Ras-MAPK Somatic Variants in Mesial Temporal Lobe Epilepsy

Abstract

Mesial temporal lobe epilepsy is the most common focal epilepsy in adults and historically attributed to acquired hippocampal injury caused by precipitating insults such as prolonged febrile seizures and head trauma. However, recent studies have identified somatic variants activating Ras–MAPK signaling in more than 40% of surgically resected MTLE hippocampi. These variants, which are enriched in cases with hippocampal sclerosis (MTLE-HS), exhibit evidence of positive clonal selection, and are localized to neuroglial lineages arising from hippocampal progenitors. Here, we review emerging evidence supporting a role for somatic Ras–MAPK mosaicism in MTLE pathogenesis and propose a two-hit model in which acquired insults promote clonal expansion of Ras–MAPK-mutant progenitors, resulting in aberrant neurogenesis, hippocampal remodeling, and epileptogenesis. These findings suggest new opportunities for disease prevention and precision therapeutics targeting Ras–MAPK signaling.

Mesial temporal lobe epilepsy (MTLE), defined by seizures arising from the hippocampus and adjacent medial temporal lobe structures, is the most common focal epilepsy and accounts for a substantial proportion of patients with drug-resistant epilepsy who require surgical treatment [1–3]. A defining histopathological finding in drug-resistant MTLE is hippocampal sclerosis (HS), characterized by varying degrees of neuronal loss and astrogliosis in the affected hippocampus [2,4]. The frequent association of HS with precipitating insults, including childhood complex febrile seizures, head trauma, and viral infections [2,5–7], has led to one of the prevailing hypotheses that MTLE may be caused by direct hippocampal injury [8]. This hypothesis is further supported by the relative lack of evidence of strong germline genetic contribution to MTLE-HS [9,10]. However, recent studies have identified a significant role for somatic variants that activate Ras-Mitogen-Activated Protein Kinase (MAPK) signaling in the pathogenesis of MTLE [11–13]. In this review, we examine the contribution of somatic Ras-MAPK mosaicism to MTLE-HS and explore the origins of these variants, as well as their potential synergistic interactions with precipitating insults during epileptogenesis, which together inform a new two-hit model for MTLE development.

Limited Contribution of Germline Variants in MTLE-HS

The overall burden of deleterious inherited and de novo germline genetic variants in MTLE, particularly in cases with HS, appears to be low [9,10,14–17]. A comprehensive analysis of germline genetic contributions to epilepsy by the EPI25K consortium found that broadly focal epilepsies show little enrichment of protein-truncating or damaging missense variants compared to controls [16,17]. These findings have been replicated in MTLE-specific cohorts [9–11,14]. However, one study focusing on patients with unilateral drug-resistant MTLE who underwent surgical resection identified a modest but significant enrichment of ultrarare germline missense variants in MTLE cases relative to neurotypical controls [9]. Additional evidence for a limited germline contribution comes from twin studies, showing higher co-occurrence of temporal lobe epilepsy in monozygotic relative to dizygotic twin pairs [10]. Nevertheless, only 36% concordance rate was observed among the monozygotic twins indicating that germline variation alone is insufficient for disease pathogenesis in most cases. Notably, MTLE cases with HS have consistently shown minimal evidence of germline genetic contribution, with the exception of individuals carrying a diagnosis of neurofibromatosis type 1 [10–12], a condition associated with a high rate of somatic mosaicism in the brain, particularly in the hippocampus [10–12]. Even among monozygotic twins, the presence of HS was more closely related to a history of prolonged febrile seizures than with shared genetic background. Together, these findings suggest that while germline genetic background may influence susceptibility to MTLE, acquired risk factors—and potentially somatic variants—likely play a more significant role in the pathogenesis of MTLE-HS.

Specificity of Ras-MAPK pathway somatic variation to temporal lobe epilepsy

Developmentally-acquired somatic variants are now widely regarded as the primary etiology underlying focal epilepsies associated with focal developmental cortical malformations [11,18–22]. The initial decade of discovery focused on somatic variants in PI3K-mTOR pathway genes, which underlie focal cortical dysplasia type II (FCD II), and account for a substantial proportion of drug-resistant pediatric neocortical epilepsies that require surgical resection [11,18–22]. More recently, brain-restricted somatic variants in Ras-MAPK pathway genes have been identified as the most common cause of lesional focal epilepsies arising from the temporal lobes [11,12,22–25]. Both simple and complex somatic genomic alterations in Ras-MAPK genes such as FGFR1, FGFR2, KRAS, BRAF, and MAP2K1 have been repeatedly identified in low-grade epilepsy-associated tumors (LEATs) such as gangliogliomas and dysembryoplastic neuroepithelial tumors [22–24,26]. Furthermore, Ras-MAPK mosaicism has been reported in non-neoplastic brain malformations and dysplasias [22,27]. Strikingly, despite ubiquitous expression of genes in these important signaling pathways across cell types and brain regions, PI3K-mTOR-associated lesions appear to have a unique predilection for frontal and parietal lobes, whereas Ras-MAPK-driven lesions are predominantly localized to the temporal lobes [11]. This regional specificity likely reflects differences in the developmental origins, cellular lineages, and pathogenic mechanisms underlying these lesions.

Enrichment of somatic Ras-MAPK variants in MTLE hippocampi with HS

Despite early associations of epilepsy-associated somatic variants with dysplasia and neoplasia [18–24], recent evidence suggests that somatic variants may also underlie other common epilepsy-associated lesions such as HS and FCD IIIa in the absence of overt malformations. FCD IIIa is predominantly defined by HS but accompanies mild temporal neocortical lamination abnormalities. Low-abundance somatic variants in the Ras-MAPK pathway were initially reported in a small subset of MTLE cases with isolated HS on histopathology [11,13,28,29] and, have now been identified in >40% of hippocampi resected for treatment of drug-resistant [12,25]. These variants, which were enriched in cases with HS pathology without dysplasia or neoplasia, were predicted to increase Ras-MAPK signaling through gain-of-function in the pathway activators such as PTPN11, SOS1, KRAS, BRAF, RAF1, RIT1, MAP2K1 and loss-of-function in the pathway repressors such as NF1, RASAL1, RASA1, LZTR1, CBL [11,12,25], analogous to the functional consequence of PI3K-mTOR variants in FCD II and Ras-MAPK variants in LEATs [20,21,23,24,26]. For example, most variants in the PTPN11 gene, which accounts for nearly half of all reported variants in MTLE-HS, appear to alter the catalytic pocket of the encoded protein, SHP2, resulting in increased phosphatase activity through a dominant gain-of-function mechanism that leads to pathway hyperactivation [11,12]. These findings suggest that hippocampal somatic Ras-MAPK variants play a defining role in the pathogenesis of MTLE with HS, and are perhaps on the same developmental spectrum as other temporal lobe-associated epileptogenic lesions such as FCD III and LEATs.

Clonal selection of Ras-MAPK-activated neuroglial clones in MTLE-HS

Somatic Ras-MAPK variants in MTLE with isolated HS are restricted or significantly enriched in the affected hippocampus and detected at very low variant allele fractions (VAFs), indicating a late developmental or even postnatal origin [11,12]. The MTLE-HS variants are commonly seen at VAFs < 1% and are found in neurons, astrocytes, and oligodendrocytes, suggesting that they arise in hippocampus-specific neuroglial progenitors [12]. In line with the increased incidence of HS with older age at the time of epilepsy surgery, the fraction of MTLE hippocampi harboring somatic Ras-MAPK variants also increases with age at the time of surgery [12], indicating a possible role for the postnatal period in the expansion and propagation of these variants in adult-onset MTLE. In contrast, Ras-MAPK variants in FCD IIIa presenting in children with early-onset drug-resistant epilepsy were present at higher VAFs, detected in the adjacent temporal neocortex, and likely originated earlier during brain development [25], highlighting an important mechanistic and developmental distinction. The age-related increase in Ras-MAPK mosaicism in MTLE-HS is in line with persistence of neural progenitors and neurogenesis in the dentate gyrus (DG) of the hippocampus after birth [30–32], indicating that postnatal clonal expansion in the DG may play a role in MTLE-HS pathogenesis.

Positive selection of non-synonymous relative to synonymous variants – a statistical approach commonly used to study clonal evolution in cancer [33] – in Ras-MAPK genes [12], provides strong evidence of dynamic selective pressures in the MTLE hippocampus that confer a competitive advantage to the variant-carrying progenitors. While deleterious somatic variants, including in Ras-MAPK genes, are found not uncommonly in the brains of neurotypical individuals [12,34], clonal competition and selection in the hippocampus appear to be a disease-specific phenomenon since neurotypical hippocampi do not exhibit the same degree of clonal evolution and most Ras-MAPK genes appear to be under neutral selection [12]. Corroborating these genetic findings, induced pluripotent stem cells (iPSCs) engineered to carry an MTLE-associated PTPN11 variant outcompete their isogenic wildtype counterparts in a mosaic culture across a wide range of mutant cell fractions [12]. Most DG stem cells and progenitors are in a quiescent state under normal conditions [30,32], which does not promote clonal evolution due to minimal cell division and neurogenesis. However, since precipitating insults stimulate DG proliferation and neurogenesis, as repeatedly demonstrated in MTLE animal models [35–37], the Ras-MAPK mutant clones with a proliferative advantage have a unique opportunity to outcompete their wildtype counterparts and undergo clonal selection.

Contribution of Ras-MAPK signaling to proliferative DG remodeling

Despite widespread cell loss in HS, histopathologic evaluation of surgically resected hippocampi from patients with MTLE often shows proliferative remodeling of the DG in the form of focal dispersion, segmental duplication, or bilamination [38], suggesting that aberrant proliferation of the DG neural progenitor cells (NPCs) may be a key disease driver. Consistent with that, analysis of mutational signatures associated with deleterious MTLE-associated variants [12], using Catalogue of Somatic Mutations in Cancer (COSMIC) [39], revealed contribution from the cellular proliferation-associated signature, SBS1 [12]. A hallmark finding in MTLE rodent models, granule cell dispersion, characterized by ectopic migration of newborn granule cells into the CA4 (dentate hilum) and molecular layer of the hippocampus [38], has been linked to aberrant proliferation, migration, and maturation of the DG [35–37]. Although lineage-tracing studies have shown that only a small fraction of proliferating NPCs give rise to most abnormal newborn granule cells in MTLE animal models [40], indicating that cell-intrinsic factors, including somatic genetic makeup, may mediate this selective clonal expansion.

MAPK signaling is a key regulator of DG stem cell maintenance and differentiation [41,42], and upregulation of this pathway through activating variants has been linked to increased cellular proliferation [43]. However, even transient seizure-induced activation of this pathway can stimulate NPC proliferation [44,45], perhaps through induction of Brain-Derived Neurotrophic Factor (BDNF), which is a key regulator of DG neurogenesis [46–48]. Phosphorylation of a downstream effector of Ras-MAPK signaling, ERK, has been shown to activate the transcription factor cAMP Response Element-Binding protein (CREB), which positively regulates BDNF expression in neurons and neural progenitors [49,50]. Neuronal activity-dependent expression and secretion of BDNF [51,52], in turn activates the receptor tyrosine kinase TrkB, resulting in NPC proliferation and neurogenesis [47,48,53,54]. Seizure-induced BDNF overexpression [55] has been proposed to be a major contributor to pathologic DG expansion and neurogenesis following status epilepticus [54], potentially creating a permissive environment for pathologic clonal selection in the hippocampus (Figure 1).

Figure 1. Genotype- and activity-dependent upregulation of Ras-MAPK signaling in MTLE.

Somatic variants causing gain-of-function (GOF) of positive regulators and loss-of-function (LOF) of negative regulators of Ras-MAPK signaling activate the pathway, resulting in increased ERK1/2 phosphorylation. Activated ERK1/2 translocates to the nucleus and activates important transcription factors such as CREB and Elk1, which promote expression of BDNF and other genes involved in neuronal proliferation, differentiation, and excitability, both cell autonomously and non-cell autonomously. BDNF and other neurotrophic factors bind to receptor tyrosine kinases (RTKs) such as TrkB, further enhancing Ras-MAPK signaling and promoting NPC proliferation and differentiation. Neural activity may also directly stimulate Ras-MAPK signaling through calcium-mediated activation of adenylyl cyclase and protein kinase A (PKA), ultimately leading to ERK1/2 phosphorylation. Dual activation of Ras-MAPK signaling by somatic variants and precipitating insults, such as febrile seizures, may drive pathologic dentate gyrus (DG) proliferation and aberrant neurogenesis, which ultimately contribute to the development of MTLE. Figure generated with Biorender.com.

A two-hit model for MTLE-HS pathogenesis

MTLE-associated precipitating insults such as status epilepticus stimulate DG proliferation and aberrant neurogenesis in animal models, producing ectopic, abnormal granule cells that contribute to disruption of hippocampal network dynamics and hyperexcitability [35–37,56,57]. These findings are mechanistically consistent with the observations in longitudinal prospective studies showing that a subset of children who develop MRI evidence of HS soon after severe, prolonged febrile status epilepticus are at high risk of developing MTLE [8]. However, most individuals who experience complex febrile seizures do not develop MTLE later in life [8], and a subset of patients who develop MTLE as adults have a history of uncomplicated childhood seizures or other brain insults without evidence of overt hippocampal injury [7]. These observations suggest that factors intrinsic to the hippocampus, such as somatic mosaicism, may be the missing link underlying this apparent heterogeneity in epileptogenesis. The enrichment of deleterious somatic Ras-MAPK variants in surgically-resected MTLE hippocampi, together with the association of MTLE with acquired insults that stimulate DG proliferation through Ras-MAPK activation in animal models [5,6,58], supports a two-hit model for MTLE pathogenesis in which epileptogenesis is the product of synergistic contributions from somatic variants and acquired insults. Based on this model, which is not yet proven, Ras–MAPK variants in hippocampal progenitors, perhaps within the DG, confer a competitive advantage that promotes pathological clonal selection, while acquired insults act as a second hit to drive aberrant proliferation and neurogenesis and consequent MTLE development (Figure 2).

Figure 2. Two-hit model for MTLE-HS pathogenesis.

Hippocampal subregions are highlighted in a coronal schematic of the human brain (A). The DG has distinct progenitors that separate from neocortical progenitors during early gestation. In typical brain development (B), a small fraction of neural stem/progenitor cells acquire somatic variants in Ras-MAPK pathway genes but largely remain quiescent and inconsequential to DG development and neurogenesis. However, DG proliferation provoked by MTLE-associated acquired insults, which serve as a second hit, facilitates positive selection of Ras-MAPK-mutant clones with a proliferative advantage, producing focal proliferative remodeling of the DG with dispersion of immature and hyperexcitable granule cells into the molecular layer and dentate hilum (CA4). SGZ: subgranular zone; GCL: granule cell layer; ML: molecular layer. Figure generated with Biorender.com.

Given evidence from animal models that ectopic DG neurons are intrinsically hyperexcitable [57,59], together with studies demonstrating that activating Ras–MAPK variants cause neuronal hyperexcitability and seizures [60–64], this two-hit model provides a unified biological mechanism for how somatic variants and acquired risk factors may both contribute to epileptogenesis in MTLE, but there remain several important unanswered questions. For example, while strongly suspected, enrichment of variant-carrying cells in the DG in MTLE-HS has not been confirmed. Furthermore, it is not yet known whether the contribution of Ras-MAPK variants to HS is causal or correlative, especially since neuronal loss in the Cornu Ammonis (CA) region of the hippocampus, which is a common histopathological finding in HS, is not directly explained by this model. We speculate that epileptogenesis in MTLE-HS is directly tied to deleterious somatic variants that drive aberrant DG proliferation and produce abnormal, hyperexcitable newborn neurons, while CA neuronal loss and inflammatory changes are sequelae of severe epilepsy. Although, Ras-MAPK overactivation may directly facilitate neuronal loss and sclerosis [25], which requires further investigation in new models with hippocampal mosaicism. Additionally, alternative hypotheses such as a role for the Human Herpes Virus-6 in febrile seizures [65] and MTLE-HS [66] should be reevaluated, as viral infections may drive clonal selection in the hippocampus, and therefore they are not incongruent with this model.

Implications for MTLE prevention and treatment

Despite the availability of many new approved anti-seizure medications, MTLE remains drug-resistant in approximately one-third of patients [3,67], and while surgical interventions offer a cure for some patients, they may be ineffective in others or result in significant morbidity [68]. There is an urgent need for novel, disease-directed therapies for MTLE, which may be enabled by the recent discovery of somatic Ras-MAPK mosaicism. No studies to date have shown any clear reduction in future epilepsy risk by prophylactic treatment with antiseizure medications after acute insults such as febrile seizures or traumatic brain injury [8,69,70], which is consistent with our proposed model that epileptogenesis is primarily driven by pathologic clonal selection of variant-carrying NPCs that is not expected to be altered by ion channel blockers. Recent studies in MAPK-altered LEATs show persistence of neural progenitor cells and immature excitatory neurons [71,72]. Combined with evidence from MTLE animal models demonstrating that ablation of ectopic, newborn granule cells may delay, mitigate, or even partially rescue epileptogenesis in the hippocampus [73–76], these data offer promise for therapies that target pathologic clonal selection in the DG as a potential epilepsy-prevention strategy following a precipitating insult. Moreover, given the strong link between Ras-MAPK overactivation and neuronal hyperexcitability [60–64], therapies that target the Ras-MAPK pathway directly may have disease-modifying and even anti-seizure benefits in individuals with MTLE. For example, targeted inhibition of this pathway has shown promising results in a mouse model of epilepsy caused by Ras-MAPK overactivation [60]. Similarly, several case reports have demonstrated substantial seizure reduction in patients with highly drug-resistant epilepsy secondary to RASopathies and LEATs after treatment with clinically available MEK inhibitors, despite their poor brain penetrance [77–79]. Overall, there is growing genetic, experimental, and clinical evidence that Ras-MAPK signaling plays a critical role in the development and mechanisms underlying drug-resistance in MTLE, highlighting the immense therapeutic potential of targeting this pathway.