Walter, 2001; Bird, 2002). Methylation loss within repetitive elements is observed in cancer cells and can lead to activation and transposition of endogenous retroviral elements, promoting genomic instability during early embryogenesis and tumor development (Feinberg et al., 2006; Feinberg and Tycko, 2004). Active genes can also harbor methylated CpGs within their transcribed regions, regulating mRNA alternative splicing (Michalak et al., 2019; Shayevitch et al., 2018).
A mechanistically complementary regulatory layer not on the chromatin template but mRNA transcripts is provided by RNA methylation (Michalak et al., 2019), specifically methylation of adenosine to N6-methyladenosine (m6A). This modification was initially the most prevalent form of DNA methylation in prokaryotes but was later also identified in eukaryote RNA (Luo et al., 2015; Gilbert et al., 2016). Although it is not considered an epigenetic mechanism, it shares conceptual features (i.e., dedicated readers, writers, and erasers, regulates gene expression in response to developmental and environmental cues). This modification has gained increased attention as it seems to be particularly crucial for brain development and function (Chen et al., 2019). M6A is more prevalent in the brain than most other tissues, especially in adulthood. It is dynamic in neurons and responds to experience. Distribution patterns differ across brain regions, but all m6A writers and reader proteins are expressed in all brain cell types. Knockout experiments demonstrate the relevance of m6A methyltransferases for normal brain development and adult neuronal function (Engel et al., 2018). Full knockout of, for example, Mettl14, the key element of the m6A methyltransferase complex, was shown embryonic lethal in mice. Conditional knockouts of this gene in neural progenitor cells showed that Mettl14 loss disrupted cortical development and led to premature death. Intriguingly, m6A was reported to have a role in synaptic plasticity affecting spine morphology cell-surface protein content, as well as synaptic transmission and excitability (Merkurjev et al., 2018; Koranda et al., 2018). The RNA demethylation enzyme, fat mass and obesity-associated (FTO), is likewise highly expressed in the brain and crucial for normal human central nervous system (CNS) development (Rowles et al., 2012). FTO deficiency in mice as well as germline loss-of-function mutations in humans have been associated with severe growth retardation and multiple malformations affecting also the brain (e.g., microcephaly, lissencephaly) (Fischer et al., 2009; Boissel et al., 2009). Patients with pathogenic FTO variants were reported with severe developmental delay and some further presented with seizures.
The long N-terminal histone tails that protrude from the nucleosome serve as a platform for regulating gene expression through numerous chemical modifications known as histone posttranslational modifications (PTMs) (Strahl and Allis, 2000). Over 150 different PTMs have been described so far, including methylation, phosphorylation, acetylation, ubiquitylation, sumoylation, and glycosylation, among many others to lysine, arginine, serine, or threonine residues (Kouzarides, 2007). The complex landscape of histone modifications on a histone tail can impact gene expression by altering higher-order chromatin structure and its accessibility to transcription factors. The idea that chromatin-DNA interactions are guided by the combination of PTMs on the histone tail and ultimately determine the state of gene expression is known as the histone code hypothesis (Allis and Jenuwein, 2016; Jenuwein and Allis, 2001).
Histone acetylation is probably the best-studied PTM and is a highly dynamic process (Fig. 35–1). Acetylation marks are capable of changing within minutes. They are established by histone acetyltransferases (HATs) and removed by enzymes known as histone deacetylases (HDACs). Acetylation is thought to regulate gene transcription by altering nucleosome-DNA or nucleosome–nucleosome interaction. It is typically added to positively charged lysine residues, which bind strongly to negatively charged DNA prior to modification. The addition of the acetyl group effectively neutralizes this reaction, reducing the binding between histones and DNA, thereby promoting a more open chromatin conformation and active gene expression.
In contrast, deacetylation is associated with closed chromatin and silencing of gene transcription. Apart from regulating gene transcription through the described physical properties, acetylation marks can recruit a family of bromodomain (BRD) reader proteins, which may act as transcription factors themselves or as a subunit in a larger chromatin-modifying complex (Owen et al., 2000; Dhalluin et al., 1999). Disruption of histone acetylation patterns has been linked to disease development, including cancer (Ropero and Esteller, 2007), neurodegeneration and aging (Peleg et al., 2016), psychiatric disorders, and epilepsy (Hauser et al., 2018; Jakovcevski and Akbarian, 2012; Graff and Tsai, 2013). Here, both mutations and transcriptional deregulation of enzymes responsible for adding or removing histone acetylation marks, as well as protein interaction modules that recognize and interpret this important PTM, can lead to disease (Khan and Khan, 2010).
Histone methylation can either be considered an active or repressive modification mark (Fig. 35–1). Histone methylation occurs mainly on the side chains of Lys and Arg residues, but methylation of other residues also occurs in mammals (Michalak et al., 2019). Lys residues may be mono-, di- or trimethylated, whereas Arg residues may be monomethylated or symmetrically or asymmetrically dimethylated. Unlike acetylation (and phosphorylation), histone methylation does not alter the charge of the histone protein. Methyl groups are added to histone tails via enzymes known as histone methyltransferases (HMTs) and can be removed by histone eraser enzymes known as histone demethylases (Greer and Shi, 2012).
The fate of histone methylation on gene transcription is highly dependent on the residue in which the modification was placed (e.g., Lysine 4 versus Lysine 27 on histone H3), how many methyl groups were added, and the existing landscape of other histone modifications on the same tail. For example, trimethylation of Lysine 4 on histone H3 (H3K4me3) is an active mark for transcription. Meanwhile, trimethylation of Lysine 9 or Lysine 27 on histone H3 (H3K9me3 and H3K27me3, respectively) is repressive. Methyl lysine marks can be read by a class of “reader” proteins known as chromodomain proteins, which initiate their own suite of transcriptional control upon environmental and developmental cues (Blus et al., 2011). Bivalent chromatin describes histone H3 tails that have both activating H3K4me3 and repressive H3K27me3 marks simultaneously. Bivalent chromatin is commonly found in regulatory regions of developmentally regulated genes or transcription factors that are maintained at low levels. Bivalency is believed to exist to “poise” genes for rapid transcriptional activation or repression during critical, for example, developmental, time windows (Vastenhouw and Schier, 2012; Bernstein et al., 2006).
Though it is clear that histone modifications play a crucial role in incorporating events into long-lived gene expression, it is difficult experimentally to ascertain a direct causative role of histone modifications in mammalian cells because histone writers can also have nonhistone targets. For instance, in glioblastoma, the histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) that usually silences gene transcription through methylation of H3K27 was reported to methylate the nonhistone target STAT3 and, in turn, cause increased STAT3 activity (Kim et al., 2013). Besides, the H3K4 histone methyltransferase SMYD3 was found to methylate vascular endothelial growth factor receptor 1 (VEGFR1) and MAP3K2 in cancer, subsequently causing increases in both proteins’ activities (Kunizaki et al., 2007; Mazur et al., 2014).
Outside of methylation and acetylation, other well-characterized histone modifications include phosphorylation, ubiquitinylation, and glycosylation. As epigenetic research continues to grow, the number of histone modifications that play a role in gene expression also continues to increase, highlighting the exquisite complexity of epigenetic changes and gene transcription cross-talk. Most recently, serotonin was found as a novel histone modification that targets histone three glutamine 5 (H3Q5ser) in the brain and gut. Interestingly, the H3Q5ser modification was found in the presence of another histone modification, H3K4me3, which is one of the main epigenetic marks associated with active transcription. This suggested that H3Q5ser may play a role in positively regulating gene expression and may be a novel example of histone modification bivalency (Farrelly et al., 2019).
The third major category of epigenetic processes is through the action of noncoding RNAs (ncRNAs; Fig. 35–1). They are a highly heterogeneous class of RNAs that function directly as structural, catalytic, or regulatory molecules rather than as templates for protein synthesis. Their identification and functional characterization changed the notion that 95% of the genome constitutes junk (Ravasi et al., 2006; Guttman et al., 2009). One can distinguish short (or small) noncoding RNAs (sncRNA; <200 nucleotides in length; Jacquier, 2009) and long noncoding RNAs (lncRNA; >200 nucleotides; Fatica and Bozzoni, 2014), though not all noncoding RNAs are involved in epigenetic processes. For example, ribosomal and transfer RNAs are involved in protein translation in the cytoplasm.
LncRNAs constitute the largest proportion of the mammalian noncoding transcriptome. They are expressed in a tissue-specific manner, and many are exclusive to the brain (Roberts et al., 2014; Hon et al., 2017). They are essential for brain function, influencing neurons’ synaptic structure and neurophysiological properties, and may account for neurologic and psychiatric disease when deregulated (Wang et al., 2015; Qureshi and Mehler, 2012). Micro RNAs (miRNAs) are the most widely studied class of short RNA that mainly function outside the nucleus to regulate gene expression negatively (Olde Loohuis et al., 2012). Aberrant expression of miRNAs can alter the global DNA or chromatin state by restricting epigenetic enzyme activity or regulating the expression of epigenetic regulators such as DNMTs and TETs. Enhancer RNAs (eRNA) are a class of short noncoding RNA that act more locally to promote or oppose transcription of specific genes. They possess some classic epigenetic factors since they can modify histones, may contribute to a more open chromatin structure, and help bring enhancers and promoters together through chromatin looping (Li et al., 2016). Natural antisense transcripts (NATs) are transcribed from the opposite strand of DNA from the coding sequence and compete locally to reduce the abundance of the coding mRNA. Functionally, NATs participate in multiple processes of gene expression regulation, from controlling epigenetic modifications to modulating posttranscriptional modifications (Pelechano and Steinmetz, 2013; Batista and Chang, 2013). These latter two classes have been explored for therapeutic potential in epilepsy.
Nucleosomes occluding genomic DNA in promoters and coding regions present a significant barrier to gene regulation, which requires sequence-specific transcription factor binding and assembly/disassembly of transcriptional machinery. Chromatin remodelers are helicase-like proteins that use the energy of ATP hydrolysis to mobilize and restructure nucleosomes (Fig. 35–1). They increase DNA accessibility by sliding, ejecting, replacing, or deleting nucleosomes, enabling transcription, chromatin assembly, DNA repair, and other processes.
Chromatin remodelers have been grouped into four families (i.e., CHD, ISWI, SWI/SNF, INO80), all with a conserved ATPase domain (Clapier and Cairns, 2009). Other functional domains are thought to be responsible for nucleosome selection and regulation of ATPase activity. However, it remains unknown how remodelers convert the energy of ATP hydrolysis into mechanical force to mobilize the nucleosome and how different remodeler complexes select which nucleosomes to move and restructure.
The structure of nucleosomes is not carved in stone. Histone variants or substitution histones can replace the canonical core histones H2A, H2B, H3, and H4. They provide the possibility to generate a specialized chromatin environment for nuclear processes in certain parts of the genome. Histone variants are different from their counterparts by containing one or a few amino acid differences (e.g., H3.3, H2A.X, H2A.Z), existing in single or low genomic copies. They can incorporate during any part of the cell cycle (S phase or replication-independent). A complete characterization of histone variant function has yet to be established. Still, thus far, histone variants have been shown to play a critical role in DNA repair processes, heterochromatin, and centromere organization. For instance, the deletion of the H2A.X gene or mutation of its Ser139 impairs the ability to recruit DNA damage repair proteins such as BRCA1 and 53BP1 (Celeste et al., 2002; Bassing et al., 2002). Moreover, H3.3 deposition into nucleosomes is crucial for maintaining chromatin integrity and restoration of transcriptional activity upon completion of DNA damage repair (Adam et al., 2013). The centromere-specific H3 variant CENP-A defines centromeric regions that allow proper chromosome segregation. In summary, histone variants add diversity to nucleosome structure and help modulate nucleosome organization in response to stress and cell cycle stage (Hauer and Gasser, 2017).
The impactful and transient nature of seizures perfectly lends itself to be a case study in epigenomics. A single seizure can give rise to long-lasting changes in essential neurological functions, such as synaptic plasticity, neurogenesis, axonal sprouting, and inflammation, which are reflected as alterations in gene expression patterns. The acquisition of novel chromatin modifications such as DNA methylation or histone modifications allows a cell to retain an inventory of what has happened and ultimately dictate its gene expression profile for the remainder of its lifetime. In nondividing cells such as neurons, acquisition of these modifications could allow injury from a seizure to persist and enact a long-lived transcriptional program that would set the landscape for a hyperexcitable network to develop or predispose networks to damage from future seizure occurrence (Hauser et al., 2018; Kobow and Blumcke, 2011).
Mutations in epigenetic enzymes and reader proteins have been linked to epileptic encephalopathies and autism spectrum disorders with seizures (Kobow et al., 2020b; Van Loo et al., 2022). Carvill and Suls et al. identified chromodomain helicase DNA binding protein 2 (CHD2), an ATP-dependent helicase with chromatin remodeling function (Marfella and Imbalzano, 2007), as a candidate gene in Dravet-like fever associated epileptic encephalopathy (Carvill et al., 2013; Suls et al., 2013). A different subtype (i.e., SWI/SNF) of chromatin remodelers, including SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a, member 2 and 4 (SMARCA2, SMARCA4), and AT Rich Interactive Domain 1B (ARID1B), has been identified in patients with various mental retardation syndromes and seizures (Tsurusaki et al., 2012; de la Serna et al., 2006; Ronan et al., 2013). ATRX, another chromatin remodeler of the SWI/SNF family, was found to be mutated in alpha-thalassemia/mental retardation (ATRX), which is associated with epilepsy in approximately 30% of patients (Guerrini et al., 2000; Picketts et al., 1996; Gibbons, 2006). Fifty percent of all ATRX syndrome mutations affect a domain required for DNMT3b binding, leading to altered DNA methylation profiles and chromatin structure. The epileptogenic mechanisms may include perturbations of inhibitory interneuron survival and differentiation and hence lead to aberrantneural networks (Medina et al., 2009; Qureshi and Mehler, 2010). In contrast, mutation of methyl-CpG-binding protein 2 (MeCP2, Rett syndrome), DNA regulatory sequence mutations (e.g., imprinting center deletions in Angelman and Prader-Willi syndrome), or triplet repeat expansions (e.g., noncoding CGG expansion in Fragile X syndrome) change the brain’s DNA methylation landscape, causing autism spectrum disorders with a high incidence of seizures (Urdinguio et al., 2009; Qureshi and Mehler, 2010; Amir et al., 1999; Bienvenu and Chelly, 2006; Santoro et al., 2012).
In focal epilepsy, often, no primary genetic origin can be identified. Transcriptional profiling over two decades identified hundreds of candidate genes with altered expression during epileptogenesis and chronic epilepsy. Still, no clear cellular pathway or function was explicitly highlighted, and little agreement or overlap was found between different studies (Lukasiuk et al., 2006; Dingledine et al., 2017; Becker et al., 2002, 2003). It was hypothesized that epigenetics and particularly DNA methylation might act as a master switch to regulate multiple genes (Kobow and Blumcke, 2011). The reversibility of epigenetic chromatin modifications would make such a mechanism an interesting new target in the treatment of otherwise drug-resistant epilepsy.
The first description of dynamic changes in promoter methylation associated with altered gene expression in response to KCl-driven neuronal hyperactivity was by Martinowich et al. (2003). Although the “high-K+” model of hypersynchronous, epileptiform activity was used to study seizure mechanisms since the early 1990s, Martinowich et al. did not discuss their findings in the context of epilepsy or seizures. As a result, the idea that synchronized neuronal hyperactivity could impart DNA methylation changes and downstream gene function remained largely unrecognized at that time point (Martinowich et al., 2003). Two other studies demonstrated decreases in spontaneous excitatory neurotransmission and network activity following DNMT inhibition by 5-Aza- or Zebularine in hippocampal slices (Levenson et al., 2006) and postnatal murine neurons (Nelson et al., 2008), providing indirect evidence for a pathogenic role of DNA methylation in epilepsy. Later, Guo et al. showed that a single electroconvulsive stimulus could modify the genomic DNA methylation landscape in vivo in the adult rodent brain and that these changes persisted over time. This suggested that DNA methylation imparts a molecular memory onto postmitotic neurons following synchronized network hyperactivity (Guo et al., 2011). Altogether, these landmark studies helped to shape a new pathogenic concept in epilepsy known as the “methylation hypothesis” of epileptogenesis (Kobow and Blumcke, 2011, 2012). It was proposed that initial precipitating injuries (e.g., inflammation, head trauma, SE) as much as seizures by themselves are a potent inducer of epigenetic alterations and thereby aggravate the epileptogenic condition resulting in structural brain lesion, drug resistance, and cognitive dysfunction. Since then, localized and global DNA methylation changes were described in experimental and human focal epilepsy at different stages of epileptogenesis, starting with the early response to a precipitating injury through to the latency period and the development of spontaneous recurrent seizures (Williams-Karnesky et al., 2013; Lusardi et al., 2015; Miller-Delaney et al., 2012, 2015; Kobow et al., 2013, 2019, 2020a; Machnes et al., 2013; Debski et al., 2016; Wang et al., 2016; Schulz et al., 2017; Xiao et al., 2018; Ozdemir et al., 2019). However, only a two studies showed a direct link between a single event of synchronized neuronal hyperactivity and subsequent orchestrated epigenetic changes that regulated epilepsy target gene expression in vitro (Kiese et al., 2017; Jablonski et al., 2021). A cellular memory of epileptogenesis, which turns normal into pro-epileptic circuits and networks and memorizes this fate, will need to be validated in future studies.
The epigenetic landscape is highly complicated, encompassing DNA methylation, the histone code, noncoding RNA, and local and higher order chromatin structure, along with DNA sequence. However, it remains an open question whether there is an epigenetic hierarchy, that is, one modification would be superior or subordinate to the others (Jin et al., 2011), and whether this dependency would be relevant to disease development. Evidence for the interrelatedness of different epigenetic mechanisms in the pathogenesis of epilepsy was provided by Miller-Delaney et al., who found differential DNA methylation in TLE-HS patients to target specific regulatory miRNAs and lncRNAs (Miller-Delaney et al., 2015). Many lncRNAs and sncRNAs are dysregulated in their expression in experimental and human epilepsy (Henshall et al., 2016; Korotkov et al., 2017; Srivastava et al., 2017; Cava et al., 2018; Lee et al., 2015), but little is known about the functions of these transcripts. The only class of sncRNAs for which we have extensive evidence of functional roles in epilepsy and which have also been tested as a therapeutic target is miRNAs (Kan et al., 2012; McKiernan et al., 2012; Roncon et al., 2015; Srivastava et al., 2017; Xiao et al., 2018; Surges et al., 2016; Yan et al., 2017; Morris et al., 2019; Tiwari et al., 2018). Among the most studied miRNAs in epilepsy is miR-134, a brain-specific miRNA that regulates dendritic spine development and which, upon silencing, produces neuroprotective and prolonged seizure-suppressive effects (Jimenez-Mateos et al., 2012, 2015; Morris et al., 2019; Reschke et al., 2021). Other targetable epilepsy-related miRNAs include miR-132, which regulates dendritic growth and arborization of newborn neurons in the adult hippocampus; miR-146a, an inflammation-associated miRNA in experimental and human TLE (Aronica et al., 2010); mi-R128, a crucial regulator for neuronal excitability and in humans associated with a locus that links to idiopathic generalized epilepsy with mild mental retardation (Tan et al., 2013; Blair et al., 2011); miR-124 (Jimenez-Mateos et al., 2011; Peng et al., 2013; Brennan et al., 2016); and miR-34a, which regulates neural stem cell differentiation and apoptosis (Henshall, 2013; Aranha et al., 2011). Taken together, many roles have emerged for noncoding RNAs in brain excitability and seizure thresholds. Research supports therapeutic approaches that manipulate miRNA to treat or prevent epilepsy. There is also evidence that miRNA may be useful diagnostic or predictive biomarkers, for example, for drug response (Leontariti et al., 2020).
Histone lysine methylation has not been widely studied in the context of epilepsy. Khan et al. recently showed that the catalytic subunit of the transcriptional silencing complex Polycomb is increased after the induction of status epileptictus (SE) by kainic acid in mice and by pilocarpine in rats (Khan et al., 2019). Interestingly, Polycomb’s canonical role in nature is to silence Hox genes during body plan development in utero and regulate processes such as X-chromosome inactivation. Polycomb carries this out by acting as a histone methyltransferase enzyme to add di- or trimethylation to Lysine 27 on histone H3 (Margueron and Reinberg, 2011; Schuettengruber et al., 2017). This developmental regulator increases after the induction of SE, pointing to a novel role for H3K27 methylation in epilepsy. Besides, Zybruda-Broda et al. found that modification H3K27me3 influenced the expression of matrix metalloproteinase-9 during epileptogenesis—a gene known to be involved in the breakdown of extracellular matrix in physiological processes such as embryonic development, reproduction, and tissue remodeling and diseases like arthritis and metastasis (Zybura-Broda et al., 2016). Outside of epilepsy, histone methylation has been characterized to play critical roles in neuronal function, such as differentiation of pluripotent stem cells into neurons and processes such as learning and memory. H3K4me3, an active mark for transcription, was found to increase 1 hour after contextual fear conditioning. Genetic deletion of the H3K4-specific histone methyltransferase, Mll, caused deficits in contextual fear conditioning, suggesting that H3K4 tri-methylation is vital for long-term consolidation of contextual fear memories (Gupta-Agarwal et al., 2012). Also, the delivery of HDAC inhibitors to HDAC2-overexpressing mice showed amelioration of learning impairments, suggesting that HDAC2 may be critical for memory formation (Guan et al., 2009). Because impairments in spatial and episodic memory are a common comorbidity in epilepsy, it is interesting to speculate the role epigenetic modifications might play in developing seizure-associated comorbidities.
Valproic acid (VPA), a commonly prescribed antiepileptic medication for absence, partial and generalized seizures, mood stabilization, and migraine headaches, was characterized in 2001 to be a potent HDAC inhibitor (Gottlicher et al., 2001). Since then, VPA has also been shown to affect DNA methylation (Detich et al., 2003; Milutinovic et al., 2007). Given VPA’s efficacy for seizure control, early epigenetic studies focused on the role of histone acetylation in seizure development. Huang et al. found gene-specific histone acetylation changes in rat CA3 hippocampal neurons as early as 3 hours after pilocarpine-induced SE, which may be part of the early molecular events underlying epileptogenesis (Huang et al., 2002). Tsankova et al. extended these findings, showing that acute and chronic electroconvulsive seizures exerted dynamic effects on gene-specific histone acetylation. Their study suggested that electroconvulsive stimulation induces epigenetic changes at particular gene loci necessary for neuronal function and provides a potential mechanism for maintaining gene expression changes over time (Tsankova et al., 2004). Histone acetylation was further affected in the rodent epileptic hippocampus following kainic acid–induced SE and was associated with increased neurogenesis and memory impairment. VPA treatment potently blocked seizure-induced neurogenesis, normalized HDAC-dependent gene expression within the epileptic dentate area and protected the animals from seizure-induced cognitive impairment in a hippocampus-dependent learning task (Jessberger et al., 2007).
Because the HDAC inhibitor VPA has shown clinical efficacy for seizure control, the question remains whether other HDAC inhibitors can also exert similar antiepileptic effects. To test this, Reddy et al. systemically administered sodium butyrate, another HDAC inhibitor, daily to mice who had experienced SE by electrical kindling. They found that butyrate treatments delayed the development of limbic epileptogenesis and reduced mossy fiber sprouting in the hippocampus (Reddy et al., 2018). In a mouse model of tuberous sclerosis complex, a genetic malformation of cortical development with frequent epileptic seizures, TSC2+/– animals exhibited abnormal synaptic plasticity aberrations, with enhanced LTP after 1X theta-burst stimulation and decreased LTD magnitude. Application of the HDAC inhibitor Trichostatin A in vitro was sufficient to rescue both plasticity deficits and restore LTP and LTD magnitudes to wild-type levels. In addition, the administration of the HDAC inhibitor SAHA increased the latency of TSC2+/– to the onset of generalized tonic-clonic seizures when tested in a flurothyl seizure model (Basu et al., 2019). While there is no direct evidence that histone acetylation is required for epileptogenesis, Rajan et al. reported that genetic deletion of HDAC4 in mice is sufficient to cause the development of epileptic seizures later in life (Rajan et al., 2009). Several groups have characterized changes in class I and II histone deacetylase levels after kainic acid or pilocarpine administration and in human epileptic tissue (Huang et al., 2002, 2012; Jagirdar et al., 2015, 2016).
There is a well-established connection between metabolism or diet, as an environmental factor, and phenotype, which is probably best summarized in the common expression “You are what you eat.” What links diet with phenotype is epigenetics. Many metabolites are essential cofactors of epigenetic enzymes and carry functional groups which are needed to modify chromatin; for example, Acetyl-CoA supplies histone actelyation; S-Adenosylmethionine, which is synthesized from essential amino acid methionine, is the methyl-group donor for histone, DNA, and RNA methylation; NAD+/NADH ratio is important for the activities of sirtuin histone deacetylases; chromatin remodelers are ATP-dependent; and α-Ketoglutarate is a co-factor for histone and DNA demethylation reactions by Jumonji demethylases and TET (Lu and Thompson, 2012). Intriguingly, stroke, SE, and seizures are linked to augmented metabolic demand or reduced metabolic supply through epigenetic mechanisms. The activation of epigenetic metabolic sensors induces alterations in epigenetic processes that then may further contribute to seizure development and epilepsy (Gano et al., 2018; Hall et al., 2017; Wu et al., 2017).
Alterations in epigenetic marks—specifically DNA methylation—have been established as molecular biomarkers that may be used in the decision-making process for disease diagnosis, prognosis, and treatment (Laird, 2003). This holds true most notably in cancer, but there is a growing number of other diseases where epigenetic biomarkers have been developed (Heyn and Esteller, 2012). Evidence for a diagnostic potential of DNA methylation in epilepsy came from a comparison of epigenetic profiles in hippocampal tissue obtained from three independent rat epilepsy models (i.e., pilocarpine-induced SE, electroconvulsive SE, and traumatic brain injury). Genomic DNA methylation distinguished injured animals from time-matched sham-treated controls in all models at 3 months after injury (Kobow et al., 2013; Debski et al., 2016). Molecular changes observed in each pathologic condition were indicative of the epileptogenic process, but at the same time, highly etiology-dependent. This finding likely reflected the known broad pathophysiological differences between the models (i.e., the degree of cellular injury in the brain, the brain structures affected, and the time course of molecular and functional changes in each model, as well as the frequency and severity of seizures). Results indicated that the mechanistic role of DNA methylation in seizure development and chronic epilepsy is complex and may be context-dependent. However, the specificity of changes within each model and the consistency that DNA methylation profiles reliably distinguished the injured (epileptogenic) phenotype from controls, independent of the genetic background, the handling laboratory, and experimental model, argued against epigenetic changes being a simple epiphenomenon.
Kobow et al. next tested whether genomic DNA methylation could help classify various histopathological entities associated with human focal epilepsy. They compared genome-wide DNA methylation profiles in surgical specimens from patients with focal cortical dysplasia (FCD), matched with other epilepsy (TLE) and nonepilepsy controls. Differential hierarchical cluster analysis of DNA methylation distinguished all histopathological entities, extending the evidence base for disease-specific methylation signatures toward focal epilepsies (Kobow et al., 2019). These findings could be confirmed and extended to other cohorts of epilepsy patients with various cortical malformations (i.e., polymicrogyria, hemimegalencephaly, FCD; Kobow et al., 2020a; Jabari et al., 2022) or low-grade epilepsy-associated tumors (Deng et al., 2020; Hou et al., 2019; Wefers et al., 2020; Capper et al., 2018; Hoffmann et al., 2023). It will be interesting to see how currently developed epigenetic disease classification schemes in lesional epilepsy can be integrated with molecular-genetic (Lee et al., 2021; Bonduelle et al., 2021; Baldassari et al., 2019) and traditional or AI-mediated (Kubach et al., 2020; Blumcke et al., 2019) histopathological diagnostic approaches to rationalize and liberalize postsurgical epilepsy diagnosis.
Peripheral epigenetic changes observed in human (Long et al., 2017) and experimental epilepsy provide hope for developing diagnostic and predictive biomarkers from blood as successfully shown in other diseases (Badhwar and Haqqani, 2020; Agha et al., 2019; Willmer et al., 2018; García-Romero et al., 2017; Walton et al., 2015).
Since the first description of altered histone acetylation following SE in 2002, epigenetic processes were increasingly deciphered and assigned to different disease stages in experimental animal seizure models and patients with focal epilepsy. They broadly affect the gene expression landscape during seizure development and progression. There are many clinical applications for these findings, including epigenetic marks as molecular biomarkers for tissue- and liquid biopsy-based diagnostics, precision medicine-based epigenetic editing of the genome, or pharmacological approaches targeting the epigenetic machinery to attenuate dysregulated gene expression in epilepsy. The next 10 years are expected further to enhance our understanding of the epigenetic origin of epilepsy and specify the diagnostic (e.g., lesion), predictive (e.g., genotype), and prognostic value (e.g., seizure recurrence, treatment response) of epigenetic biomarkers.
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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