Abstract

Mutations in genes regulating the mechanistic target of rapamycin (mTOR) signaling pathway have emerged as a common cause of childhood epilepsy, including focal cortical dysplasia, hemimegalencephaly, and tuberous sclerosis complex. Dysregulation of mTOR signaling is also implicated in nongenetic causes of epilepsy, such as following status epilepticus or traumatic brain injury. In this chapter, we describe the evidence linking the mTOR pathway to genetic and acquired epilepsies, the variety of animal models developed to examine mTOR pathway signaling in the brain, and the general patterns of brain pathology produced by pathway disruption. We also examine potential mechanisms by which disrupted mTOR signaling regulates brain development, neuronal excitability, and epileptogenesis. The variety of tissues and cellular functions regulated by mTOR, and the breadth of epilepsies linked to disrupted mTOR signaling, all implicate the pathway as an important modulator of epileptogenesis.

The mTOR Signaling Pathway

The mechanistic target of rapamycin (mTOR) is a serine/threonine-protein kinase that is expressed throughout the body in mammals. It is evolutionarily ancient, being present from yeast to human (Heitman et al., 1991). The mTOR protein itself is a key component of the larger mTOR signaling pathway (Fig. 4–1), which can be activated by both extracellular trophic factors and intracellular factors, such as nutrients. It mediates its effects through multiple levels, from protein-protein interactions to regulation of autophagy to translation. The pathway is a critical regulator of cell metabolism and generally promotes growth (Sabatini, 2017). It is implicated in the regulation of lifespan in mammals, following a pattern in which enhanced growth is associated with reduced longevity (Harrison et al., 2009, Leontieva et al., 2012, Garratt et al., 2016). In the periphery, the pathway also promotes immune function (Jones and Pearce, 2017) and wound healing (Ekici et al., 2007). In the brain, the pathway regulates neurogenesis, neuronal development, neuronal survival, microglial function, and synaptic plasticity (Sarbassov et al., 2005, Switon et al., 2017). While interest in the mTOR pathway in the neurosciences has exploded in recent years, mTOR pathway genes have long been known to be among the most commonly mutated in cancer (Zou et al., 2020). In general, single and double hits to the pathway lead to neurological disease and noninvasive tumors (Martin et al., 2017), whereas multiple hits produce malignant tumors.

Figure 4–1.

The mTOR signaling pathway.

The mTOR pathway acts through two signaling arms mediated by mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 signaling requires the adaptor protein Raptor (Regulatory Associated Protein of mTOR) to function, while mTORC2 requires Rictor (Rapamycin Insensitive Component of mTOR). The two arms are important, as they mediate distinct functions and show differential sensitivity to pharmacological inhibitors—in particular, mTOR’s namesake, rapamycin. Rapamycin originates from a bacterium discovered on Rapa Nui (Easter Island) in the 1970s (Vézina et al., 1975). Rapamycin inhibits mTOR signaling by preventing assembly of mTORC1, although the drug is not a pure mTORC1 antagonist and can also inhibit mTORC2 under certain conditions (Beretta et al., 1996, Sarbassov et al., 2006, Urbanska et al., 2012). Rapamycin (sirolimus) and its analogs (e.g., everolimus) serve both as experimental tools to identify mTOR dependent cellular functions and to treat diseases caused by mTOR pathway hyperactivation.

Clinical Disorders Caused by mTOR Pathway Mutations

Genetic Models of mTORopathies

An Overview of Models

Understanding mechanisms of epileptogenesis and seizure generation in mTORopathies depends largely on having relevant preclinical and animal models of these disorders. Given the expanding number of mTORopathies and variety of associated genetic, pathological, and epileptogenic mechanisms, there has been a recent explosion in animal models of mTORopathies. These include over 20 brain-specific models of TSC and an increasing number of models targeting other mTOR pathway genes (Table 4–1). While most models of mTORopathies share the central feature of demonstrating increased mTOR pathway activation, no animal model perfectly recapitulates all aspects of human disease. However, each model has specific advantages and limitations in mimicking the various phenotypes of mTORopathies, including genetic mechanisms, pathological features, and seizure semiology. Rather than attempting to describe all relevant models, we provide an overview of the different strategies for generating models of mTORopathies. The corresponding advantages and limitations of different models, as well as the key lessons learned, will also be discussed.

Table 4–1

Summary of Genetic Rodent Models of mTORopathies.

Constitutive Knockout Models

Given the role of mTOR in critical physiological and developmental functions, homozygous constitutive knockout of most mTOR pathway genes in mice is embryonic lethal, thus limiting their usefulness as models of mTORopathies. Heterozygous constitutive knockouts are viable and represent realistic models on the genetic level for disorders involving germline mutations, particularly TSC. However, the pathological and behavioral phenotypes of heterozygous TSC models, such as the Eker rat and Tsc1+/– or Tsc2+/– mice, are relatively limited. The Eker rat with a spontaneous Tsc2 mutation has been reported to have lesions resembling cortical tubers and subependymal hamartomas, but these are very rare (one rat with a tuber, two rats with subependymal hamartomas, out of 19 Eker rats) (Mizuguchi et al., 2000). In terms of an epilepsy phenotype, there are no known reports of the Eker rat having spontaneous seizures, though they do have a modestly decreased threshold to chemically induced seizure kindling (Waltereit et al., 2006). Similarly, adult Tsc1+/– and Tsc2+/– mice have minimal to no pathological abnormalities or spontaneous seizures, although they do have learning deficits (Goorden et al., 2007, Ehninger et al., 2008), and pre-weanling Tsc1+/– mice have been reported to have seizures in a limited developmental period that apparently resolve with age (Gataullina et al., 2016). The relatively mild neurological phenotypes of heterozygous models of TSC may reflect the absence of “second hits,” which may be required to induce more robust pathological manifestation and seizures, at least within animal models.

Conditional Knockout Models

Conditional knockout models target gene deletion selectively to subsets of cells (e.g., neurons, astrocytes), thus limiting the effects of the genetic manipulation and potentially testing the role of that gene in the targeted cell type. In addition, the timing of the gene inactivation can be varied by using promoters expressed at specific developmental time points or by utilizing inducible gene inactivation systems. There have been numerous conditional knockout mouse models of mTORopathies, most frequently involving Tsc1 or Tsc2 gene inactivation, but also other mTOR pathway genes, particularly negative regulators of mTOR, such as Pten and Depdc5 (Table 4–1). The phenotypic features depend on the spatial and temporal properties and targeted cells of the gene inactivation, but most models exhibit evidence of abnormal mTOR pathway activation, similar pathological features, and epilepsy, regardless of which mTOR pathway gene is inactivated. Conditional knockout mice targeting neurons, glia, or neuroglial progenitor cells typically exhibit cytomegaly, astrogliosis, and megalencephaly, which tend to be diffuse, without focal tuber-like abnormalities (Meikle et al., 2007, Uhlmann et al., 2004, Goto et al., 2011, Way et al., 2009, Yuskaitis et al., 2018, Backman et al., 2001). Most of these conditional knockout mice also exhibit epilepsy, which is often progressive, leading to premature death. mTOR inhibitors are uniformly effective against seizures in these models, including prevention of epilepsy when started early, which along with prevention of the underlying pathological abnormalities, is most likely consistent with an antiepileptogenic rather than simply seizure-suppressing effect (Meikle et al., 2008a, Yuskaitis et al., 2019, Ljungberg et al., 2009, Zeng et al., 2008).

The similarities in the phenotype of these conditional knockout models regardless of the mTOR regulator involved confirm the clinical overlap between the mTORopathies and suggest that mTOR activation is the primary pathophysiological mechanism causing the phenotype. Furthermore, as only homozygous, not heterozygous, conditional knockout mice develop severe neurological manifestations, these models support the importance of a “two-hit” mechanism for brain manifestations of mTORopathies, at least those involving negative regulators of the mTOR pathway, such as Tsc1/Tsc2, Pten, and Depdc5. A major limitation of the CKO knockout strategy in modeling human disease is the diffuse nature of the pathological abnormalities in animals and the absence of focal lesions resembling tubers or FCD, which is better addressed by viral or in utero electroporation approaches.

Viral Deletion and In Utero Electroporation Strategies

These models provide a means to recapitulate focal lesions more precisely in TSC and FCDII. One such TSC mouse model combined exogenous injection of a Cre-expressing viral vector in Tsc1^{floxed/floxed} pups. By controlling the dose of the viral vector, investigators achieved mosaic Tsc1 loss in a localized region similar to those observed in human TSC (Prabhakar et al., 2013). Another approach that has emerged within the last decade is based on in utero electroporation (IUE). This technique allows for in vivo manipulation of specific cell types in select brain regions (LoTurco et al., 2009). Using this technique, plasmid DNA targeting PI3K-mTOR or GATOR1 components was microinjected into the ventricles of embryonic rodent brains and transfected via electrical pulses into neural progenitor cells (i.e., radial glia) that line the ventricles. The position of the electrodes that generate the electrical pulses, which is manually determined by the investigator, directs plasmid expression into the brain region of interest. Current IUE-based models of mTORopathies include expression of Pi3k, Akt, Rheb, and mTOR gain-of- function variants (mTORC1 activators) or suppression of Pten, Tsc1, Tsc2, Stradα, Depdc5, and Nprl3 (mTORC1 negative regulators) (Table 4–2). Some of the gain-of-function variants are mutations that have been identified in patients with malformations of cortical development (MCD) and epilepsy while others are experimental mutations predicted to be pathogenic. Suppression of the negative regulators is achieved by CRISPR/Cas9 gene editing, which leads to gene knockout, or short hairpin RNA (shRNA)-mediated methods, which leads to gene knockdown. Additionally, two studies used a two-hit model where a Cre plasmid was expressed by IUE in Tsc1^{floxed/mutant}  or Depdc5^{floxed/mutant} mice. The studies all targeted cortical pyramidal neurons of layer (L) 2/3 as these principal cells are proposed to be most severely affected in MCDs (Blumcke et al., 2009; Hadjivassiliou et al., 2010; D’Gama et al., 2017).

Table 4–2

Summary of IUE-Based Rodent Models of mTORopathies.

It is important to note that although IUE targets radial glia that give rise to both neurons and astrocytes, episomal plasmids that are used in standard IUE will only be expressed in neurons due to a plasmid dilution effect that occurs in dividing cells (Chen and LoTurco, 2012, Feliciano et al., 2011). In contrast, CRISPR/Cas9 plasmids integrate into the genome of the electroporated radial glia and are expected to label all cell types in the radial glial cell lineage (i.e., neurons and astrocytes). IUE has allowed for quantification of the cytoarchitectural abnormalities, including cell misplacement leading to mislamination, increased neuronal soma size, and dendritic arborization. In addition, multinucleated cells have also been reported (Feliciano et al., 2011). In a few studies, IUE has been followed by video-EEG recordings in >2-month-old mice. Mice displayed Racine grade 4/5 convulsive seizures that occurred almost daily at a rate of 3–6 per day (as a mean) depending on the degree of mTORC1 activation. Overall, IUE offers the flexibility to vary the region, timing, and cell-type specificity of plasmid expression in a relatively simple manner, facilitating analyses of their impact on cellular alterations and seizure activity.

Patient-Derived Induced Pluripotent Stem Cell and Organoid Models

Given some of the limitations of rodent models in recapitulating human disease, patient-derived cellular models offer some advantages for mechanistic and preclinical studies, including preservation of patient-specific genetic backgrounds and mechanisms. Induced pluripotent stem cell (iPSC)-derived neurons in culture or three-dimensional organoids have been generated from patients with TSC, STRADA, or DEPDC5 mutations (Dooves et al., 2021, Nadadhur et al., 2019, Winden et al., 2019, Klofas et al., 2020, Dang et al., 2021). While there are limitations in the ability of these reduced systems to generate seizures, they do show evidence of hyperexcitable neuronal network activity, as well as pathological features of cytomegaly (Nadadhur et al., 2019, Winden et al., 2019, Dooves et al., 2021). Further studies may better define mechanisms related to epileptogenesis in these patient-specific models.

Molecular Mechanisms of Epileptogenesis due to mTOR Hyperactivation

Morphological and Pathological Effects of mTOR Hyperactivation on Cell Structure

Ectopic neuronal placement and the presence of dysmorphic, cytomegalic neurons are major histopathological findings in TSC and FCDII (Blumcke et al., 2011; Wong, 2008). Animal models based on IUE provide a precise method to quantify the cellular alterations that are outlined below. All gene variants (see Table 4–2) consistently reproduce these phenotypes (Fig. 4–2), supporting the notion that the underlying mechanisms for these alterations involve the mTOR pathway and its downstream processes. Additional histopathological phenotypes, such as the presence of balloon cells, have not been commonly reported in murine studies. Gliosis, including astrocyte and microglia reactivity, is regularly observed but seems to track with seizure activity (Feliciano et al., 2011, Nguyen et al., 2019) and is not discussed here.

Figure 4–2.

Biocytin-filled hippocampal dentate granule cells from a control and a Pten knockout (KO) mouse. The KO granule cell shows pronounced hypertrophy. Scale = 100 µm.

Cortical pyramidal neurons destined to L2/3 are generated from radial glia in the ventricular zone at E14-15 in mice. Newborn neurons migrate from the ventricular zone shortly thereafter and reach their final position in the cortex by postnatal day (P) 7 (Greig et al., 2013; Kast and Levitt, 2019). In line with this timeframe, IUE at E13.5–15.5 results in neuronal migration defects and misplacement for all evaluated gene variants (Table 4–2). Despite being misplaced in other cortical layers, these neurons preserved the molecular identity of L2/3 pyramidal neurons, as evidenced by positive staining for the upper layer markers Cux1 or Satb2 and negative staining for the deeper layer marker Ctip2 (Baek et al., 2015; Iffland et al., 2020; Onori et al., 2020; Park et al., 2018; Tarkowski et al., 2019; Tsai et al., 2014; Zhong et al., 2021; Moon et al., 2015; Lin et al., 2016). Prenatal treatment with rapamycin to reduce mTORC1 activity prevented the migration defects and led to correct placement of the neurons in the cortex, supporting that mTORC1 hyperactivity contributes to these defects (Baek et al., 2015; Tsai et al., 2014; Onori et al., 2020; Kim et al., 2019; Parker et al., 2013; Ribierre et al., 2018; Iffland et al., 2020; Kassai et al., 2014b). Postnatal rapamycin treatment after P7, when cortical neuronal migration is complete, is not expected to rescue neuronal misplacement. Indeed, this expectation was confirmed in the Pi3k E545 variant (Zhong et al., 2021). Additionally, two studies used a conditional Rheb mutant plasmid with a tamoxifen-inducible Cre plasmid to show that mTORC1 hyperactivation has no effect on neuronal placement once migration is complete (Hsieh et al., 2016; Onori et al., 2020). Thus, the impact of mTORC1 hyperactivation on neuronal placement occurs during a specific developmental timeframe, and cortical mislamination can be prevented in a limited temporal window.

Neuronal cytomegaly is consistently reported for the evaluated gene variants (Table 4–2). Furthermore, increasing mTORC1 activity levels with increasing concentrations of the activating Rheb S16H plasmid resulted in dose-dependent neuronal hypertrophy, supporting a direct relationship between mTORC1 activity level and soma size (Nguyen et al., 2019). In contrast to migration defects, mTORC1-induced neuronal dysmorphogenesis is not restricted to an early developmental window, as hyperactivating mTORC1 signaling via conditional Rheb S16H expression at P7 resulted in enlarged neurons (Hsieh et al., 2016). Neuron soma size was reduced with both pre- and postnatal rapamycin (or everolimus) treatment (Baek et al., 2015; Lim et al., 2015; Tsai et al., 2014; Zhong et al., 2021; Hsieh et al., 2016; Lim et al., 2017; Zhang et al., 2019; Park et al., 2018; Hu et al., 2018; Kassai et al., 2014b; Iffland et al., 2020). Moreover, postnatal rapamycin treatment reduced soma size regardless of whether treatment began in neonatal or adult ages, suggesting mTORC1 hyperactivation impacts neuron size via a dynamic, modifiable process. Dendrite morphology was assessed in the IUE studies targeting Pi3k, Pten, Rheb, and Depdc5, and all revealed dendritic overgrowth involving (for some) increased dendrite thickness and complexity (Zhong et al., 2021; Hsieh et al., 2016; Lin et al., 2016; Chen et al., 2015; Sokolov et al., 2018; Zhang et al., 2019; Zhang et al., 2020; Onori et al., 2020; Ribierre et al., 2018; Dawson et al., 2020). Rescue of dendrite morphology by postnatal rapamycin treatment was reported in the Pi3k E545 and Rheb S16H variants, and rapamycin likely has the same effect for the other variants (Zhong et al., 2021; Zhang et al., 2019). Data on axons in the IUE models are more limited. One study reported accelerated axon growth in L2/3 neurons at P0 following Rheb S16H expression (Gong et al., 2015). Axon overgrowth to the contralateral cortex of L2/3 neurons at P45 has also been reported in mice expressing the Rheb P37L variant (Onori et al., 2020).

The effectiveness of rapamycin in restoring neuronal migration and morphology defects supports that these phenotypes are mTORC1-dependent processes. mTORC1 regulates several downstream intracellular pathways, in particular altered translational control via 4E-BP1/2 and S6K1/2, and defective neuronal ciliogenesis in the context of autophagy, that contribute to some of the cellular alterations. mTORC1 controls mRNA translation via direct phosphorylation of two major translational regulators, S6K1/2 and 4E-BP1/2 (Ma and Blenis, 2009). 4E-BP1/2 serves as a translational repressor that inhibits eIF4F function. Expressing a mutant 4E-BP1/2 which resists inactivation by mTORC1 or knockdown of eIF4E prevented mutant Rheb and mTOR-induced neuronal misplacement (Lin et al., 2016; Kim et al., 2019). Furthermore, knockdown of 4E-BP2 alone, which leads to increased cap-dependent translation, led to ectopic neuronal placement, suggesting that increased cap-dependent translation is necessary and sufficient to induce neuronal misplacement (Lin et al., 2016). With regards to morphology, expression of constitutively active 4E-BP1 or knockdown of eIF4E reduced soma enlargement caused by Rheb or mTOR mutants. Additionally, knockdown of S6K1/2 reduced the increased soma size associated with a hyperactivating rat mTOR mutant (Kassai et al., 2014b). In terms of axons, studies by Gong et al. showed that enhancing 4E-BP1 or reducing S6K1/2 activity prevented Rheb mutant-induced axon overgrowth in vivo (Gong et al., 2015), supporting in vitro studies demonstrating both 4E-BP1/2 and S6K1/2 involvement in axon development (Choi et al., 2008; Li et al., 2008; Morita and Sobue, 2009). With respect to another major mTORC1-dependent pathway, it was reported that neuronal autophagy-mediated primary cilia defects cause mTORC1-induced neuronal misplacement (Park et al., 2018). More specifically, impaired autophagy following mTOR mutant expression led to OFD1 accumulation, a regulator of neuronal ciliogenesis, and disrupted cilia formation. Neuronal cilia are essential organelles that integrate many important signaling pathways, such as Wnt, and defective neuron cilia-mediated signaling can lead to abnormal migration and ectopic neuronal placement (Guemez-Gamboa et al., 2014). Knockdown of Ofd1 or expression of Wnt5a restored neuronal placement in the mTOR mutant condition, supporting that disrupted ciliogenesis underlies these alterations. Importantly, defective ciliogenesis was sensitive to rapamycin treatment. Overall, these studies demonstrate that defects in neuronal migration and morphology are conserved phenotypes across distinct PI3K-mTOR and GATOR1 gene variants, and the mechanisms that underlie these alterations likely converge on mTORC1 and its downstream effectors.

Genetic Effects: Not All mTORopathy Genes Are Equal

While pathological variants in the PI3K-mTOR pathway and GATOR1 complex all lead to mTORC1 hyperactivity, they can also differentially modulate other intracellular signaling pathways independently of mTORC1. These parallel pathways include (but are not limited to) AKT-GSK3β, AKT- FOXG1-reelin, and TSC/RHEB-MAPK/ERK-FLNA. Other examples, such as the noncanonical function of TSC/RHEB on the notch signaling pathway, have been reviewed elsewhere (Neuman and Henske, 2011). One of the best established functions of AKT is phosphorylation and inhibition of the glycogen synthase GSK3β (Bellacosa et al., 2004). GSK3β regulates many neuronal processes, including neuronal polarization, axon growth, and axon branching (Kim et al., 2011). Studies in the cancer field have shown that gain-of-function mutations in Pi3k and Akt, as well as Pten loss-of-function mutations, upregulate AKT and lead to decreased GSK3β activity (Duda et al., 2020). By contrast, Tsc1 loss-of-function or Rheb gain-of-function mutations indirectly downregulate AKT via a negative homeostatic feedback loop through mTORC1-S6K1/2, thereby increasing GSK3β activity (Meikle et al., 2008a; Gong et al., 2015; Shah et al., 2004). It is expected that variants of the GATOR1 complex would lead to decreased AKT activity through the same feedback mechanism, but this needs to be examined. Interestingly, despite Pten and Rheb mutants having opposite effects on GSK3b activity, Pten^–/– and Rheb S16H neurons both displayed increased axon growth (Kwon et al., 2006), suggesting that too little or too much GSK3β activity leads to a similar axonal phenotype in hyperactive mTORC1 conditions. It was also reported that Rheb S6H leads to MAPK activation independent of mTOR that ultimately increases an actin-crosslinking molecule FLNA (Zhang et al., 2020). Knockdown of FLNA prevented neuronal misplacement and reduced soma and dendrite hypertrophy in Rheb S16H-expressing mice, supporting a role for FLNA in cortical mislamination and neuronal dysmorphogenesis in mTORopathies (Zhang et al., 2020). Further, this reduced seizure activity. Although alterations in FLNA were observed in Rheb and Tsc^–/– conditions, it was not conserved with mTOR variants.

Perhaps the greatest divergence across gene variants is for synaptic activity. The diverging effects of Pi3k, Pten, Tsc1, Rheb, mTOR, and Depdc5 variants on synaptic activity emphasize a critical need to systematically investigate the functional effects of these variants side by side in the same system (i.e., IUE), cell type, cortical region, and age to parse out the sources of the differences (i.e., whether the observed effects are due to variant-specific functional differences giving rise to distinct effects or variable experimental conditions). Such side-by-side comparisons to investigate the effects of Pten and Tsc1 knockout on hippocampal neurons have been performed in vitro by Weston and colleagues (Weston et al., 2014). The authors found that Pten deletion led to an increase in both excitatory and inhibitory synaptic transmission, while Tsc1 deletion reduced inhibitory but did not affect excitatory synaptic transmission within the same culture system. These findings support diverging effects of Pten and Tsc1 on synaptic transmission, which may be accounted for by the differential impact of Pten and Tsc1 deletion on AKT activation (discussed above). In addition, Pten is present in the nucleus, where it modulates a range of functions, including chromosome stability and DNA replication (Chow and Salmena, 2020). Pten is also present at synapses and in synaptic spines, where it has been shown to regulate long-term depression (Wang et al., 2006; Jurado et al., 2010; Arendt et al., 2014; Sánchez-Puelles et al., 2020) and may play a role in memory impairment in Alzheimer’s disease (Knafo and Esteban, 2017; Knafo et al., 2016).

mTOR Hyperactivation in Acquired Epilepsy

Evidence establishing mTOR pathway mutations as a cause of genetic epilepsy is unambiguous and compelling. Somewhat unexpectedly, however, the mTOR signaling pathway is also implicated in the development of acquired epilepsy. Acquired epilepsy can result from a range of neurological injuries, such as status epilepticus, head trauma, or stroke. A role for mTOR signaling in acquired epilepsy is not necessarily intuitive, because acquired epilepsies are not associated with mTOR pathway mutations. However, enhanced mTOR signaling is evident in almost all acquired epilepsy models—presumably as part of the injury response. For example, mTOR hyperactivation occurs following systemic kainic acid-induced status epilepticus (Zeng et al., 2009; Macias et al., 2013), intrahippocampal kainic acid-induced status epilepticus (Tang et al., 2018; Gericke et al., 2020), systemic pilocarpine-induced status epilepticus (Huang et al., 2010; Okamoto et al., 2010), electrically induced status epilepticus (Drion et al., 2016), traumatic brain injury (Chen et al., 2006; Guo et al., 2013), and hypoxia (Talos et al., 2012b; Sun et al., 2013). Increased mTOR pathway activation following these injuries is typically assessed by immunostaining for proteins activated by the mTOR signaling cascade, such as phosphorylated S6 (Fig. 4–1). Increased signaling is evident within hours of injury, and elevated levels can persist for weeks (Zeng et al., 2008). Although human data for acute periods of epileptogenesis are not available, enhanced mTOR signaling is evident in tissue from patients with chronic idiopathic temporal lobe epilepsy (Sha et al., 2012; Sosunov et al., 2012; Talos et al., 2018).

Potential physiological and morphological effects of enhanced mTOR signaling after epileptogenic brain injury are less pronounced than the changes observed following genetic hyperactivation of mTOR but follow the same trends. Specifically, acquired epilepsy is associated with neuronal hypertrophy, dendritic overgrowth, spine changes, axonal sprouting, altered neuroplasticity and network hyperexcitability—all of which are features disrupted in mTOR pathway mutants. Use of rapamycin in animal models is beginning to reveal which changes are mTOR dependent. Most prominently, rapamycin has consistently been found to block hippocampal granule cell mossy fiber sprouting (Buckmaster et al., 2009; Yamawaki et al., 2015; Hester et al., 2016), in which granule cell axons form de novo connections with granule cell dendrites in the dentate inner molecular layer. Mossy fiber sprouting is a consistent pathological finding in human temporal lobe epilepsy, and in animal models of the disease. The recurrent excitatory connections created by sprouted mossy fibers have long been proposed as a mechanism of epileptogenesis (Sutula and Dudek, 2007; Hendricks et al., 2019), although their functional significance is debated (Buckmaster, 2014). Rapamycin treatment has also been found to prevent dendritic change in rodent models of epilepsy (Brewster et al., 2013), regulate changes in ion channel expression controlling neuronal excitability (Sosanya et al., 2015), and block increases in spontaneous excitatory postsynaptic current frequency (Tang et al., 2012).

Evidence for mTOR hyperactivation in animal models of acquired epilepsy has led to a host of studies aimed at determining whether blocking mTOR signaling with mTOR antagonists, like rapamycin, can suppress spontaneous seizures. Encouragingly, antagonists have been found to reduce spontaneous seizure frequency in many of these studies (Zeng et al., 2009; Huang et al., 2010; Raffo et al., 2011; Talos et al., 2012b; Russo et al., 2013; Guo et al., 2013; Butler et al., 2015; Drion et al., 2016; Chi et al., 2017). The literature also includes, however, many negative results (Buckmaster and Lew, 2011; Sliwa et al., 2012; Heng et al., 2013; Shima et al., 2015; Gericke et al., 2020). An additional unresolved question regards the extent to which rapamycin exerts true antiepileptogenic/disease modifying effects versus transient seizure-suppressive effects, like all existing antiseizure medications (ASMs). Studies showing recurrence of seizures after rapamycin withdrawal suggest it may act more as an ASM (Huang et al., 2010; Drion et al., 2016; Löscher, 2020); however, interpretation is complicated, as treatment may not eliminate underlying disease mechanisms (i.e., cell loss or injury) that could drive excess mTOR signaling, thus allowing the disease processes to resume once the drug is withdrawn. This would certainly appear to be the case for genetic mTORopathies, where treatment cannot fix the causal genetic lesion, requiring chronic treatment of patients with TSC (Franz and Capal, 2017).

The consistent ability of rapamycin to prevent disease in genetic models of mTORopathies, versus the variable effects in acquired epilepsies, is notable and suggests differences in underlying disease mechanisms. That treatment works well in models with genetic hyperactivating mutations of the mTOR pathway is not particularly surprising and supports the conclusion that excess mTOR signaling is the principal disease-driving mechanism. Mixed effects in acquired epilepsy, on the other hand, suggest that excess mTOR signaling is one of several epileptogenic mechanisms. Indeed, rapid loss of interneurons (hours to days) is prominent in many acquired epilepsy models and is hypothesized to be a key mechanism of epileptogenesis (Houser, 2014; Buckmaster et al., 2017). There is no evidence that mTOR inhibition could restore this nonregenerative cell population. By contrast, interneuron loss has been observed as a delayed, secondary effect in a Pten knockout mouse model of epilepsy (LaSarge et al., 2021), and therefore potentially preventable with early mTOR inhibition.

Inhibition of compensatory interneuron sprouting may be another mechanism that limits the efficacy of rapamycin in acquired epilepsy. Parvalbumin-expressing interneurons undergo extensive sprouting in epileptic rodents (Christenson Wick et al., 2017) and humans (Ábrahám et al., 2020). Similarly, somatostatin-expressing interneurons exhibit increased soma size, longer dendrites, axonal sprouting, and enhanced connectivity with excitatory cells in rodent epilepsy models (Buckmaster and Dudek, 1997; Zhang and Buckmaster, 2009; Halabisky et al., 2010; Hunt et al., 2011; Long et al., 2011; Drexel et al., 2012; Peng et al., 2013; Butler et al., 2017). In human temporal lobe epilepsy, somatostatin neurons also undergo sprouting (Mathern et al., 1995; DeLanerolle, 2012). Evidence suggests that interneuron sprouting and plasticity are mTOR dependent. In the pilocarpine model, for example, rapamycin blocks sprouting of inhibitory somatostatin neurons (Buckmaster and Wen, 2011). Rapamycin also reduces excitatory input to somatostatin neurons in the closed cortical impact model (Butler et al., 2017), and it reduces inhibitory input to granule cells (Butler et al., 2016). Directly supporting the idea that rapamycin can block protective interneuron sprouting, Jiang and colleagues (Jiang et al., 2018) demonstrated that rapamycin treatment blocked sprouting of somatostatin neurons in a mutant calcium channel model of epilepsy, increasing seizure severity. Together, these findings provide an instructive example of how systemic inhibition of mTOR could exert simultaneous anti- and pro-epileptogenic effects. Given the many roles and ubiquitous expression of mTOR, competing effects are likely to be common and represent a challenge for developing effective therapies.

Challenges and Opportunities

The mTOR pathway is emerging as an important regulator of epileptogenesis. Hyperactivating mutations in mTOR pathway genes consistently cause epilepsy in animal models and are linked to numerous childhood syndromes causing a range of neurological deficits, including epilepsy. Enhanced mTOR signaling is also evident in acquired epilepsy in both animal models and human temporal lobe epilepsy. Studies using mTOR inhibitors in animal models of acquired epilepsy support the conclusion that the pathway could promote epileptogenesis in some cases.

Significant challenges and unanswered questions, however, remain. While disease-causing mTOR mutations produce many common phenotypes, differential gene effects are also evident and remain incompletely characterized. The number of affected cells, mosaicism, and cell type-specific effects are also likely sources of significant variation. Given the diverse functions and ubiquitous expression of mTOR, involvement of organ systems outside the CNS may also impact epilepsy phenotypes. Finally, while many pro-excitatory effects of enhanced mTOR signaling have been identified, whether and how specific changes promote epileptogenesis remains an area of active investigation. Despite these challenges, the mTOR signaling pathway is an extremely promising target for the development of antiepileptogenic therapies. Initial successes in treating TSC with mTOR antagonists are encouraging, and clinical trials assessing the ability of early treatment with mTOR inhibitors to prevent epilepsy, not just reduce existing seizures, in TSC patients are in progress. As knowledge of mTOR signaling in epilepsy is expanded and refined, expansion of treatments to other epilepsies may be on the horizon.

Acknowledgments

Disclosure Statement

References

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