Role of mTOR inhibitors in epilepsy treatment

Pharmacological Reports 67 (2015) 636–646

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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep

Review article

Role of mTOR inhibitors in epilepsy treatment Krzysztof Sadowski a,*, Katarzyna Kotulska-Jo´z´wiak b, Sergiusz Jo´z´wiak b a b

‘‘Living with Parkinson disease’’ Foundation, Warszawa, Poland Department of Neurology and Epileptology, The Children’s Memorial Health Institute, Warszawa, Poland

A R T I C L E I N F O

Article history: Received 13 September 2014 Received in revised form 24 December 2014 Accepted 30 December 2014 Available online 14 January 2015 Keywords: Epilepsy Epileptogenesis mTOR inhibitors Tuberous sclerosis Rapamycin

A B S T R A C T

In spite of the fact, that subsequent new antiepileptic drugs (AEDs) are being introduced into clinical practice, the percentage of drug-resistant epilepsy cases remains stable. Although a substantial progress has been made in safety profile of antiepileptic drugs, currently available substances have not been unambiguously proven to display disease-modifying effect in epilepsy and their mechanisms of action influence mainly on the end-stage phase of epileptogenesis, namely seizures. Prevention of epileptogenesis requires new generation of drugs modulating molecular pathways engaged in epileptogenesis processes. The mammalian target of rapamycin (mTOR) pathway is involved in highly epileptogenic conditions, such as tuberous sclerosis complex (TSC) and represents a reasonable target for antiepileptogenic interventions. In animal models of TSC mTOR inhibitors turned out to prevent the development of epilepsy and reduce underlying brain abnormalities. Accumulating evidence from animal studies suggest the role of mTOR pathway in acquired forms of epilepsy. Preliminary clinical studies with patients affected by TSC demonstrated seizure reduction and potential disease-modifying effect of mTOR inhibitors. Further studies will determine the place for mTOR inhibitors in the treatment of patients with TSC as well as its potential antiepileptogenic effect in other types of genetic and acquired epilepsies. This review presents current knowledge of mTOR pathway physiology and pathology in the brain, as well as potential clinical use of its inhibitors. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mTOR signaling network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mTOR protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Upstream of mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Downstream of mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . mTOR inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of the mTOR inhibitors in animal models of epileptogenesis TSC, cortical dysplasias, and other TORopathies . . . . . . . . . Other forms of epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infantile spasms. . . . . . . . . . . . . . . . . . . . . . . . . . . Status epilepticus . . . . . . . . . . . . . . . . . . . . . . . . . Temporal lobe epilepsy . . . . . . . . . . . . . . . . . . . . . Traumatic brain injury . . . . . . . . . . . . . . . . . . . . . Other etiologies of epilepsy . . . . . . . . . . . . . . . . . Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. E-mail address: [email protected] (K. Sadowski). http://dx.doi.org/10.1016/j.pharep.2014.12.017 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.

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Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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immunosuppressive properties were detected, which later led to the recognition of rapamycin as an immunosuppressant. In the 1980s, rapamycin was also found to have anticancer activity although the exact mechanism of action remained unknown until many years later [6]. mTOR gene isolation and protein identification as well as elucidation of the mechanism of action of rapamycin started an avalanche of new discoveries [7–9]. Swiss researchers from Basel who isolated rapamycin-resistant mutants of yeast and discovered corresponding mutations named the affected genes TOR1 and TOR2, in honor of the Spalentor in their home town [10]. Several years later, in 1994 the mammalian target of rapamycin (mTOR) was identified and found to be the ortholog of the yeast Tor1/2 proteins and defined as the rapamycin target in mammals [8]. It was found in subsequent years that rapamycin inhibited cellular proliferation and cell cycle progression, thus further studies on rapamycin use in oncology were initiated. New clinical indications for mTOR inhibitors keep arising far beyond the scope of immunosuppressive activity. An increasing number of human diseases have been linked to mTOR pathway dysregulation, including many types of cancer, cardiovascular diseases, immunological disorders, metabolic disorders, and neurological disorders [11–13]. Intriguingly, most of these conditions are related to hyperactivity of mTOR pathway. Experimental and clinical trials reveal that mTOR pathway inhibition is an attractive therapeutic option which might modify the process of epileptogenesis in affected individuals. The goal of this study is to review recent advances concerning mTOR pathway inhibition in epilepsy treatment.

Introduction According to WHO data around 50 million people worldwide have active epilepsy requiring continuous treatment and about 30% of affected individuals remain drug-resistant [1]. The rapid increase in the number of available AEDs contributed mainly to the progress in safety profile of antiepileptic treatment. Disappointingly, the percentage of cases refractory to treatment remained stable. Currently available AEDs have not been proven to display disease-modifying effect and their mechanisms of action influence mainly on the end-stage phase of the complex process of epileptogenesis, namely seizures. The term epileptogenesis is associated with the time period between the occurrence of proepileptogenic insult (e.g. brain injury, status epilepticus, genetic defect) and the appearance of the first spontaneous seizure [2]. The mechanisms involved in epileptogenesis include neurodegeneration, neurogenesis, gliosis, axonal damage or sprouting, dendritic plasticity, blood–brain barrier damage, inflammation, reorganization of the extracellular matrix as well as reorganization of the architecture of individual neurons [2]. The molecular background of epileptogenesis is complex, and in many cases conditionspecific, but some mechanisms seem to be involved in many types of epilepsy. Accumulating data reveal that abnormal activity of mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by many different factors [3] (Fig. 1). The mTOR pathway regulates a variety of neuronal functions, including cell proliferation, survival, growth, metabolism, and plasticity (Fig. 2). Given that dysregulation of the mTOR pathway is emerging as a common theme in diverse human diseases drugs that target mTOR have many substantial therapeutic uses [4]. The discovery of mTOR is closely related to the discovery of its inhibitor, rapamycin [5]. Rapamycin was isolated by Canadian scientists in 1975 in a soil sample from Easter Island, also known as Rapa Nui, from which its name is derived. Rapamycin is a macrolide antibiotic obtained from Streptomyces hygroscopius showing some antifungal properties. Shortly after its discovery, its

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Genetic alterations Brain injury Status epilepticus

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Ion channel activation Post-translational changes Immediate early genes

mTOR protein mTOR is a well-conserved 289 kDa PI 3-kinase (PIKK) present in all eukaryotic organisms (Fig. 3). The C-terminal domain is conserved most highly and is characterized by serine-threonine kinase activity [14]. An intact PIKK domain is indispensable for all

Minutes

INSULT

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Transcriptional events Neuronal death Inflammation

Axonal sprouting Network reorganization Neurogenesis & gliosis

FIRST SEIZURE

Level of activity

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mTOR signaling network

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SEIZURES

Days

Weeks

Months/Years

LATENT PERIOD

CLINICAL SEIZURES

EEG deterioration Fig. 1. Basic mechanisms of epileptogenesis.

Mental retardation Drug-resistant epilepsy Autism-spectrum disorders

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GROWTH FACTORS

STRESS

GLUCOSE

AMINO ACIDS

Cell membrane PI3K Akt AMP

PTEN mTORC2 mTOR Ric

AMPK

TSC1 TSC2

GβL

LKB1

Rheb

mSin1

mTORC1 mTOR Rap

ACTIN ORGANISATION

S6K1

GβL mLST8

4EBP1

RIBOSOME BIOGENESIS

TRANSLATION

PROTEIN SYNTHESIS

GROWTH Fig. 2. Signaling network of the mTOR pathway and its main regulators. Detailed description in the text.

Fig. 3. Functional domains of mTOR and mechanism of action of rapamycin. Detailed description in the text.

known activities of the protein [15]. The FKBP 12-rapamycinbinding (FRB) domain is an 11 kDa region. Rapamycin binds FRB to form a sandwich-like structure [16]. The FRB domain is present only in mTOR protein, not in other members of PIKK family, thus rapamycin highly specifically inhibits mTOR. The N-terminal part consists of about 20 tandem repeats of 37–43 amino-acids. These so called HEAT (an acronym for: huntingtin, elongation factor 3, protein phosphatase 2A, TOR) repeats form a rod-like helical structure [17], which interacts with cofactors and kinase substrates [18]. mTOR forms two distinct complexes: mTOR complex 1 (mTORC1) consisting of mTOR, mammalian lethal with

SEC13 protein 8 (mLST8), regulatory-associated protein of mTOR (Raptor) and G protein beta subunit-like (GbL), and mTOR complex 2 (mTORC2) composed of mTOR, mLST8, GbL, rapamycininsensitive companion of mTOR (Rictor) and mammalian stressactivated protein kinase interacting protein 1 [19]. mTORC1 is the better characterized of the two complexes. mTORC1 responds to nutrient signals, trophic and metabolic factors and increases protein translation. mTORC2 activation, which is less understood, is regulated by growth factors like insulin [20]. mTORC2–ribosome interaction is also postulated to play a significant role in mTORC2 activation [21]. mTORC2 is involved in actin organization as well as

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cell metabolism and survival by Akt kinase activation [22,23]. Recent findings showed that mTORC2 plays a role in genome stability maintenance under oxidative and replicative stress [24]. Rapamycin was initially thought to inhibit only mTORC1, but recently new data indicate the effect of rapamycin on mTORC2. It turned out that chronic administration of rapamycin reduces mTORC2 signaling by suppressing mTORC2 assembly [25]. Upstream of mTOR The ‘‘canonical’’ pathway for mTOR activation starts with activation of receptor tyrosine kinases by mitogens, trophic factors (like BDNF) or hormones (e.g. insulin). Those input signals are mediated by the activation of PI 3-kinase [26]. In neurons mTOR activity is also modulated by glutamate and dopamine receptors [27]. Active PI 3-kinase phosphorylates phosphatidylinositol (4,5)biphosphate, whose concentration is regulated negatively by tumor suppressor PTEN (phosphatase and tensin homolog). As a consequence, Akt protein is activated. Akt phosphorylates and inactivates TSC1–TSC2 complex [28]. Tumor suppressor genes TSC1 and TSC2 as well as PTEN, STRADalpha (STE20-related kinase adaptor alpha) and NF1 (neurofibromin) are main negative regulators of mTOR pathway [29,30]. TSC2 has GTPase-activiting properties. A small G protein Rheb (Ras homolog enriched in brain) is a direct downstream target of TSC1–TSC2 and a positive regulator of mTOR function [31], which induces a conformational change of mTOR that results in activation and phosphorylation of protein effectors [32]. Nutrients might influence mTOR pathway through energy production in the form of ATP. AMP-activated protein kinase (AMPK) detects ATP:AMP ratio and phosphorylates TSC2 in energy deprivation conditions. The result is mTOR pathway inhibition [33]. Phosphorylation of TSC2 by AMPK is required for translation regulation and cell size control in response to energy deprivation. Furthermore, TSC2 and its phosphorylation by AMPK protect cells from energy deprivation-induced apoptosis. Hypoxia inhibits mTOR through TSC via REDD1 (also known as DDIT4-DNAdamage-inducible transcript 4) and REDD2 homologous proteins [34]. DNA damage may also induce AMPK-TSC pathway through p53 protein [35]. The direct activation of mTORC2 kinase activity by PI-3 was observed in vitro and in cultured cells [36]. An exhaustive description of mTOR signaling pathway was published by Laplante and Sabatini [37]. Downstream of mTOR mTOR is a key regulatory protein for both catabolic and anabolic processes. In optimal growth conditions mTOR upregulates ribosome biogenesis and protein translation leading to an increase in cell size and mass as well as proliferation. In addition to the effects on translational processes, mTOR regulates gene expression on a transcriptional level [38]. Although neurons are post-mitotic cells, the size of neuronal soma is also influenced by mTOR pathway [39]. In diseases related to mTOR dysregulation hypertrophic neural cells are a frequent pathologic feature. mTOR activates ribosomal kinase S6K1, which phosphorylates the ribosomal protein S6 promoting ribosomal biogenesis and protein translation [40,41]. Inhibition of the elongation factor 4E binding protein 1 (4EBP1) and the subsequent release of inhibition of the mRNA elongation initiation factor 4E (eIF4E) is a parallel pathway inducing protein synthesis [42]. Nucleophosmin (B23) is a nucleolar phosphoprotein, that has been assigned to participate in numerous cellular processes such as interactions with nucleolar components of newly synthesized ribosomes to promote ribosome nuclear export. Nucleophosmin accumulation is dependent on mTOR activation [43] thus, providing a link between TSC1/mTOR signaling, nucleophosmin-mediated nuclear export of ribosome

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subunits, protein synthesis levels and cell growth. The WNT (Wingless – a family of embryonic growth factors) signaling pathway controls gene expression through b-catenin. Due to the regulation of transcription of several proteins responsible for cell cycle control, such as cyclin D1 or c-Myc, WNTs strongly influence cell proliferation. TSC1–TSC2 complex participates in b-catenin degradation joining two important cell growth pathways – WNT and mTOR [44]. mTOR signaling plays an important role in autophagy and apoptosis. In general, mTOR inhibits autophagy. Experimental data suggest, that autophagy is increased in neuronal cells after neonatal hypoxia-ischemia serving as regulatory mechanism [45]. In such circumstances mTOR inhibitors might have a neuroprotective effect. mTOR plays a critical role in regulation of antigen-presenting cells and T-cells [46]. The mechanism of immunosuppressive action of rapamycin is commonly assigned to inhibition of growth factor-induced T cell proliferation. Downstream and upstream mTOR pathways play a significant role in regulation of neuroinflammatory processes. Recently rapamycin was shown to dampen and decrease LPS-induced neuroinflammatory cytokines release in animal model of the absence epilepsy [47]. Also, a role of mTOR pathway in the reduction of microgliamediated neuroinflammation was recently raised [48]. All abovementioned mechanisms may potentially contribute to neuromodulatory and antiepileptogenic effects of mTOR inhibitors. Basic interactions in the mTOR pathway are illustrated in Fig. 2. An exhaustive description of mTOR signaling pathway was published by Laplante and Sabatini [37]. mTOR inhibitors Rapamycin (sirolimus) is a prototype drug for all mTOR inhibitors. Limitations in pharmacokinetic properties of rapamycin resulted in identification of rapamycin analogs (rapalogs) enabling, for example, intravenous administration [49]. Temsirolimus (CCI779) is an ester pro-drug of rapamycin with higher water solubility. Everolimus (RAD-001), is the 40-O-(2-hydroxyethyl) derivative of rapamycin that has been developed for oral administration. Deforolimus (AP23573, MK-8669) or Ridaforolimus has C43 secondary alcohol moiety of the cyclohexyl group of Rapamycin that was substituted with phosphonate and phosphinate groups [50]. Ridaforolimus is stable in organic solvents, aqueous solutions at various pHs, plasma and whole blood. Regardless of the subtle differences described above rapamycin and its analogs share the same mechanism of action. Rapamycin and rapalogs are small-molecule kinase inhibitors. The mode of action of this group is to bind FKBP12 The rapamycin–FKBP12 complex inhibits the mTOR pathway by directly binding to mTORC1. New drugs modulating mTOR pathway appear on the horizon. Second generation of dual mTORC1/mTORC2 inhibitors are known as ATP-competitive mTOR kinase inhibitors. They inhibit all of the kinase-dependent functions of mTORC1 and mTORC2 and therefore, block the feedback activation of PI3K/AKT signaling [50]. PF-4708671, first downstream of mTOR drug, is a highly specific inhibitor of p70 ribosomal S6 kinase [51]. A better understanding of molecular mechanisms of both epileptogenesis and their action as well as clinical trials will reveal if these drugs could be applied to epilepsy treatment. Role of the mTOR inhibitors in animal models of epileptogenesis TSC, cortical dysplasias, and other TORopathies TSC is caused by inactivation of either of the tumor suppressor genes TSC1 (locus 9q34) or TSC2 (locus 16p13.3) [52]. Their protein

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products hamartin and tuberin, respectively, form a heterodimer (TSC1–TSC2 complex) and play an important role in the regulation of cell proliferation and differentiation processes through negative mTOR pathway regulation [53]. The hallmark of the disease is the formation of benign tumors, and in the course of TSC the brain, kidneys, liver, heart, retina of the eye, lungs, and skin might be affected. With up to 85% patients having seizures and the incidence of TSC estimated to be 1:6000 [54], this disorder is a major genetic cause of childhood epilepsy [55]. It is well known, that in many patients, diagnosis of TSC can be set before the onset of seizures [56], sometimes even prenatally, because of multiple cardiac tumors and brain hamartomas that can be revealed in fetuses with TSC. Recent studies showed that clinical seizures in TSC are preceded by several days with progressive deterioration of EEG recording and that process can be followed with serial EEGs. First clinical seizures are usually subtle and focal, but in most cases epilepsy evolves to more severe forms, including infantile spasms [55]. Drug-resistant epilepsy is common in TSC, and many patients cannot be effectively treated with available methods. Epilepsy is regarded as a major risk factor for mental retardation and autism in TSC [57]. Thus, TSC may serve as a model disease to initially test the antiepileptogenic potential of mTOR inhibitors. Although mTOR abnormalities were reported to play role in the development of many different epilepsies, the results obtained in TSC model need to be carefully validated in other epilepsies. As discovered in TSC tuberin-hamartin heterodimer acts as a complex inhibiting mTOR signaling pathway. mTOR disinhibition consequently leads to tumor or hamartoma formation in multiple organs. Lee and collaborators [58] used the Tsc2+/ mutant mice, which are heterozygous for deletion of exons 1–2. Animals were treated with Temsirolimus. The authors observed improved survival and decreased tumor growth (kidney cystadenomas) in treated animals. The experiment demonstrated that targeted therapy with an mTOR inhibitor markedly decreased the severity of disease in a TSC mouse model. The link between tumorogenesis and mTOR signaling is obvious. It was also reasonable to assume, that mTOR inhibition may impact the process of epileptogenesis. Abnormal cell growth and proliferation might affect the excitability of neuronal circuits and evoke seizures. The influence of mTOR pathway on essential neuronal functions, such as neurotransmitter and ion channel expression as well as synaptic plasticity [59,60] could influence a variety of mechanisms involved in epileptogenesis. In a mouse neuronal model of TSC in which Tsc1 gene is ablated in most neurons during cortical development rapamycin treatment improved median survival [61]. Behavior, phenotype, and weight gain were all also markedly improved. Neurofilament abnormalities, myelination, and cell enlargement were all improved by the treatment but dysplastic neuronal features persisted, and there were only modest changes in dendritic spine density and length. Mice treated with rapamycin for 23 days (postnatal days 7–30) displayed a persistent improvement in phenotype, with median survival of 78 days. Zeng and colleagues [62] provided the first clear evidence that rapamycin may have antiepileptogenic effect in animal model of TSC. The authors investigated Tsc1(GFAP)CKO mice – animals with conditional inactivation of the Tsc1 gene primarily in glia, developing abnormal glial proliferation, progressive epilepsy and premature death. Rapamycin treatment was started at postnatal day 14 (early treatment) or 6 weeks of age (late treatment), corresponding to the onset of neurological abnormalities. Late treatment with rapamycin suppressed seizures and prolonged survival and, strikingly, early treatment with rapamycin prevented the development of epilepsy and premature death. A potentially clinically significant limitation observed in this study is that after rapamycin treatment was stopped, neurological and histological abnormalities reappear within few weeks. Mice with a

heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/) mice) show synaptic plasticity disturbances and deficits in learning and memory tests, such as spatial learning tasks and contextual discrimination [63,64]. A brief treatment with rapamycin in adult mice rescued not only the synaptic plasticity, but also the behavioral deficits in this model of TSC [65]. Talos et al. [66] revealed that treatment with rapamycin immediately before and after seizures reversed early increases in glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior. In a more recently developed model mosaic induction of Tsc1 loss in neural progenitor cells of mouse embryos by doxycycline lead to multiple neurological symptoms, including severe epilepsy and premature death. Tsc1null neural progenitor cells develop into highly enlarged giant cells with enlarged vacuoles resembling pathological findings in TSC [67]. Postnatal rapamycin treatment not only prevented affected animals from seizures and premature death but also reversed cellular abnormalities. In the face of the fact that the hallmark brain pathology of TSC, cortical tubers and giant cells, are fully developed at late gestational ages, Anderl et al. [68] aimed to examine the benefit of prenatal rapamycin in a new fetal brain model of TSC. In this Tsc1cc Nes-cre+ mouse model, recombination and loss of Tsc1 in neural progenitor cells leads to brain enlargement, hyperactivation of mTOR, and neonatal death due to reduced pup–maternal interaction. A single dose of prenatal rapamycin given to pregnant dams rescued the lethality of mutant mice. This treatment scheme did not prevent brain enlargement and eventual later lethality of the mutation. Rapamycin was ascribed to reduce anxiety- and depression-like phenotypes in a mouse model of TSC [69]. Way et al. [70] described combined preand postnatal rapamycin treatment scheme in Tsc2-hGFAP neuroglial mouse model of TSC. The treatment resulted in almost complete histologic rescue, however the animals treated with the combined therapy did not perform as well as postnatally-treated animals in learning and memory tasks. Focal cortical dysplasias belong to the most frequent malformations of brain development causing epilepsy [71]. According to ILAE classification [72], there are three different types of FCD, with type II presenting with neuronal cytomegaly (FCDIIA) and balloon cells (FCDIIB), which are significant similarities between FCD and TSC-associated lesions, suggesting similar pathomechanisms. mTOR hyperactivity influence on cellular abnormalities in FCD has been intensively investigated [73]. While known diseasecausing mutations for TSC have not yet been reported in FCD, an increase in allelic variants or polymorphisms in the TSC1 or TSC2 genes has been found in some series of patients with FCD [74,75] but not in other studies [76]. According to Grajkowska et al. [77] hamartin and tuberin expression is decreased in FCDIIB cases, supporting the relation between TSC and FCD. On the other hand, giant cells in TSC and balloon cells in FCD were reported to display different patterns of genetic expression, raising doubts about mTOR disinhibition in FCD [73]. It is likely that other upstream modulators of the mTOR pathway, besides the TSC genes, are affected in these malformations, leading to abnormal mTOR activation. In a small experimental study on resected epileptogenic cortex tissue samples from TSC, FCD, and other non-FCD lesions rapamycin blocked paroxysmal activity induced by 4-aminopyridine in both TSC and FCD samples [78]. Due to recent findings human papillomavirus expression during fetal brain development in some cases may influence mTOR pathway and FCDIIB formation [79]. Liu et al. [80] observed mTOR activation in a range of epilepsy surgical pathologies, including FCD, present in dysmorphic neurons, microglia and immature cell types. This study suggests that mTOR dysregulation may be implicated in pathogenesis of various acquired forms of epilepsy, particularly in the presence of dysmorphic cytopathology.

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PTEN mutations cause clinical syndromes exhibiting a wide range of phenotypes, including macrocephaly, mental retardation, seizures, isolated cancer as well as tumor/hamartoma syndromes, for example Proteus syndrome [81]. mTOR inhibitors have been shown to reverse the neuronal hypertrophy and macrocephaly in PTEN knock-out mice [82,83], demonstrating the importance of the mTOR pathway in this spectrum of disorders. PMSE (polyhydramnios, megalencephaly, symptomatic epilepsy syndrome) – a rare condition observed in Old Order Mennonite population, is related to the mutation in STRADalpha gene, which normally facilitates AMPK pathway inhibition of the mTOR pathway [30]. Parker et al. [84] showed that disrupted pathfinding in migrating mouse neural progenitor cells in vitro caused by STRADalpha depletion is prevented by mTORC1 inhibition with rapamycin or inhibition of its downstream effector p70 S6 kinase (p70S6K) with the drug PF-4708671. Rapamycin can rescue aberrant cortical lamination and heterotopia associated with STRADalpha depletion in the mouse cerebral cortex. According to Peter Crino’s definition the group of cortical malformations described above (TSC, FCD, PMSE and syndromes related to PTEN mutations) constitute a spectrum of ‘‘TORopathies’’ causing epilepsy [85]. There is growing evidence that Sturge-Weber syndrome and neurofibromatosis type 1 might also be classified as TORopathies. Neurofibromin is a negative regulator of mTOR pathway [86], which can account for glioma formation in a TSC-independent manner [87]. The mTOR pathway was also activated in specimens from vascular lesions taken from patients diagnosed with SturgeWeber syndrome [88]. The pathological features of FCD and tubers in TSC are welldescribed, whereas the exact mechanism of epileptogenesis remains a matter of debate. Probably seizures might start not within the lesion itself, but in the surrounding area. Although in general the results of resective surgery in appropriately selected patients are positive [89,90], a number of patients carry on having seizures. In electrocorticographic studies tubers are electrically silent and epileptiform activity arises from the perituberal cortex [91]. Another issue is whether epileptogenesis involves primarily circuit abnormalities or cellular/molecular defects. Potential circuit-level mechanisms include a loss of GABAergic inhibitory neurons or aberrant excitatory connections within local networks. Cellular or molecular defects promoting epileptogenesis may involve changes in expression of neurotransporter receptors or ion channels. mTOR signaling might become an ideal candidate for a common molecular pathway just in the center of epileptogenic processes. Data from animal models provide solid evidence that mTOR inhibitors could be beneficial for epilepsy due to TORopathies [92]. Other forms of epilepsy Infantile spasms Infantile spasms (IS) are described as clusters of extension/ flexion tonic spasms in infants, frequently as a component of West syndrome together with hypsarrhythmia and psychomotor delay [93]. Various mechanisms may be responsible for generation of IS. Aside from acquired forms, there is a growing number of genetically determined conditions related to the development of IS [94]. The relatively high comorbidity of IS and TORopathies suggests a common denominator on molecular level [55]. Raffo et al. [95] tested the therapeutic potential of rapamycin in a multiple-hit rat model of symptomatic IS. Postnatal day 3 rats were treated with stereotactic infusions of doxorubicin and LPS. Early treatment with rapamycin was introduced after the onset of spasms. The authors observed dose-dependent effect with very high doses of rapamycin suppressing IS. A 3-day pulse protocol was

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able to stop spasms permanently and improve cognitive outcome. Suppression of spasms correlated with the ability of rapamycin to normalize TORC1 activity in perilesional cortical neurons. The authors postulated, that pathologic overactivation of mTOR pathway is time-limited to the period when spasms manifest. Other types of seizures were still present. The results suggest that IS and other types of seizures are caused by different types of neural networks. As IS in this model were ACTH-resistant, rapamycin pulse treatment may be an effective alternative treatment in clinical conditions. In another study a betamethasone and postnatal trigger of spasms by N-methyl-D-aspartate (NMDA) rat model pretreatment with vigabatrin, but not rapamycin in low doses, suppressed the spasms [96]. In this study rats were prenatally injected with betamethasone on gestational day 15 and injected postnatally with NMDA on 10–15 day. Vigabatrin was administered in a single dose 24 h prior to NMDA injection. Rapamycin was given 24 h prior to NMDA challenge or from P7 to P14. Different lesion and treatment protocols as well as different ages of animals at the moment of treatment could indicate that the dose used in the older animals may not have been equivalent to that used in the younger rats. The development of reliable animal model of IS faces difficulties. Little is known about the biological basis of IS. As the etiologies of IS are diverse, the multiple causes must converge into a final common pathway that results in this specific epilepsy phenotype. Finding a model to test this final pathway is necessary. According to an NINDS/NIH-sponsored workshop on Models of Pediatric Epilepsies an ‘‘ideal’’ model should fulfill minimal requirements (age dependency, response to ACTH and/or vigabatrin, cognitive problems), as well as a set of optimal features such as spontaneous clinical spasms, sleep-wake clustering, hypsarrhythmia, cognitive decline [97]. Nowadays it is not certain that all are required features of an appropriate animal model. ACTH-refractory cases of IS are seen in clinical practice. Although hypsarrhythmia is a characteristic and bad prognostic feature of human West syndrome, infants with IS may have abnormal EEG patterns without meeting the criteria for hypsarrhythmia [98]. Development of consecutive clinically relevant phenotypes in animal models will be essential in improving future therapies [99,100]. Status epilepticus Mossy fiber sprouting reduction appears to be the main protective mechanism of mTOR pathway inhibitors in status epilepticus (SE) models [101,102]. Macias et al. [103] revealed that mTOR signaling was activated by kainic acid (KA)-induced status epilepticus through several brain areas, including the hippocampus and cortex. Two distinct waves of mTOR activation were identified: an early wave (2 h) occurring in neurons and a late wave (24 h) in astrocytes. Unexpectedly, the authors found that longterm treatment with rapamycin gradually sensitized animals to KA likely by changing threshold for KA-induced epileptic discharges and induced gross anatomical changes in the brain (hippocampal size reduction). These findings might be related to chronic mTOR inhibition inducing changes in blood–brain barrier permeability or decreased threshold of neuronal excitability and, at the same time, proconvulsive stimulation with KA. Brewster and collaborators [104] examined the effects of rapamycin on the early hippocampal-dependent spatial learning and memory deficits associated with an episode of pilocarpine-induced SE. Rapamycin-treated SE rats performed significantly better than the vehicle-treated rats. Rapamycin decreased the SE-induced mTOR activation and attenuated microgliosis which was mostly localized within the CA1 area. In another study mTOR inhibition led to strong reduction of seizure development despite the presence of microglia activation, suggesting that effects of rapamycin on seizure development are not due to a control of inflammation [105]. Since

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results from different studies diverge, no definite conclusion can be achieved concerning the efficacy of mTOR inhibition in SE. Temporal lobe epilepsy Several research groups published studies revealing that rapamycin might have antiepileptic and, probably, antiepileptogenic effect in post-SE rat models of temporal lobe epilepsy (TLE) [102,106]. The reduction of mossy fiber sprouting (late treatment) and neuronal cell loss (early treatment) are most frequently mentioned as a potential mechanisms of anti-seizure effect. However, in the study of Buckmaster et al. [107] rapamycin suppressed mossy fiber sprouting but seizure frequency reduction was not observed. The authors postulated that hilar neuron loss and ectopic granule cells might contribute to TLE. S´liwa et al. [108] tested whether rapamycin post-treatment influences epileptogenesis in the amygdala stimulation model of temporal lobe epilepsy in rats. Animals were treated with rapamycin (6 mg/kg) or vehicle daily for 2 weeks, beginning 24 h after stimulation. There was no significant difference between groups in the area occupied by mossy fibers. The authors postulated that antiepileptogenic effect of rapamycin treatment is not an universal phenomenon. In all TLE studies published so far chronic rapamycin treatment was necessary to maintain the antiepileptogenic effect, since a complete reversal of epileptic phenotype has not been achieved. Further studies are necessary. Traumatic brain injury Influence on microglial activation following traumatic brain injury was observed in the study by Erlich and colleagues [109]. Rapamycin injection 4 h following closed head injury significantly improved functional recovery. In rodent models of controlled cortical impact, mTOR inhibition reduced neuronal death and mossy fibers sprouting and, as a result, improved cognitive outcome [110]. Single dose of rapamycin was injected immediately before cortical impact. Dual Akt and mTOR inhibition improved the outcome to a greater degree than single drug administration. Other etiologies of epilepsy mTOR activation in hippocampus and neocortex was described in animal model of neonatal hypoxia-ischemia by Talos et al. [66,111]. The authors hypothesized that seizures occurring at a developmental stage coinciding with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in later life. Seizures were evoked at postnatal day 10. At the 24 h peak of the post-seizure activation mTOR pathway was predominantly induced in the dendritic portion of glutamatergic neurons, consistent with its known role in synaptic function. Rapamycin administration immediately before and after seizures significantly suppressed mTOR activation in both hippocampus and neocortex. Autistic-like social deficits were less frequent in the animals treated with rapamycin. In a genetic rat model (WAG/Rij rats) of the absence epilepsy Russo et al. [112] tested different schedules of rapamycin administration (i.e. early chronic – 17 weeks, sub-chronic – 7 days, and acute-single dose). The authors found rapamycin to exert antiepileptogenic effect. Chronic scheme revealed prodepressive action, whereas acute or sub-acute regimens had antidepressive influence. Anti-inflammatory action of rapamycin mediated by microglia inhibition and cytokines was postulated as the possible mechanism accounting for the acute treatment effects of rapamycin on the absence seizures. The same research group reported a role for persistent mTOR activation and consequent change in hippocampal progenitor cell proliferation during the epileptogenic process leading to the development of the absence seizures in rats [113].

Clinical studies In clinical settings, rapamycin and rapalogs were used in healthy individuals only during the early phases of clinical trials, according to GCP. Now, beyond TSC, both rapamycin and everolimus in children are used mainly in transplant recipients. Many reports show the possible adverse events of these drugs in pediatric transplantation patients. However, it should be noted in these patients, mTOR inhibitors are used together with other immunosuppressive drugs and this, in addition to underlying disorder, likely changes the safety profile of the drugs. Moreover, TSC patients present constitutive overexpression of mTOR and the use of mTOR inhibitor might be associated with different risks in such situation. For example, there are reports that mTOR inhibition in children after transplantations is associated with significant decrease in weight and height gain [114,115]. Our study [116] did not show this effect in TSC children receiving everolimus for SEGA. Recent clinical guidelines recommend vigabatrin as a treatment of choice in infantile spasms and partial seizures related to TSC [117]. Interestingly, vigabatrin was reported to inhibit mTOR [118]. Recently published clinical studies support the role that mTOR inhibitors – both rapamycin and everolimus play in the control of subependymal giant cell astrocytomas (SEGA) [119,120], and renal angiomyolipomas and, to a lesser degree, pulmonary lymphangioleiomyomatosis in both TSC and non-TSC patients [121,122]. Recent clinical recommendations for the management of SEGAs in TSC state that mTOR inhibitor therapy can be considered when adults and children require therapeutic intervention but are not amenable to surgery [123]. Data on the effect of mTOR inhibitors in TSC-related epilepsy are more limited. Muncy et al. [124] described for the first time rapamycin treatment in drug-resistant epilepsy. A 10-year-old girl with TSC after ineffective tuberectomy has been receiving rapamycin for 10 months. At the dose of 0.15 mg/kg/d there was a dramatic reduction in seizure frequency. Krueger et al. [120] analyzed 28 patients with SEGA related to TSC. 16 patients presented with active epilepsy. The patients received everolimus orally, at a dose of 3.0 mg/m2 for 6 months. Aside from meaningful reduction in volume of SEGAs, seizure frequency for the 6-month study period (vs. the previous 6month period) decreased in 9, did not change in 6, and increased in 1 patient. In a 10-year old boy with huge SEGA regrowth after conventional treatment, everolimus treatment of 4.5 mg/m2/day resulted in significant improvement within 3 months [125]. Seizures ceased completely within first 6 weeks of treatment. Twelve months after treatment introduction further neurological improvement was observed and the patient continues to be seizurefree. No adverse events were observed. Kotulska and collaborators [116] analyzed results in patients under the age of 3 who received everolimus for SEGAs associated with TSC. The mean follow-up was 35 months. In 6 out of 8 children, at least a 50% reduction in SEGA volume was observed. In 1 child with drug-resistant epilepsy, everolimus treatment resulted in cessation of seizures and in 2 other children, at least a 50% reduction in the number of seizures was noted. The incidence of adverse events was similar to that observed in older children and adults. In an open-label trial Krueger et al. [126] observed that seizures were reduced in 17 of the 20 patients with TSC, by a median reduction of 73%. This trial was the first one with seizure frequency as the primary outcome. Significant reductions in seizure duration and improvement in parent-reported behavior and quality of life were also observed. In the follow-up of this study everolimus, titrated to trough serum levels of 5–15 ng/ml, was effective and safe in reducing tumor size in patients with SEGA secondary to TSC for a median of 34 months [127]. EXIST-1 study was a phase III clinical trial focusing on the efficacy and safety of everolimus in patients with SEGAs associated with TSC [121]. The median change from baseline in seizure

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Table 1 Clinical studies with mTOR inhibitors. Author

Ref.

Disease

Number of patients

Age of patients

Drug

Effective dose

Follow-up

Antiepileptic effect

Type of study

Muncy et al. (2009)

[124]

TSC

1

10 y

Rapamycin

0.15 mg/kg/day

10 months

Case study

Krueger et al. (2010) Perek-Polnik et al. (2012) Krueger et al. (2013)

[120]

TSC

>3 y

Everolimus

4.7–5.6 mg/m2/day

6 months

11 y

Everolimus

2

4.5 mg/m /day

3 months

5 mg/m2/day, then titrated to obtain serum level 5–15 ng/ml 4.5 mg/m2/day, then titrated to obtain serum level 5–15 ng/ml 4.5–8 mg/m2/day (serum level 5–15 ng/ ml)

12 weeks

Significant improvement. Seizure clusters stopped. 1–5 short <2 min seizures daily. Seizure reduction in 9/16 pts. Seizures ceased completely. In 17/20 pts a median reduction of 73%. Inconclusive results.

[125]

TSC

28 (16 with epilepsy) 1

[126]

TSC

20

>2 y

Everolimus

Franz et al. (2013)

[121]

TSC

78

0–65 y

Everolimus

Kotulska et al. (2013)

[116]

TSC

8 (5 with epilepsy)

<3 y

Everolimus

Wiegand et al. (2013)

[128]

TSC

6

2–12 y

Everolimus

3–6 mg/day

36 weeks

Parker et al. (2013)

[84]

PMSE

5

7 m–5 y

Rapamycin

1–5 mg/m2/day

6 months

Cardamone et al. (2014)

[129]

TSC

13 (7 with active epilepsy)

>3 y

Rapamycin, Everolimus

6–36 months (median 18 months)

Wiemer-Kruel et al. (2014)

[130]

TSC

1

13,5 y

Everolimus

Everolimus: 2.5 mg (1.2 m2), 5 mg (1.3– 2.1 m2), 7.5 mg (2.2 m2) per day Rapamycin: 1 mg/m2/ day 5 mg/m2/day

frequency in 24 h with video-EEG monitoring was 0 in the everolimus and placebo groups, but a large proportion of patients did not have any seizures at baseline 24 h video-EEG. Though, the analysis of change in seizure frequency in this trial was inconclusive. In another study 6 patients with intractable epilepsy due to TSC underwent everolimus treatment [128]. A reduction of seizures was observed in 4 of 6 patients with a reduction of 25– 100%. In addition, the percentage of seizure-free days increased in 3 of these patients. In 2 of 6 patients, no alteration of seizure frequency was noted. Most frequent side effects in all aforementioned studies were aphthous ulcers, fatigue, rash, mucositis, anorexia, diarrhea and nausea, arthralgias, thrombocytopenia and effects on lipid metabolism, most of them being transient and not requiring drug withdrawal. Recently Cardamone et al. [129] reported a series of 13 children and adolescents with TSC treated with everolimus. 7 patients had intractable seizures. By 12 months of treatment 1 patient had >90% reduction, 4 had 50–90% reduction, and 2 had <50% reduction. Three reported subjective improvements in learning. A single case of seizures aggravation after everolimus treatment for SEGA has been reported [130]. The clinical significance of this observation is unclear. Actual clinical recommendations for TSC-related epilepsy management state that however available clinical data are promising, though mTOR inhibitor treatment may need to be continuous and long-term to maintain seizure control and questions about the long-term safety of mTOR inhibitors remain open [117]. In the light of current encouraging results concerning safety issues in long-term everolimus treatment [116,127] these criteria may require re-evaluation. Consecutive trials of everolimus for the treatment of TSC-related epilepsy in a phase 2 trial

6 months

33–38 months (mean 35 months)

12 days

Open label study Case study Phase I/II trial

Phase III trial

Seizures ceased in 1 case, in 2 cases reduction of seizures number. 25–100% seizure reduction in 4/6 patients. Reduced seizure frequency and improved receptive language. 1 patient had >90% reduction, 4 had 50–90% reduction, and 2 had <50% reduction.

Series of cases

Seizure aggravation.

Case study

Series of cases

Series of cases

Series of cases

(NCT01070316; www.clinicaltrials.gov) and on neurocognitive deficits in a phase 3 trial (NCT01289912; www.clinicaltrials.gov) are underway. Clinical data related to other TORopathies are limited. Five patients diagnosed with PMSE were treated with rapamycin without complication [84]. The authors observed a reduction in seizure frequency and an improvement in receptive language. Clinical trials with mTOR inhibitors are summarized in Table 1. Future perspectives A recently proposed definition of antiepileptogenic treatment states that such treatment should either prevent or delay the onset of epilepsy or alleviate seizure severity, prevent or reduce epilepsy progression or pharmacoresponsiveness if given after epilepsy onset [131]. None of available antiepileptic drugs fits into this definition. There is strong evidence that mTOR inhibitors may prevent epilepsy development, especially in genetic TORopathies. However, some important issues have to be investigated. Shall we treat epilepsy with mTOR inhibitors in the same way as we treat with other AEDs? As most of the beneficial effects of mTOR inhibition might cease after drug discontinuation, a life-long treatment might be necessary. The effect of long-term mTOR inhibition on developing brain structure and function is still not fully understood. Although first studies on everolimus safety brought promising results, long-term side effects have to be taken into consideration. Interactions between mTOR inhibitors and available antiepileptic drugs are not fully known and will have to be analyzed. Further experimental studies have to clarify whether mTOR inhibitors have direct anticonvulsant effect. The perinatal or

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prenatal diagnosis of TSC open up a possibility for early, diseasemodifying therapy. According to recent studies preventive antiepileptic treatment without documented seizures could be an attractive option to reduce the damaging effect of early-onset seizures on mental development [132]. Is there a place for mTOR inhibition? The ongoing EPISTOP project funded by the European Commission (www.epistop.eu) will focus on the prospective observation of epilepsy development in children with TSC along with neuroimaging, genetic and biochemical analysis of potential markers of epileptogenesis and drug-resistant epilepsy. New data on epileptogenesis biomarkers might change the way we treat our patients. One might hypothesize, that future treatment of TSCrelated epilepsy will enclose early intervention in high-risk individuals not only with vigabatrin but also with mTOR inhibitor. The understanding of the role of mTOR pathway in acquired epilepsies is in its infancy and requires further experimental studies, however preliminary results are promising.

[14] [15]

[16]

[17] [18]

[19] [20]

[21] [22]

Conclusions [23]

The role of mTOR inhibitors in epilepsy treatment still needs further research and an extension of clinical trials to non-TSC populations. Despite these limitations recent developments in mTOR pathway modulation open up new perspectives for future therapy of epilepsy. Conflict of interest

[24]

[25]

[26] [27]

The authors declare that there are neither financial nor personal relationships that could inappropriately influence this study.

[28] [29]

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