Epileptogenesis in tuberous sclerosis complex-related developmental and epileptic encephalopathy.

Abstract

Epileptogenesis in infants with tuberous sclerosis complex (TSC) is a gradual and dynamic process, leading to early onset and difficult-to-treat seizures. Several cellular, molecular and pathophysiologic mechanisms, including mammalian target of rapamycin (mTOR) dysregulation, GABAergic dysfunction and abnormal connectivity, may play a role in this epileptogenic process and may also contribute to the associated developmental encephalopathy. Disease-specific antiseizure medications or drugs targeting the mTOR pathway have proved to be effective in TSC-associated epilepsy. Pre-symptomatic administration of vigabatrin, a GABAergic drug, delays seizure onset and reduces the risk of a subsequent epileptic encephalopathy, such as infantile spasms syndrome or Lennox–Gastaut syndrome. Everolimus, a rapamycin-derived mTOR inhibitor, reduces seizure frequency, especially in younger patients. This evidence suggests that everolimus should be considered early in the course of epilepsy.

Future trials are needed to optimize the use of everolimus and determine whether earlier correction of mTOR dysregulation can prevent progression to developmental and epileptic encephalopathies or mitigate their severity in infants with TSC. Clinical trials of several other potential antiseizure drugs (cannabidiol and ganaxolone) that target contributing mechanisms are also underway.

This review provides an overview of the different biological mechanisms occurring in parallel and interacting throughout the life course, even beyond the epileptogenic process, in individuals with TSC. These complexities highlight the challenges faced in preventing and treating TSC-related developmental and epileptic encephalopathy.

Untitled section

Keywords: tuberous sclerosis complex, epileptogenesis, developmental and epileptic encephalopathy, animal model, mTOR

Teaser

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder associated with early-onset and difficult-to-treat seizures. Aronica et al. review the cellular and molecular mechanisms of epileptogenesis in TSC and related developmental and epileptic encephalopathies, and the implications for treatment of TSC-related epilepsy.

Article notes

Untitled section

Received 2022 Sep 18; Revised 2022 Dec 18; Accepted 2023 Feb 3; Collection date 2023 Jul.

Author notes

Introduction

Developmental and epileptic encephalopathies (DEE) refer to a group of disorders characterized by early onset, difficult-to-treat epileptic seizures with developmental impairment, which is mainly related to the underlying aetiology and possibly worsened by the epileptiform activity.^1–3 Many pathogenic tuberous sclerosis complex (TSC) gene variants are indicated by accumulating evidence to cause complex neurodevelopmental disorders (NDD), with the superimposed epileptic encephalopathy further affecting the developmental outcome.⁴

Tuberous sclerosis complex is a multisystem genetic disorder characterized by age-related formation of benign tumours throughout the body (including brain, kidneys, heart, skin and eyes). TSC is caused by inactivating mutations in either TSC1 or TSC2 genes.⁵ These genes encode proteins that form an inhibitory complex for RHEB (Ras homolog enriched in brain), thus mTORC1 (mammalian target of rapamycin complex 1) hyperactivation represents a key feature of TSC.^5,6 mTOR-signalling deregulation is also observed in a large spectrum of epileptogenic developmental pathologies (termed ‘mTORopathies’; mTOR pathway-related malformations of cortical development, such as hemimegalencephaly and focal cortical dysplasia, FCD type II).^7–9

More than 2000 pathogenic TSC1/TSC2 variants have been described,¹⁰ which has challenged the correlation of genotypes with the highly variable forms of TSC that exist between patients, even within families with an inherited form of TSC.^5,11–13 Generally, the TSC2 mutation seems to be associated with a more severe phenotype than the TSC1 mutation, including earlier seizure onset, a lower cognitive level and a greater tuber load.^14–16 However, patients with a milder phenotype may also have TSC2 mutations.^17–19

Multiple genetic, epigenetic, as well as acquired and environmental, factors can influence dynamically the phenotypical outcome throughout the life course. Early seizure onset, the occurrence of epileptic spasms, neurosurgery, anti-seizures treatments and age-dependent somatic mutation rate may affect, and thus complicate, genotype–phenotype associations.^12,20–23

Several NDD appear with epileptic seizures, or in some patients, even precede clinical seizure onset.²⁴ Of particular interest is the bridge between these neurological and neuropsychiatric aspects.²⁵ TSC offers the unique opportunity to probe the age-dependent mechanisms of mTOR pathway-related epileptogenesis, involving dynamic and complex combinations with mTOR-dependent and mTOR-independent processes.^9,26,27

Diverse in vitro and in vivo models of TSC, more recently including human stem cell-based models, have improved the understanding of mTOR pathway deregulation on brain development and epileptogenesis. This review will focus on cellular and molecular mechanisms of TSC-associated epileptogenesis and related DEE, discussing the rationale for current therapeutic options for TSC-related epilepsy.

Search strategy

We searched PubMed for peer-reviewed publications published between 1 January 2015 and 31 August 2022, with the term ‘tuberous sclerosis’. Searching for the term ‘tuberous sclerosis’ in PubMed returned 3449 possible articles (accessed 31 August 2022). We then refined our search terms to ‘tuberous sclerosis’ AND (as individual combinatory terms) ‘epilepsy’, ‘epileptogenesis’, ‘diagnostic criteria’, ‘mTOR inhibitors’, ‘neurobiology’, and ‘treatment’. Selection criteria from full-text outputs were novelty of study findings and their relevance to neurologists, with inclusion decided collectively by all authors. Relevant historical references outside the search timeframe were also included.²⁸

Cellular and molecular mechanisms of TSC associated epileptogenesis

Epileptogenesis is currently viewed as a continuum that underlies the development of spontaneous seizures and continues after epilepsy diagnosis.^29,30 The development of drug resistance as well as neurological and neuropsychiatric comorbidities are all part of epileptogenesis. This broad definition for epileptogenesis extends the therapeutic opportunities for intervention beyond the prevention of epilepsy onset to disease modification, including both antiepileptogenesis and modification of concomitant NDD.^31,32

Although some commonalties in epileptogenic processes exist,³³ few temporal dynamics of mechanisms in acquired epilepsies apply to genetic epilepsies, in which gene-, pathway- and age-specific mechanisms (as discussed below) are likely to be involved.

Tuberous sclerosis complex affects the immature brain and so is particularly challenging, since we are often dealing with an ‘immediate’ epileptogenesis. Such early development of the epileptogenic network is supported by imaging, neuropathological and EEG clinical studies.^24,34–37 Foetal brain MRI shows cortical and subcortical lesions in the large majority of TSC patients evaluated, and prenatal detection of such lesions correlate with NDD and autism at 2 years.³⁴ Moreover, longitudinal observational studies provide evidence of early epileptiform EEG activity in infants with TSC.^37,38

Understanding the temporal evolution of the epileptogenic process in TSC represents a key step towards the development of stage-specific therapeutic strategies. Furthermore, genetic models of TSC can be equally informative on the behavioural complexities associated with the epileptogenic network as on the temporal dynamics of mTOR-related epileptogenesis.

Insights from human pathology and genetic models

mTORC1 signalling and its roles in normal brain development

mTOR is a serine/threonine protein kinase that acts through two distinct protein complexes, mTORC1 and mTORC2.^6,39 mTOR signalling influences survival and proliferation of neural stem cells, neuronal migration and axon and dendritic formation and outgrowth. Thus, balanced spatiotemporal mTOR signalling is crucial for the proper development of human cortical structure and organization.^6,9 In particular, mTOR regulates the morphology and migration of a specific population of neural stem cells prevalent in the developing human cortex (the outer radial glial cells).⁴⁰

Morphological and functional alterations in TSC models

Investments have been made into developing models that recapitulate or mimic the brain lesions observed in TSC patients and enable the association of specific cell types to morphological and functional alterations.

Loss of Tsc1 or Tsc2 in germline knockout-mouse models is associated with embryonic lethality, whereas monoallelic loss of TSC genes fails to result in cortical tuber-like formation in rodent brains. Nevertheless, Tsc2^+/− rats and mice, as well as Tsc1^+/− mice, display aberrations in neuronal function, resulting in impaired learning and social behaviour, even in the absence of apparent cerebral pathology and spontaneous seizures.^41–43 Hence, haploinsufficiency for the TSC genes can lead to cognitive deficits independently from seizure activity, at least in these models.

Cognitive deficits in Tsc2^+/− mutant mice could be rescued by rapamycin treatment.⁴³ Sato et al.⁴⁴ reported that impaired social interaction of both Tsc1^+/− and Tsc2^+/− mice lessened with rapamycin treatment. However, in the Tsc2-hGFAP mouse model (Tsc2 removal in embryonic neural progenitor cells)—which is characterized by cortical abnormalities, seizures and cognitive deficits—differential effects on neurodevelopmental defects of rapamycin treatment were reported depending on prenatal and/or postnatal administration.⁴⁵

Recently, developmental status epilepticus was induced in Tsc2^+/− (Eker) and wild-type rats by 12 days after birth.⁴⁶ In this study, both Tsc2^+/− mutations and developmental status epilepticus may have caused social behaviour deficits and epileptiform EEG abnormalities; however, the mTOR inhibitor everolimus improved only the autistic-like behaviours related to Tsc2 haploinsufficiency.⁴⁶ These observations suggest that seizures during early postnatal development can lead in later life to autism spectrum disorder (ASD) symptoms that cannot be ameliorated by mTOR inhibitors.

Forebrain-specific Tsc1 deletion in mice (Tsc1^flox/flox; CaMKIIα-cre) causes both epilepsy and autism-like behaviours.⁴⁷ With this model, McMahon et al.⁴⁷ show that epileptiform activity spreads to the brainstem, resulting in seizure-dependent hyperactivation of mTOR in serotonergic neurons—indicating the role of the serotonergic system dysfunction in TSC. Moreover, mTOR hyperactivation targeted to serotonergic neurons (Tsc1^flox/flox; Slc6a4-cre mice) produced autism-like behaviours only, which rapamycin treatment reversed.⁴⁷

Wu et al.⁴⁸ have recently generated a Tsc1 conditional knock-out mouse model in which Tsc1 inactivation in late-embryonic radial glia produces cytomegalic pyramidal neurons with development of both spontaneous seizures and social/cognitive impairment. To mimic persistent mTOR activation at different level of activity, a constitutively active Rheb (RhebCA; the canonical activator of mTORC1) was expressed in cortical neurons of mouse embryos using in utero electroporation (IUE) with dose ranging. Intriguingly, mTOR-hyperactivity levels correlated with the severity of epilepsy and associated neuropathology in this model.⁴⁹

To study mTORC1-driven epileptogenesis, a brain-specific inducible Tsc1 mouse model has been developed (Tsc1^fl/fl-Camk^2a-Cre^ERT2). Deletion of Tsc1 resulted in strong activation of the mTORC1 pathway in this model, and both epileptogenesis and lethality could be prevented by inhibitors of the mTOR pathway.⁵⁰ Time-dependent changes were observed in the transcriptome and neuronal excitability, with an increase in the excitation-to-inhibition ratio in the hippocampus (but not in the cortex) later in epileptogenesis.⁵¹

The high co-occurrence and link between early onset epilepsy, especially infantile spasms, and neurodevelopmental outcomes in TSC has long been emphasized.^37,52 However, despite the development of multiple animal models of TSC and epilepsy, few models have reported spasm-like seizures or age-dependent seizure patterns that mimic infantile spasms.^53,54 Gataullina et al.⁵³ described age-dependent electrographic discharge patterns (including early onset ‘spike clusters’, ‘spasm-like’ and ‘tonic–clonic like’ patterns) with Tsc1^+/− mouse pups, reporting a sequence similar to TSC patients. Although the Tsc1^GFAPCKO mouse model does not have spontaneous infantile spasms, it has been reported to have increased NMDA-induced spasms (with a reduced threshold for these), supporting the concept of a ‘two-hit’ model for infantile spasms that may explain their development in a subset of TSC patients.⁵⁴ One hypothesis is a requirement for an inflammatory insult.⁵⁵

Zebrafish models reproduce several human-like disease features and have proven useful for transcriptomic and pharmacologic analyses—exploring the mechanisms of drugs [i.e. mTOR inhibitors or cannabidiol (CBD)] and their effects on TSC-related phenotypes, including behavioural effects.^56,57 In-depth analysis of changes in brain connectivity of the TSC^2vu242 mutant zebrafish during development has implicated TrkB signalling (now a potential therapeutic target) in the complex TSC pathology, providing a mechanistic link between brain anatomy and human NDD.⁵⁸

Other recent models include patient-derived induced pluripotent stem cells (iPSC) and cortical organoids. These models have the advantage of studying the earliest stages of neural development and so may discern the contribution of specific cell populations and cell type-specific dysfunction to the disease network in TSC and related mTORopathies.^40,59–62 Notably, a recent study in iPSC-derived neurons from TSC patients suggested that mTOR-independent processes for impairing axon extension or guidance could contribute.⁶³ Concordantly, the neural network connectivity abnormalities of these TSC patients cannot be ameliorated by mTOR inhibitors.⁶³

Morphological and functional alterations in human brain

mTOR signalling pathway hyperactivation represents a shared pathogenic mechanism for a group of developmental malformations with similar histopathological abnormalities.^9,64 Brain lesions with evidence of mTORC1 activation can be detected prenatally in TSC patients.^36,65 Second-hit loss of either TSC1 or TSC2, inducing mTORC1 activation, has been identified in various TSC neoplastic lesions, such as subependymal giant cell astrocytoma (SEGA), whereas TSC inactivation in cortical tubers is less frequently detected.^22,66 Still debated is whether only a small, poorly detectable, portion of cells is affected for tuber development or whether a causal complex is required between mono-allelic mutation and additional molecular mechanisms (dependent or independent of mTOR).²²

Cortical tubers are focal developmental malformations and represent the neuropathological basis of the epileptogenic cellular network in patients with TSC. The availability of surgical specimens has provided a unique opportunity to dissect the cellular contributors to seizure development and progression and associated comorbidities.^9,24

Neural circuit dysfunctions and altered neurotransmission

Functional studies indicate that focal seizures and interictal epileptiform discharges in TSC arise within the epileptogenic tubers and may propagate in the perituberal cortex and other epileptogenic tubers.⁶⁷ Electrophysiological recording in ex vivo slices of surgical specimens has demonstrated that the neurons display hyperexcitable intrinsic membrane properties that may contribute to the mechanisms of epileptogenesis in different mTORopathies.^68,69 Several studies in both models and human tissues provide evidence of neuronal network alterations resulting in a disrupted excitatory-inhibitory balance as the underlying mechanism of epileptogenesis (Fig. 1).^9,48,60

Cellular and molecular mechanisms underlying mTOR-related DEE in TSC. Overview of the complex cellular and molecular mechanisms contributing to the epileptogenic process and developmental disabilities in TSC. mTORC1 signalling plays a central role during brain development, regulating many basic cellular functions, such as energy metabolism and protein synthesis to regulate cell growth, proliferation and migration. mTORC1 is inhibited by the multiprotein complex consisting of TSC1 and TSC2. Loss-of-function mutations in either TSC1 or TSC2 result in prenatal mTOR overactivation; mTOR inhibitors (mTORi; such as everolimus and sirolimus) are powerful inhibitors of mTORC1 activity (for a more detailed illustration of the mTOR pathway, see Curatolo et al.26). Knowledge about the complex cellular and molecular consequences of mTORC1 overactivation, affecting different cell types, during brain development is rapidly increasing. Early and sustained oxidative stress and inflammation (with enhanced pro-inflammatory cytokines production and recruitment of the peripheral immune cells), accompanied by blood–brain barrier (BBB) dysfunction/leakage and alterations in the composition of the ECM are observed in TSC brain. Crosstalk among these multiple cellular and molecular can contribute to network dysfunction during brain development. Dysregulation of mTOR signalling itself results in developmental alterations of the balance between excitation and inhibition (i.e. GABAergic system dysfunction). Moreover, mTOR hyperactivity affects the oligodendroglial turnover (with failure to produce proper myelin), further contributing to abnormal cell-signalling and premature activation of mechanisms of neurodegeneration. Glu = glutamate; NFTs = neurofibrillary tangles; ROS = reactive oxygen species. — Figure 1 **Cellular and molecular mechanisms underlying mTOR-related DEE in TSC**. Overview of the complex cellular and molecular mechanisms contributing to the epileptogenic process and developmental disabilities in TSC. mTORC1 signalling plays a central role during brain development, regulating many basic cellular functions, such as energy metabolism and protein synthesis to regulate cell growth, proliferation and migration. mTORC1 is inhibited by the multiprotein complex consisting of TSC1 and TSC2. Loss-of-function mutations in either *TSC1* or *TSC2* result in prenatal mTOR overactivation; mTOR inhibitors (mTORi; such as everolimus and sirolimus) are powerful inhibitors of mTORC1 activity (for a more detailed illustration of the mTOR pathway, see Curatolo *et al.*²⁶). Knowledge about the complex cellular and molecular consequences of mTORC1 overactivation, affecting different cell types, during brain development is rapidly increasing. Early and sustained oxidative stress and inflammation (with enhanced pro-inflammatory cytokines production and recruitment of the peripheral immune cells), accompanied by blood–brain barrier (BBB) dysfunction/leakage and alterations in the composition of the ECM are observed in TSC brain. Crosstalk among these multiple cellular and molecular can contribute to network dysfunction during brain development. Dysregulation of mTOR signalling itself results in developmental alterations of the balance between excitation and inhibition (i.e. GABAergic system dysfunction). Moreover, mTOR hyperactivity affects the oligodendroglial turnover (with failure to produce proper myelin), further contributing to abnormal cell-signalling and premature activation of mechanisms of neurodegeneration. Glu = glutamate; NFTs = neurofibrillary tangles; ROS = reactive oxygen species.

Alterations in the expression of ionotropic glutamate receptor subunits (iGluRs; such as NMDA, GluN and AMPA, GluA receptors) have been reported in TSC and related mTORopathies. These alterations involve an increase in the GluA1-to-GluA2 subunit ratio and in GluN2B-containing GluN receptor expression, which may contribute to increased network excitability.^26,70–73 The Tsc1^+/−-mouse model exhibited an mTOR-dependent increase in GluN-mediated excitatory activity due to an upregulation of the GluN2C subunit, and these findings were also seen in human surgical TSC resection samples.^74,75 Also reported in relation to both epilepsy and co-occurring NDD in TSC is abnormal synaptic transmission through alterations of expression and function of group I metabotropic glutamate receptors (i.e. mGluR5).^76,77

GABAergic deficit/imbalance is also implicated in the dysfunctional neural circuitry underlying the epileptogenesis in a large variety of NDD characterized by marked genetic and phenotypic heterogeneity (see the ‘Age-dependent pathophysiological mechanisms and GABA signalling’ section).^78,79

Non-neural mechanisms

Astrocytes may contribute to epileptogenesis due to either impairment of their homeostatic function or gain of aberrant properties.⁸⁰ In TSC models (e.g. Tsc1^GFAPcKO mouse), increased astroglial proliferation has been observed at the time of onset of spontaneous seizures, and both astrocyte-mediated glutamate and K⁺ reuptake were impaired.^81,82 Interestingly, postnatal reduction of TSC1 (Tsc1^GFAP-CreER mouse) was sufficient to cause both astrogliosis and spontaneous seizures.⁸³ Astroglial morphological and functional changes concordant with the intrinsic epileptogenicity of the tuber (including decreased homeostatic function related to ion homeostasis and neurotransmitter metabolism) are evident in resected TSC cortical tissue.^27,84

Increased density and activation of microglia cells are also observed in both TSC models and resected tubers, suggesting supportive roles of these cells in the pathogenesis of seizures in TSC.^27,85–88 Particularly interesting is the crosstalk between microglia and astrocytes to maintain a pro-inflammatory environment with the induction of pro-epileptogenic inflammatory pathways in TSC brain (Fig. 1).^27,87

Immunohistochemical and large-scale transcriptomic studies in brain tissue from TSC patients showed induction of various inflammatory pathways, some of which (i.e. interleukin-1 receptor/toll-like receptor and complement pathways) may contribute to epileptogenesis and associated comorbidities.^27,89–92 Notably, prenatal activation was evident for inflammatory pathways,³⁶ as were transcription factors (such as SPI1/PU.1) involved in the pro-inflammatory gene expression observed even in developing TSC brain lesions,⁹⁰ supporting the hypothesized role of immune–inflammatory responses to early epileptogenic processes. Specific small non-coding RNAs—and in particular microRNAs, such as miR-146a, miR147b and miR155—have been shown to contribute to the regulation of the astrocytic inflammatory phenotype in TSC.^93–95

Astrocytes release cytokines that activate receptors on endothelial cells and pericytes of micro-vessels. In this way, perivascular astrocytes may contribute to blood–brain barrier (BBB) dysfunction, with increased BBB permeability and facilitated leukocyte diapedesis.⁹⁶ Brain extravasation of serum albumin and its uptake into astrocytes has been reported in brain tissue from TSC patients (Fig. 1).⁸⁵ Reactive astrocytes represent also an import source of the metalloproeinases (MMPs) upregulated in TSC brain tissue, the release of which contributes to extracellular matrix (ECM) remodelling and the pathological network underlying epilepsy and/or co-occurring NDDs in TSC (Fig. 1 and Table 1).^91,97–99

Table 1Risk factors for epilepsy and TSC-related DEE

	Risk factor for epilepsy	Risk factor for co-occurring NDDs	References
TSC2 mutation	X	X	Farach et al.,^15,19 Mongrain et al.,¹⁶ Ogorek et al.¹⁴
Structural abnormalities: tubers and microlesions	X	X	Curatolo et al.,²⁴ Catlett et al.,⁶³ Hulshof et al.,^34,100 Pagani et al.¹⁰¹
Myelin pathology		X	Scholl et al.,¹⁰² Prohl et al.,¹⁰³ Peters et al.,¹⁰⁴ Sato et al.¹⁰⁵
Neural circuit dysfunctions and altered neurotransmission: glutamatergic transmission	X	X	Wu et al.,⁴⁸ Catlett et al.,⁶³ Cepeda et al.,⁶⁸ Talos et al.,⁷² Lozovaya et al.,⁷⁴ Gataullina et al.,⁷⁵ Catania et al.⁷⁶
Neural circuit dysfunctions and altered neurotransmission: GABAergic deficit/imbalance	X	X	Eichmuller et al.,⁶² Ruffolo et al.,⁷³ Katsarou et al.,⁷⁸ Amegandjin et al.,¹⁰⁶ van Andel et al.¹⁰⁷
Non-neural mechanisms: astrocytes	X	X	Zou et al.,⁸³ Sosunov et al.,⁸⁴ Zimmer et al.²⁷
Non-neural mechanisms: microglia	X	X	Zhang et al.,^87,88 Koike-Kumagai et al.,¹⁰⁸ Zimmer et al.²⁷
Inflammation, oxidative stress and BBB dysfuntion	X	X	Eichmuller et al.,⁶² Boer et al.,⁸⁵ Arena et al.,⁸⁹ Zimmer et al.,^90,95 Mills et al.,⁹¹ van Scheppingen et al.,⁹³ Gorter et al.⁹⁶
ECM remodelling	X	X	Mills et al.,⁹¹ Long et al.,⁹⁷ Bongaarts et al.,⁹⁸ Broekaart et al.,⁹⁹ Lewis et al.¹⁰⁹
Neurodegeneration progression: tauopathy	–	X	Iyer et al.,¹¹⁰ Kovacs et al.,¹¹¹ Sarnat et al.,¹¹² Hwang et al.,¹¹³ Liu et al.^114,115

Strong interdependence between inflammation and oxidative stress in TSC has been revealed recently.^27,89 Involving NF-κB signalling, extent of oxidative stress is suggested to predict the neuroinflammatory state of the brain.^27,89 Moreover, oxidative stress is closely linked to iron metabolism and may act synergistically to exacerbate cell dysfunction or death.^95,116

The activation of adaptive immune responses with recruitment of the peripheral immune system is another feature of TSC pathology that further contributes to the sustained inflammation and related pro-epileptogenic mechanisms.^85,86,91,117 The presence of T cells has recently been correlated with myelin pathology, suggesting an involvement of the adaptive immune response in the pathogenesis of hypomyelination (even beyond the white matter) that has previously been linked to cognitive dysfunction in TSC patients (Fig. 1).¹¹⁸

mTORC1 is essential for the differentiation of oligodendrocytes (myelin-producing cells).¹¹⁹ Thus, the maturation of oligodendrocytes and production of a proper myelin sheath is also impaired as a result of mTOR pathway disturbance in TSC.¹²⁰ Evidence of specific interactions between oligodendrocytes and inhibitory interneurons has been reported,¹²¹ which raises an interesting hypothesis for impaired bi-directional communication that results in a pathological network.¹²² This hypothesis deserves further investigation in the context of epileptogenesis in TSC-related DEE. Most non-neuronal mechanisms mentioned above also play a role in other epilepsy syndromes, suggesting that TSC may also be targeted with non-mTOR specific antiseizure drugs as mentioned later.

Age-dependent pathophysiological mechanisms and GABA signalling

Increasing evidence supports the concept of GABAergic dysfunction as a unifying mechanism underlying the variety of DEE.^{79,106,107,123–125} Several studies support the link between mTOR dysregulation and the development of GABA signalling. Experimental studies indicate that mTOR dysregulation affects the maturation and function of the GABAergic system, even beyond postnatal neurodevelopment.^{106,126–129} Conditional knock-out mice with selective deletion of the Tsc1 gene in GABAergic interneuron progenitor cells show alterations in interneuron development and function, along with a concomitantly decreased seizure threshold.¹²⁶ A key role of mTORC1 signalling in the development of parvalbumin interneurons is supported by Amegandjin et al.,¹⁰⁶ using conditional TSC1-mutant mice and single-cell genetics in cortical organotypic cultures. This study also identified a critical developmental period during which deficits in both parvalbumin interneuron-connectivity and social behaviour of mice can still be rescued by rapamycin.¹⁰⁶

Using the human cerebral organoid model of TSC, a recent study has identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells, suggesting that dysregulation of specific interneuron generation may plausibly be a mechanism underlying vulnerability to pathology in TSC.⁶² Evaluation of neuronal networks derived from ASD-patient iPSCs with a TSC2 mutation showed abnormal network connectivity, resulting from an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses.¹²⁹

The link between the GABAergic system and mTOR dysregulation is further supported by studies indicating a delay (or lasting impairment) of the physiological maturation of GABAergic signalling in TSC. Expression of GABA_A-receptor subunits and cation-chloride cotransporters (NKCC1 and KCC2) are altered, leading to alterations in excitatory/inhibitory (E/I) balance at the network level.^73,130,131 The concept of GABAergic ‘immaturity’ may represent another common mechanism underlying mTOR-related epileptogenesis and NDD in TSC. Interestingly, CBD at low doses acts as positive allosteric modulator on GABA_A receptors.¹³²

Biomarkers for TSC-related developmental and epileptic encephalopathies

Clinically relevant biomarkers for TSC-related DEE would greatly facilitate appropriate patient selection for combination drug therapy and/or epilepsy surgery (treatment personalization) and, ultimately, improve quality of life. Particularly important is the identification of individuals at risk of developing neurological and neuropsychiatric comorbidities.

Several types of biomarkers (genetic, imaging, EEG, molecular and behavioural) are established and could be implemented in early diagnostic protocols. Early assessment of gene variants can inform on the risk of seizure development and co-occurring NDDs. Therefore, TSC infants and young children can potentially benefit the most from early assessment and more timely, appropriate pharmacological and/or behavioural intervention.¹⁴

Structural and functional MRI may improve prognostication of co-occurring NDDs in patients with TSC.^133,134 Early MRI characteristics are predictive of neurologic manifestations and neurodevelopmental outcome at 2 years.^34,100 Several clinical studies have had promising results in support of the value of EEG-based biomarkers of epileptogenesis and co-occurring NDDs.^38,135–141

Serum-based biomarkers, such as circulating microRNAs (miRNAs) and their isoforms (isomiRs), have recently shown potential to aid standard clinical testing in the early risk assessment of ASD and intellectual disability development in TSC patients.¹⁴² Future studies are needed to elucidate further potential applications of circulating miRNAs to predict and monitor treatment efficacy.¹⁴³

Early behavioural biomarkers also have the potential to be utilized in several aspects of clinical care in children, targeting the earliest symptoms of abnormal neurodevelopment.^144–146 Future studies of integrative biomarker research, using machine learning techniques, could be important to further understanding the relationships among different biomarkers and to establish and provide superior prognostic information in individuals with TSC-related DEE.

Mechanisms of developmental encephalopathy

mTOR dysregulation has been observed as a possible mechanism in idiopathic ASD.¹⁴⁷ Furthermore, evidence of a pathogenetic role for hyperactive mTOR signalling in TSC-associated ASD has been reported, in addition to the reversal of impaired social interaction with rapamycin in a mouse model of TSC (see the ‘Cellular and molecular mechanisms of TSC associated epileptogenesis’ section).^44,147

As discussed above (see the ‘Age-dependent pathophysiological mechanisms and GABA signalling’ section), dysfunction of cortical GABA interneurons are hypothesized to contribute to the large variety of NDDs and further investigations are required in TSC-related DEE.^148,149Figure 1 and Table 1 provide an overview of the convergent cellular and molecular mechanisms contributing to both the epileptogenic process and co-occurring NDDs in TSC, some of which are highlighted below.

Contributing mechanisms in TSC

Inflammation

The evidence of early inflammation and its long-term effects on brain development and function could provide a means by which multiple mechanisms associated with epilepsy may lead to co-occurring NDD.^150–152

Extracellular matrix and cell adhesion

ECM remodelling and dysfunctional cell adhesion have been implicated in the pathogenesis of NDD.^97,153 ECM/cell adhesion could also contribute to the pathological network underlying TSC and co-occurring NDD. In concordance, changes in the expression of genes associated with cell adhesion have been observed in cortical tubers.⁹¹ Notably, lower expression of the cell-adhesion molecule contactin-3 in TSC brain during the early postnatal period is a hypothetical pathophysiological mechanism.¹⁰⁹

Myelin pathology

Myelin pathology represents a major feature of TSC brain pathology, linked to the hyperactivation of the mTOR pathway (‘Cellular and molecular mechanisms of TSC associated epileptogenesis’ section).^9,26 Several imaging studies have further emphasized hypomyelination in TSC, supporting its contribution to behavioural and cognitive dysfunctions in TSC patients.^154–157 Dysfunctional white matter, responsible for clinical manifestations of TSC, including co-occurring NDD, has been investigated in a plethora of studies, supporting this as one mechanism underpinning a network disorder.^{102–105,118,120}

Early neurodegeneration progress: tauopathy

The link between neurodevelopmental and neurodegenerative mechanisms is well supported, with developmental disorders showing evidence of premature neurodegeneration associated with deregulation of the mTOR pathway, including TSC.^9,110–113 Both apoptotic cell death and ferroptosis-mediated cell death could also contribute.^95,110 Particularly interesting are studies that point to accelerated (early) neurodegeneration with tau dysregulation in TSC.

Tau is microtubule-associated protein involved in a group of neurodegenerative diseases (called ‘tauopathies’), including infantile disorders with enhanced levels of phosphorylated tau (phosphor-Tau immunoreactivity).¹⁵⁸ Enhanced levels of phosphorylated tau have also been reported in different mTORopathies, such as hemimegalencephaly, FCD type II and TSC.^110,112,159 Clinical evidence points to the overlap between TSC and frontotemporal dementias.¹¹⁵ Moreover, adult patients with TSC have recently been reported to have elevated aggregation of phosphorylated tau isoforms (3R/4R tau);¹¹⁴ hence, co-occurring NDD could represent a novel 3R/4R tauopathy, independent of amyloid plaque formation, linked to the hyperactivation of the mTOR pathway and accelerated (early) neurodegeneration.¹¹⁴ A recent study has provided additional evidence for a specific pattern of post-translational modifications in TSC (with differences between TSC1 and TSC2 mutation carriers), suggesting that individuals with TSC may have increased risk for tauopathy in mid-life.¹¹³

Untangling the complex interplay between genotype and resulting phenotype in a dynamic disease network is crucial to the characterization and subtyping of TSC phenotypes. Figure 2A–C illustrates the complexities within genotype–phenotype associations and the hypothetical temporal dynamics of disease progression. Table 1 provides an overview of the risk factors (convergent cellular and molecular mechanisms) for epilepsy and/or co-occurring NDDs in TSC.

The early development of a disease network in TSC. (A) Schematic illustration of the levels of complexity from DNA to RNA and proteins, to cells, network and phenotype. The black lines show the relationships between levels from genotype (top) to phenotype (bottom) with emergence of developmental encephalopathy (DE) or DEE. (B) The convergent and divergent relationships between genetic defects (TSC1 and TSC2 mutations) and the abnormal network with hidden levels of complexity at the level of the genetic information (protein-coding or non-coding genes) and structural/cellular and functional abnormalities influencing the phenotype. (C) Hypothetical temporal dynamics of disease progression in TSC (early and late epileptogenesis), highlighting the establishment of an mTOR-related epileptogenesis associated with co-occurring NDDs and accelerated (early) neurodegeneration. ID = intellectual disability. — Figure 2 **The early development of a disease network in TSC**. (A) Schematic illustration of the levels of complexity from DNA to RNA and proteins, to cells, network and phenotype. The black lines show the relationships between levels from genotype (*top*) to phenotype (*bottom*) with emergence of developmental encephalopathy (DE) or DEE. (B) The convergent and divergent relationships between genetic defects (*TSC1* and *TSC2* mutations) and the abnormal network with hidden levels of complexity at the level of the genetic information (protein-coding or non-coding genes) and structural/cellular and functional abnormalities influencing the phenotype. (C) Hypothetical temporal dynamics of disease progression in TSC (early and late epileptogenesis), highlighting the establishment of an mTOR-related epileptogenesis associated with co-occurring NDDs and accelerated (early) neurodegeneration. ID = intellectual disability.

Abnormal functional connectivity

Networks of abnormal functional connectivity are increasingly supported as underlying the comorbidity between TSC and ASD. Identifying the pathological brain connectivity patterns in TSC individuals with ASD may yield neurophysiological markers, facilitating early intervention.^101,105,160 In particular, the study by Sato et al.¹⁰⁵ suggests that white-matter microstructural integrity is associated with connectivity dysfunction, underlying co-occurring NDD. Evidence that large-scale network aberrations are associated with both ASD and mTOR-related connectopathy (characterized by fronto-cortico-striatal hyperconnectivity and rescued by inhibition of mTOR) has recently been reported using resting-state fMRI, electrophysiology and in silico modelling in Tsc2 haplo-insufficient mice.¹⁰¹

Sleep disorders are a common neurological symptom and a cause of decreased quality of life in TSC patients.¹⁶¹ Exploration of the abnormal functional connectivity in TSC may provide a link to the novel mechanisms for sleep dysfunction recently reported in experimental models.¹⁶²

Mechanism of action of drugs and targeted therapeutic options

Mechanisms of drug resistance

Inflammatory mediators and release of glutamate by perivascular astrocytes may contribute to up-regulation of multidrug transport proteins on BBB endothelial cells in TSC.^163,164 Multidrug transport proteins (notably p-glycoprotein) are generally overexpressed at the luminal side of endothelial cells and astrocytic endfeet but are further upregulated in TSC brain lesions.^163,165 p-Glycoprotein overexpression may limit the access of several antiseizure medications to the intended brain targets, reducing their therapeutic effects and contributing to drug resistance.^163,164 For mTOR inhibitors, several additional molecular mechanisms of resistance have been explored previously within the field of oncology.¹⁶⁶ Interestingly, other compounds interacting with the mTOR pathway have been tested for the treatment of TSC-associated AML, SEGA and epileptic seizures, such as metformin,¹⁶⁷ however, it deserves further investigation.

Table 2 summarizes how approved therapeutic options for the treatment of TSC-associated epilepsy are currently being used.

Table 2Current and emerging treatment options: mechanism of action and level of evidence

Drug	Mechanism of action	Level of evidence	FDA/EMA approval	Studies in TSC	Minimal effective dose	Time to response	References
Vigabatrin	Irreversible inhibitor of GABA-T, it reduces glutamate/glutamine cycling between astrocytes and neurons, inhibits mTOR pathway	1b	FDA: Aug 2009 EMA: Oct 1999 (epilepsy)	Randomized trial comparing VGB and hydrocortisone in infantile spasms due to TSC	50–80 mg/kg/day	1 week	Chiron et al.,¹⁶⁸ Ounissi,¹⁶⁹ Rodrigues et al.¹⁷⁰
Cannabidiol	Block of GPR55 receptors, desensitization of TRPV1 channels, inhibition of ENT1 adenosine reuptake pumps, reduction of mTOR pathway signalling	1b	FDA: Aug 2020 EMA: Jan 2021 (TSC)	Placebo-controlled phase 3 trial GWPCARE6 (NCT02544763)	25 mg/kg/day	4 weeks	Thiele et al.¹⁷¹
Everolimus	Potent mTOR (mechanistic target of rapamycin) inhibitor	1b	FDA Apr 2018 EMA Jul 2020 (epilepsy in TSC)	EXIST-3 (NCT01713946), randomized, double-blind, multicentre trial	3–7 ng/ml	8 weeks	French et al.,¹⁷² Franz et al.¹⁷³
Ganaxolone	Positive allosteric modulator of GABA_A receptors synthetic analog of allopregnanolone	1b	FDA Mar 2022 (epilepsy in CDD)	Phase 2 CALM study (NCT0485346) Phase 3 TRUSTTSC study (NCT05323734)	Study ongoing in TSC	Study ongoing in TSC	NCT0485346

Vigabatrin

Vigabatrin (VGB) can be considered as a neuromodulator agent: its major effect is to influence the activity of neurons. VGB is an irreversible inhibitor of GABA-transaminase (GABA-T),¹⁷⁴ an enzyme that degrades GABA, resulting in elevated brain GABA levels.¹⁷⁵ Higher concentrations of GABA terminate seizure activity. Additionally, vigabatrin may facilitate the synaptic release of GABA and prevent its neuronal uptake.¹⁷⁶ It may also inhibit glial uptake of GABA.¹⁷⁷ Beyond GABA mechanisms, VGB may reduce glutamate⁄glutamine cycling between astrocytes and neurons, and the antiseizure effects may also be related to this glutamatergic effect.¹⁷⁸

VGB is effective when treating patients with TSC with both focal seizures and infantile spasms. VGB inhibits mTOR pathway activity,¹⁷⁹ which could represent a further mechanism of action that may contribute to the distinctive efficacy of VGB in TSC. VGB also showed an inhibitory effect on glial proliferation, at least in hippocampus,¹⁷⁹ and reduced astrocyte numbers in the neocortex in the mouse model. Effects of VGB on glial proliferation can likely be attributed to mTOR pathway inhibition.

In practice, a minimal effective dose should be considered to limit the risk of serious adverse events, including MRI changes and visual field abnormalities.¹⁶⁹

Cannabidiol

CBD has broad spectrum anticonvulsive efficacy in animal model of seizures and epilepsy (including acute pentylenetetrazol and maximal electroshock for generalized seizures, acute pilocarpine and penicillin for focal seizures, audiogenic model of genetic epilepsy, chronic lithium-pilocarpine for generalized epilepsy and Scn1a^+/− for developmental and epileptic encephalopathy).^180–182 Additionally, CBD has improved survival and behavioural comorbidity outcomes in a mouse model of Dravet syndrome¹⁸¹ and in a rat model of temporal lobe epilepsy.¹⁸³

The mechanism of action of CBD remains to be fully elucidated. CBD has very poor affinity for CB1 receptors and lacks euphoric side effects, and no direct effects seem to be mediated via cannabinoid receptors.^180,184,185 CBD is hypothesized to act at a synaptic level by reducing excitatory neurotransmission through at least three possible mechanisms: blocking GPR55 receptors, desensitization of TRPV1 channels or inhibition of ENT1 adenosine reuptake pumps.^186,187

Other potential mechanisms for CBD in epilepsy include the reduction of signalling in the mTOR pathway observed in zebrafish.¹⁸⁸ Different effects of CBD on signalling within the mTOR pathway have been reported across diverse experimental models. This may suggest a potential pro-homeostatic mechanism for CBD, whereby it counteracts the disease-associated perturbation in mTOR signalling.¹⁸⁹

Everolimus

Everolimus, a derivative of rapamycin (sirolimus), is a potent mTOR inhibitor. Its major effects are related to immunosuppression and antiangiogenic properties, and it was first approved as an immunosuppressant to prevent the rejection of organ transplants.¹⁹⁰

Everolimus acts only on the mTORC1 protein and not on the mTORC2 protein.¹⁹¹ It binds with high affinity to the FK506 binding protein-12 (FKBP-12), forming a drug complex that can inhibit the activation of mTOR,¹⁹² resulting in inhibition of T-lymphocyte activation and proliferation.¹⁹³

Reduction of neuronal excitability by everolimus (the antiepileptic activity) is mediated by a prolonged opening of Ca²⁺ and K⁺ channels, via an increased expression of K_v1.1 in cortical and hippocampal neurons and reduced expression of AMPA receptors.^69,194,195 Everolimus also has some neuroprotective activities exerted via the modulation of synaptic plasticity, regulation of neuronal death and regulation of neurogenesis.^196,197 Chronic treatment leads to changes in synaptic membranes, with reduced excitability and increased GABA-mediated synaptic activity.⁶⁹

Prevention of epilepsy has been suggested in a mouse model of TSC. Early treatment with rapamycin itself (age postnatal Day 14) showed prevention of seizures and premature death, whereas late treatment (age 6 weeks) only induced suppression of seizures and prolonged survival in a Tsc1-GFAP CKO mouse model.¹⁹⁸ Different TSC mouse models have shown concordant results for antiepileptogenic effects; however, rapamycin has also been shown to restore cell size and myelination, indicating a possible disease-modifying effect.^199–202 Recently, rapamycin has been shown to relieve seizures and neuropsychiatric symptoms, except for ASD, and this effect seems to be related to the regulation of microglia polarity in the TSC mouse model.¹⁰⁸

Everolimus improves white matter microstructural integrity, particularly in younger patients during a period of a rapid white matter maturation.²⁰³ Furthermore, due to the targeted mechanism of action, everolimus is also effective in reducing several tumour manifestations associated with TSC (including subependymal giant cell astrocytoma and renal angiomyolipomas); hence, may represent a systemic treatment for this devasting condition.²⁰⁴

In the randomized controlled trial EXIST-3, both low and high exposure to adjunctive everolimus treatment in patients with drug resistant epilepsy showed a clinically meaningful reduction of seizure frequency in comparison with the placebo arm at the end of the core phase.¹⁷² This response remained sustained in the post-extension phase. Furthermore, in a post hoc analysis, the response was particularly high in the younger subgroup (children <6 years).²⁰⁵ The long-term safety of everolimus was assessed in an interventional post authorization safety study of 179 patients who received everolimus for the licensed indications in the European Union.²⁰⁶ The most frequent treatment-related adverse events were stomatitis (6.7%) and mouth ulcers (5.6%). Serious adverse events, such as grade 2–3 pneumonia, were reported in 3% of patients, reflecting the safety and tolerability of everolimus in the management of TSC in real-world routine clinical practice.

Interactions with CBD have been reported, with an increase of everolimus blood levels.²⁰⁷ Detection and further evaluation of potential drug interactions in patients using the combination of everolimus with CBD should be considered to increase patient safety.²⁰⁸

Ganaxolone

Ganaxolone (GNX) received the first approval in March 2022 within the USA for the treatment of seizures associated with CDKL5 deficiency disorder (CDD).²⁰⁹ A phase III study named MARIGOLD (NCT03572933) documented its efficacy in patients with CDD. A phase III study (TRUSTTSC) is ongoing in patients with TSC. GNX is a synthetic analog of allopregnanolone, a metabolite of progesterone, with a methyl substitution at the 3β position that prevents back-conversion to any active intermediates.²¹⁰ GNX also acts as a positive allosteric modulator of GABA_A receptors in the CNS via receptor binding at different sites, all of them different from the benzodiazepine binding sites.²¹¹

Discussion

Epileptogenesis in TSC is a multi-layered and dynamic process, and the epileptogenic network evolves over time (Fig. 2A and B). Overactivation of mTOR signalling is associated with cellular effects, and altered excitatory/inhibitory balance may be an important mechanism promoting epilepsy.²¹² Mechanisms manifesting epilepsy, such as altered interneuron development, may also co-manifest in autism-like features.^{148,149,212–215}

Recently, altered expression of mTOR and MAPK pathways—both key regulators of synaptogenesis and protein synthesis—were identified in children affected by idiopathic autism.¹⁴⁷ Progress in understanding the molecular basis of DEE and discerning the pathogenetic mechanisms that trigger both the epileptic and developmental encephalopathy components may help to find the most appropriate treatment.

Close EEG monitoring may allow early identification of pre-symptomatic EEG patterns and thus allow immediate commencement of treatment against seizures. This early seizure recognition and treatment is crucial to minimizing the risk of a poor neurological outcome.²¹⁶ Pre-symptomatic diagnosis of TSC may allow early identification of patients at high risk of developing drug-resistant epilepsy and DEE. EEG, MRI and genetic biomarkers have all been used successfully to identify infants at high risk of developing epilepsy and autism.^{13–15,34,37,144} There is an optimum time window for planning and implementing therapeutic intervention; this should include not only the use of specific antiseizure medications (such as vigabatrin or CBD) but also targeted medications such as mTOR inhibitors.

Early abnormalities in developmental trajectories up to 6–12 months of age also predict a higher risk for autism.¹⁴⁴ Despite a solid biological rationalization based on animal models and preclinical data for the improvement of learning disabilities^43,217 and autism,^44,46,218 clinical trials of everolimus targeting mTOR overactivation have not yet yielded unequivocal positive results in TSC-associated intellectual disability and autism; however, some improvements have been seen in the 3–6-year-old age group.^172,219 The timing of therapeutic intervention may be crucial in reshaping brain development and normalizing its function.^214,220

Genetic and acquired risk factors during critical and sensitive periods of synaptic plasticity and circuit development may have a significant impact on developmental trajectories.²²¹ The study of developmental trajectories in TSC infants may enable the discovery of biomarkers that have the potential to help in identifying infants at high risk of ASD before the onset of the first behavioural abnormality and consequently help to find targeted therapies for DEE.²²²

Epilepsy in young Tsc1^+/− mice exhibits age-dependent expression that mimics that of human TSC.⁵³ Animal models show that mTOR inhibitors are potentially effective not only in reducing seizure frequency but also in improving cognitive function. These outcomes have not, however, been reflected by clinical experience, where the age-dependent profile and appropriate dose need consideration.²²³ Future clinical studies need to include more patients aged under 2 years; for example, rapamycin has been tested as a preventative treatment in TSC patients.²²⁴

Evidence is growing to support that co-occurring NDD is not only a consequence of epilepsy and epileptiform abnormalities but may also reflect a common branched effect of TSC1 and TSC2 genetic variants. Medically reducing seizures still has only minimal impact on cognitive and behavioural symptoms.

The concept of EEG monitoring with pre-symptomatic treatment has changed clinical practice within the past few years. In a recent study, the epilepsy rate in a subgroup of patients receiving preventative treatment was much lower when compared with the conventional treatment approach.²²⁵ Despite the use of preventative treatments, even when the delay between seizure onset and vigabatrin initiation is short, a reduction in the risk of epileptic encephalopathy is by no means a certainty; the effect of seizures may be minimized, but not the effect had on developmental encephalopathy.

Different biological mechanisms should be considered to explain these findings. Firstly, the current treatment approach is able to modify the shape of neurons but has no effect on the dyslamination that starts prenatally. Again, time dependency of treatment is critical, even if the results are equivocal with effectiveness of mTOR inhibition in ASD remaining controversial.^44,101

Selection of the children who are likely to benefit from mTOR therapy requires predictive biomarkers. Yet, no interventional trials of mTOR inhibitors in patients with epilepsy and autism exist that would enable us to evaluate both the effects on seizure frequency as well as the symptoms behind ASD. Further studies are needed to optimize mTOR inhibitor use, the safety and efficacy with long term use of rapalogs, and to determine whether early drug therapy combined with behavioural cognitive intervention can prevent progression to DEE or mitigate DEE severity.

Interestingly, increased EEG connectivity has been shown to precede the onset of epileptic spasm in TSC infants, indicating the establishment of a progressive pathological network synchronization.¹³⁹

Despite getting closer, targeted treatment is still not available. In TSC, the outcome with respect to seizures, cognitive dysfunction and comorbid conditions is highly variable, even within the same family. Considering the great heterogeneity of TSC, an integrative approach is essential, and clinical trials are needed on biologically homogeneous subgroups.

The advances made in our understanding of the mechanisms underlying epileptogenesis in infants with TSC have led to the emergence of new concepts in the management of TSC-related epilepsy. A predictive and preventive approach can help both to delay seizure onset and improve seizure response, offering new avenues for targeted medicine. Novel treatment options should be explored that target the cellular and molecular pathway alterations which contribute to epileptogenesis.

Acknowledgements

Editing of the English text was by David Macari PhD (freelance medical writer), whose services were entirely funded by the authors.

Contributor Information

Eleonora Aronica, Department of Neuropathology, Amsterdam Neuroscience, Amsterdam UMC, University of Amsterdam, Amsterdam 1105 AZ, The Netherlands; Stichting Epilepsie Instellingen Nederland (SEIN), Heemstede 1105 AZ, The Netherlands.

Nicola Specchio, Full Member of European Reference Network EpiCARE, Clinical and Experimental Neurology, Bambino Gesù Children's Hospital, IRCCS, Rome 00165, Italy.

Mark J Luinenburg, Department of Neuropathology, Amsterdam Neuroscience, Amsterdam UMC, University of Amsterdam, Amsterdam 1105 AZ, The Netherlands.

Paolo Curatolo, Child Neurology and Psychiatry Unit, Systems Medicine Department, Tor Vergata University, Rome 00133, Italy.

Funding

M.J.L. is supported by EpilepsieNL (project number 2020-02) and A.E. by the ZonMw (The Netherlands Organisation for Health Research and Development).

Competing interests

E.A. has received speaker honoraria from Novartis, Nutricia and UCB; has served as an investigator for UCB and Nutricia; and has served on scientific advisory boards for Novartis and UCB. N.S. has served on scientific advisory boards for GW Pharma, BioMarin, Arvelle, Marinus and Takeda; has received speaker honoraria from Eisai, Biomarin, Livanova, Sanofi; and has served as an investigator for Zogenix, Marinus, Biomarin, UCB and Roche. P.C. has served on scientific advisory boards for Novartis. M.L. reports no competing interests.

References

Untitled section

References

1. Scheffer IE, Berkovic S, Capovilla G, et al. ILAE Classification of the epilepsies: Position paper of the ILAE commission for classification and terminology. Epilepsia. 2017;58:512–521.
2. Scheffer IE, Liao J. Deciphering the concepts behind “epileptic encephalopathy” and “developmental and epileptic encephalopathy”. Eur J Paediatr Neurol. 2020;24:11–14.
3. Specchio N, Wirrell EC, Scheffer IE, et al. International league against epilepsy classification and definition of epilepsy syndromes with onset in childhood: Position paper by the ILAE task force on nosology and definitions. Epilepsia. 2022;63:1398–1442.
4. Specchio N, Curatolo P. Developmental and epileptic encephalopathies: What we do and do not know. Brain. 2021;144:32–43.
5. Northrup H, Aronow ME, Bebin EM, et al. Updated international tuberous sclerosis Complex diagnostic criteria and surveillance and management recommendations. Pediatr Neurol. 2021;123:50–66.
6. Switon K, Kotulska K, Janusz-Kaminska A, Zmorzynska J, Jaworski J. Molecular neurobiology of mTOR. Neuroscience. 2017;341:112–153.
7. Lai D, Gade M, Yang E, et al. Somatic variants in diverse genes leads to a spectrum of focal cortical malformations. Brain. 2022;145:2704–2720.
8. Iffland PH, Everett ME, Cobb-Pitstick KM, et al. NPRL3 Loss alters neuronal morphology, mTOR localization, cortical lamination, and seizure threshold. Brain. 2022;145:3872–3885.
9. Mühlebner A, Bongaarts A, Sarnat HB, Scholl T, Aronica E. New insights into a spectrum of developmental malformations related to mTOR dysregulations: Challenges and perspectives. J Anat. 2019;235:521–542.
10. Rosset C, Netto CBO, Ashton-Prolla P. TSC1 And TSC2 gene mutations and their implications for treatment in tuberous sclerosis Complex: A review. Genet Mol Biol. 2017;40:69–79.
11. Curatolo P, Moavero R, Roberto D, Graziola F. Genotype/phenotype correlations in tuberous sclerosis Complex. Semin Pediatr Neurol. 2015;22:259–273.
12. Vanclooster S, Bissell S, van Eeghen AM, et al. The research landscape of tuberous sclerosis complex–associated neuropsychiatric disorders (TAND)—A comprehensive scoping review. J Neurodev Disord. 2022;14:13.
13. Capal JK, Williams ME, Pearson DA, et al. Profile of autism Spectrum disorder in tuberous sclerosis Complex: Results from a longitudinal, prospective, multisite study. Ann Neurol. 2021;90:874–886.
14. Ogórek B, Hamieh L, Hulshof HM, et al. TSC2 Pathogenic variants are predictive of severe clinical manifestations in TSC infants: Results of the EPISTOP study. Genet Med. 2020;22:1489–1497.
15. Farach LS, Pearson DA, Woodhouse JP, et al. Tuberous sclerosis Complex genotypes and developmental phenotype. Pediatr Neurol. 2019;96:58–63.
16. Mongrain V, van Doesburg NH, Rypens F, et al. A case report of severe tuberous sclerosis complex detected in utero and linked to a novel duplication in the TSC2 gene. BMC Neurol. 2020;20:324.
17. Jansen AC, Sancak O, D’Agostino MD, et al. Unusually mild tuberous sclerosis phenotype is associated with TSC2 R905Q mutation. Ann Neurol. 2006;60:528–539.
18. van Eeghen AM, Nellist M, van Eeghen EE, Thiele EA. Central TSC2 missense mutations are associated with a reduced risk of infantile spasms. Epilepsy Res. 2013;103:83–87.
19. Farach LS, Richard MA, Lupo PJ, et al. Epilepsy risk prediction model for patients with tuberous sclerosis Complex. Pediatr Neurol. 2020;113:46–50.
20. Capal JK, Bernardino-Cuesta B, Horn PS, et al. Influence of seizures on early development in tuberous sclerosis complex. Epilepsy Behav. 2017;70(Pt A):245–252.
21. Specchio N, Di Micco V, Trivisano M, Ferretti A, Curatolo P. The epilepsy-autism spectrum disorder phenotype in the era of molecular genetics and precision therapy. Epilepsia. 2022;63:6–21.
22. Martin KR, Zhou W, Bowman MJ, et al. The genomic landscape of tuberous sclerosis complex. Nat Commun. 2017;8:15816.
23. Viñuela A, Brown AA, Buil A, et al. Age-dependent changes in mean and variance of gene expression across tissues in a twin cohort. Hum Mol Genet. 2018;27:732–741.
24. Curatolo P, Specchio N, Aronica E. Advances in the genetics and neuropathology of tuberous sclerosis complex: Edging closer to targeted therapy. Lancet Neurol. 2022;21:843–856.
25. Curatolo P, Moavero R, de Vries PJ. Neurological and neuropsychiatric aspects of tuberous sclerosis complex. Lancet Neurol. 2015;14:733–745.
26. Curatolo P, Moavero R, van Scheppingen J, Aronica E. mTOR dysregulation and tuberous sclerosis-related epilepsy. Expert Rev Neurother. 2018;18:185–201.
27. Zimmer TS, Broekaart DWM, Gruber V-E, van Vliet EA, Mühlebner A, Aronica E. Tuberous sclerosis Complex as disease model for investigating mTOR-related gliopathy during epileptogenesis. Front Neurol. 2020;11:1028.
28. Page MJ, McKenzie JE, Bossuyt PM, et al. The PRISMA 2020 statement: An updated guideline for reporting systematic reviews. BMJ. 2021;372:n71.
29. Pitkänen A, Engel J. Past and present definitions of epileptogenesis and its biomarkers. Neurotherapeutics. 2014;11:231–241.
30. Pitkänen A, Lukasiuk K, Dudek FE, Staley KJ. Epileptogenesis. Cold Spring Harb Perspect Med. 2015;5:a022822.
31. Löscher W. The holy grail of epilepsy prevention: Preclinical approaches to antiepileptogenic treatments. Neuropharmacology. 2020;167:107605.
32. Galanopoulou AS, Löscher W, Lubbers L, et al. Antiepileptogenesis and disease modification: Progress, challenges, and the path forward—Report of the preclinical working group of the 2018 NINDS-sponsored antiepileptogenesis and disease modification workshop. Epilepsia open. 2021;6:276–296.
33. Klein P, Dingledine R, Aronica E, et al. Commonalities in epileptogenic processes from different acute brain insults: Do they translate? Epilepsia. 2018;59:37–66.
34. Hulshof HM, Slot EMH, Lequin M, et al. Fetal brain magnetic resonance imaging findings predict neurodevelopment in children with tuberous sclerosis Complex. J Pediatr. 2021;233:156–162.e2.
35. Cavalheiro S, da Costa MDS, Richtmann R. Everolimus as a possible prenatal treatment of in utero diagnosed subependymal lesions in tuberous sclerosis complex: A case report. Childs Nerv Syst. 2021;37:3897–3899.
36. Prabowo AS, Anink JJ, Lammens M, et al. Fetal brain lesions in tuberous sclerosis Complex: TORC1 activation and inflammation. Brain Pathol. 2013;23:45–59.
37. De Ridder J, Verhelle B, Vervisch J, et al. Early epileptiform EEG activity in infants with tuberous sclerosis complex predicts epilepsy and neurodevelopmental outcomes. Epilepsia. 2021;62:1208–1219.
38. De Ridder J, Kotulska K, Curatolo P, et al. Evolution of electroencephalogram in infants with tuberous sclerosis complex and neurodevelopmental outcome: A prospective cohort study. Dev Med Child Neurol. 2022;64:495–501.
39. Saxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. Cell. 2017;168:960–976.
40. Andrews MG, Subramanian L, Kriegstein AR. mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex. Elife. 2020;9:e58737.
41. von der Brelie C, Waltereit R, Zhang L, Beck H, Kirschstein T. Impaired synaptic plasticity in a rat model of tuberous sclerosis. Eur J Neurosci. 2006;23:686–692.
42. Goorden SMI, van Woerden GM, van der Weerd L, Cheadle JP, Elgersma Y. Cognitive deficits in Tsc1^+/− mice in the absence of cerebral lesions and seizures. Ann Neurol. 2007;62:648–655.
43. Ehninger D, Han S, Shilyansky C, et al. Reversal of learning deficits in a Tsc2+/- mouse model of tuberous sclerosis. Nat Med. 2008;14:843–848.
44. Sato A, Kasai S, Kobayashi T, et al. Rapamycin reverses impaired social interaction in mouse models of tuberous sclerosis complex. Nat Commun. 2012;3:1292.
45. Way SW, Rozas NS, Wu HC, et al. The differential effects of prenatal and/or postnatal rapamycin on neurodevelopmental defects and cognition in a neuroglial mouse model of tuberous sclerosis complex. Hum Mol Genet. 2012;21:3226–3236.
46. Petrasek T, Vojtechova I, Klovrza O, et al. mTOR inhibitor improves autistic-like behaviors related to Tsc2 haploinsufficiency but not following developmental status epilepticus. J Neurodev Disord. 2021;13:14.
47. McMahon JJ, Yu W, Yang J, et al. Seizure-dependent mTOR activation in 5-HT neurons promotes autism-like behaviors in mice. Neurobiol Dis. 2015;73:296–306.
48. Wu X, Sosunov AA, Lado W, et al. Synaptic hyperexcitability of cytomegalic pyramidal neurons contributes to epileptogenesis in tuberous sclerosis complex. Cell Rep. 2022;40:111085.
49. Nguyen LH, Mahadeo T, Bordey A. mTOR hyperactivity levels influence the severity of epilepsy and associated neuropathology in an experimental model of tuberous sclerosis Complex and focal cortical dysplasia. J Neurosci. 2019;39:2762–2773.
50. Koene LMC, van Grondelle SE, Proietti Onori M, et al. Effects of antiepileptic drugs in a new TSC/mTOR-dependent epilepsy mouse model. Ann Clin Transl Neurol. 2019;6:1273–1291.
51. Koene LM, Niggl E, Wallaard I, Proietti-Onori M, Rotaru DC, Elgersma Y. Identifying the temporal electrophysiological and molecular changes that contribute to TSC-associated epileptogenesis. JCI Insight. 2021;6:e150120.
52. Nabbout R, Belousova E, Benedik MP, et al. Historical patterns of diagnosis, treatments, and outcome of epilepsy associated with tuberous sclerosis Complex: Results from TOSCA registry. Front Neurol. 2021;12:697467.
53. Gataullina S, Lemaire E, Wendling F, et al. Epilepsy in young Tsc1^+/− mice exhibits age-dependent expression that mimics that of human tuberous sclerosis complex. Epilepsia. 2016;57:648–659.
54. Rensing N, Johnson KJ, Foutz TJ, Friedman JL, Galindo R, Wong M. Early developmental electroencephalography abnormalities, neonatal seizures, and induced spasms in a mouse model of tuberous sclerosis complex. Epilepsia. 2020;61:879–891.
55. Scantlebury MH, Galanopoulou AS, Chudomelova L, Raffo E, Betancourth D, Moshé SL. A model of symptomatic infantile spasms syndrome. Neurobiol Dis. 2010;37:604–612.
56. Kim S-H, Speirs CK, Solnica-Krezel L, Ess KC. Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin. Dis Model Mech. 2011;4:255–267.
57. Scheldeman C, Mills JD, Siekierska A, et al. mTOR-related neuropathology in mutant tsc2 zebrafish: Phenotypic, transcriptomic and pharmacological analysis. Neurobiol Dis. 2017;108:225–237.
58. Kedra M, Banasiak K, Kisielewska K, Wolinska-Niziol L, Jaworski J, Zmorzynska J. Trkb hyperactivity contributes to brain dysconnectivity, epileptogenesis, and anxiety in zebrafish model of tuberous sclerosis Complex. Proc Natl Acad Sci U S A. 2020;117:2170–2179.
59. Blair JD, Hockemeyer D, Bateup HS. Genetically engineered human cortical spheroid models of tuberous sclerosis. Nat Med. 2018;24:1568–1578.
60. Afshar Saber W, Sahin M. Recent advances in human stem cell-based modeling of tuberous sclerosis Complex. Mol Autism. 2020;11:16.
61. Dang LT, Vaid S, Lin G, et al. STRADA-mutant human cortical organoids model megalencephaly and exhibit delayed neuronal differentiation. Dev Neurobiol. 2021;81:696–709.
62. Eichmüller OL, Corsini NS, Vértesy Á, et al. Amplification of human interneuron progenitors promotes brain tumors and neurological defects. Science. 2022;375:eabf5546.
63. Catlett TS, Onesto MM, McCann AJ, et al. RHOA Signaling defects result in impaired axon guidance in iPSC-derived neurons from patients with tuberous sclerosis complex. Nat Commun. 2021;12:2589.
64. Nguyen LH, Bordey A. Convergent and divergent mechanisms of epileptogenesis in mTORopathies. Front Neuroanat. 2021;15:664695.
65. Gelot AB, Represa A. Progression of fetal brain lesions in tuberous sclerosis Complex. Front Neurosci. 2020;14:899.
66. Bongaarts A, Giannikou K, Reinten RJ, et al. Subependymal giant cell astrocytomas in tuberous sclerosis Complex have consistent TSC1/TSC2 biallelic inactivation, and no BRAF mutations. Oncotarget. 2017;8:95516–95529.
67. Kannan L, Vogrin S, Bailey C, Maixner W, Harvey AS. Centre of epileptogenic tubers generate and propagate seizures in tuberous sclerosis. Brain. 2016;139(Pt 10):2653–2667.
68. Cepeda C, André VM, Yamazaki I, et al. Comparative study of cellular and synaptic abnormalities in brain tissue samples from pediatric tuberous sclerosis complex and cortical dysplasia type II. Epilepsia. 2010;51(Suppl 3):160–165.
69. Cepeda C, Levinson S, Yazon V-W, et al. Cellular antiseizure mechanisms of everolimus in pediatric tuberous sclerosis complex, cortical dysplasia, and non–mTOR-mediated etiologies. Epilepsia Open. 2018;3(S2):180–190.
70. Boer K, Crino PB, Gorter JA, et al. Gene expression analysis of tuberous sclerosis complex cortical tubers reveals increased expression of adhesion and inflammatory factors. Brain Pathol. 2010;20:704–719.
71. Finardi A, Gardoni F, Bassanini S, et al. NMDA Receptor composition differs among anatomically diverse malformations of cortical development. J Neuropathol Exp Neurol. 2006;65:883–893.
72. Talos DM, Kwiatkowski DJ, Cordero K, Black PM, Jensen FE. Cell-specific alterations of glutamate receptor expression in tuberous sclerosis complex cortical tubers. Ann Neurol. 2008;63:454–465.
73. Ruffolo G, Iyer A, Cifelli P, et al. Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions. Neurobiol Dis. 2016;95:93–101.
74. Lozovaya N, Gataullina S, Tsintsadze T, et al. Selective suppression of excessive GluN2C expression rescues early epilepsy in a tuberous sclerosis murine model. Nat Commun. 2014;5:4563.
75. Gataullina S, Galvani G, Touchet S, et al. Glun2c selective inhibition is a target to develop new antiepileptic compounds. Epilepsia. 2022;63:2911–2924.
76. Catania MV, D’Antoni S, Bonaccorso CM, Aronica E, Bear MF, Nicoletti F. Group I metabotropic glutamate receptors: A role in neurodevelopmental disorders? Mol Neurobiol. 2007;35:298–307.
77. Boer K, Troost D, Timmermans W, et al. Cellular localization of metabotropic glutamate receptors in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex. Neuroscience. 2008;156:203–215.
78. Katsarou A-M, Moshé SL, Galanopoulou AS. Interneuronopathies and their role in early life epilepsies and neurodevelopmental disorders. Epilepsia open. 2017;2:284–306.
79. Cherubini E, Di Cristo G, Avoli M. Dysregulation of GABAergic signaling in neurodevelomental disorders: Targeting cation-chloride co-transporters to Re-establish a proper E/I balance. Front Cell Neurosci. 2022;15:813441.
80. Verhoog QP, Holtman L, Aronica E, van Vliet EA. Astrocytes as guardians of neuronal excitability: Mechanisms underlying epileptogenesis. Front Neurol. 2020;11:591690.
81. Uhlmann EJ, Wong M, Baldwin RL, et al. Astrocyte-specific TSC1 conditional knockout mice exhibit abnormal neuronal organization and seizures. Ann Neurol. 2002;52:285–296.
82. Jansen LA, Uhlmann EJ, Crino PB, Gutmann DH, Wong M. Epileptogenesis and reduced inward rectifier potassium current in tuberous sclerosis complex-1-deficient astrocytes. Epilepsia. 2005;46:1871–1880.
83. Zou J, Zhang B, Gutmann DH, Wong M. Postnatal reduction of tuberous sclerosis complex 1 expression in astrocytes and neurons causes seizures in an age-dependent manner. Epilepsia. 2017;58:2053–2063.
84. Sosunov AA, Wu X, Weiner HL, et al. Tuberous sclerosis: A primary pathology of astrocytes? Epilepsia. 2008;49(Suppl 2):53–62.
85. Boer K, Jansen F, Nellist M, et al. Inflammatory processes in cortical tubers and subependymal giant cell tumors of tuberous sclerosis complex. Epilepsy Res. 2008;78:7–21.
86. Prabowo AS, Iyer AM, Anink JJ, Spliet WGM, van Rijen PC, Aronica E. Differential expression of major histocompatibility complex class I in developmental glioneuronal lesions. J Neuroinflammation. 2013;10:12.
87. Zhang B, Zou J, Han L, et al. The specificity and role of microglia in epileptogenesis in mouse models of tuberous sclerosis complex. Epilepsia. 2018;59:1796–1806.
88. Zhang B, Zou J, Han L, Rensing N, Wong M. Microglial activation during epileptogenesis in a mouse model of tuberous sclerosis complex. Epilepsia. 2016;57:1317–1325.
89. Arena A, Zimmer TS, van Scheppingen J, et al. Oxidative stress and inflammation in a spectrum of epileptogenic cortical malformations: Molecular insights into their interdependence. Brain Pathol. 2019;29:351–365.
90. Zimmer TS, Korotkov A, Zwakenberg S, et al. Upregulation of the pathogenic transcription factor SPI1/PU.1 in tuberous sclerosis complex and focal cortical dysplasia by oxidative stress. Brain Pathol. 2021;31:e12949.
91. Mills JD, Iyer AM, van Scheppingen J, et al. Coding and small non-coding transcriptional landscape of tuberous sclerosis complex cortical tubers: Implications for pathophysiology and treatment. Sci Rep. 2017;7:8089.
92. Gruber V-E, Luinenburg MJ, Colleselli K, et al. Increased expression of complement components in tuberous sclerosis complex and focal cortical dysplasia type 2B brain lesions. Epilepsia. 2022;63:364–374.
93. van Scheppingen J, Mills JD, Zimmer TS, et al. Mir147b: A novel key regulator of interleukin 1 beta-mediated inflammation in human astrocytes. Glia. 2018;66:1082–1097.
94. van Scheppingen J, Iyer AM, Prabowo AS, et al. Expression of microRNAs miR21, miR146a, and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures. Glia. 2016;64:1066–1082.
95. Zimmer TS, Ciriminna G, Arena A, et al. Chronic activation of anti-oxidant pathways and iron accumulation in epileptogenic malformations. Neuropathol Appl Neurobiol. 2020;46:546–563.
96. Gorter JA, Aronica E, van Vliet EA. The roof is leaking and a storm is raging: Repairing the blood-brain barrier in the fight against epilepsy. Epilepsy Curr. 2019;19:177–181.
97. Long KR, Huttner WB. The role of the extracellular matrix in neural progenitor cell proliferation and cortical folding during human neocortex development. Front Cell Neurosci. 2022;15:804649.
98. Bongaarts A, de Jong JM, Broekaart DWM, et al. Dysregulation of the MMP/TIMP proteolytic system in subependymal giant cell astrocytomas in patients with tuberous sclerosis Complex: Modulation of MMP by MicroRNA-320d in vitro. J Neuropathol Exp Neurol. 2020;79:777–790.
99. Broekaart DWM, Scheppingen J, Anink JJ, et al. Increased matrix metalloproteinases expression in tuberous sclerosis complex: Modulation by microRNA 146a and 147b in vitro. Neuropathol Appl Neurobiol. 2020;46:142–159.
100. Hulshof HM, Kuijf HJ, Kotulska K, et al. Association of early MRI characteristics with subsequent epilepsy and neurodevelopmental outcomes in children with tuberous sclerosis Complex. Neurology. 2022;98:e1216–e1225.
101. Pagani M, Barsotti N, Bertero A, et al. mTOR-related synaptic pathology causes autism spectrum disorder-associated functional hyperconnectivity. Nat Commun. 2021;12:6084.
102. Scholl T, Mühlebner A, Ricken G, et al. Impaired oligodendroglial turnover is associated with myelin pathology in focal cortical dysplasia and tuberous sclerosis complex. Brain Pathol. 2017;27:770–780.
103. Prohl AK, Scherrer B, Tomas-Fernandez X, et al. Early white matter development is abnormal in tuberous sclerosis complex patients who develop autism spectrum disorder. J Neurodev Disord. 2019;11:36.
104. Peters JM, Sahin M, Vogel-Farley VK, et al. Loss of white matter microstructural integrity is associated with adverse neurological outcome in tuberous sclerosis complex. Acad Radiol. 2012;19:17–25.
105. Sato A, Tominaga K, Iwatani Y, et al. Abnormal white matter microstructure in the limbic system is associated with tuberous sclerosis Complex-associated neuropsychiatric disorders. Front Neurol. 2022;13:782479.
106. Amegandjin CA, Choudhury M, Jadhav V, et al. Sensitive period for rescuing parvalbumin interneurons connectivity and social behavior deficits caused by TSC1 loss. Nat Commun. 2021;12:3653.
107. van Andel DM, Sprengers JJ, Oranje B, Scheepers FE, Jansen FE, Bruining H. Effects of bumetanide on neurodevelopmental impairments in patients with tuberous sclerosis complex: An open-label pilot study. Mol Autism. 2020;11:30.
108. Koike-Kumagai M, Fujimoto M, Wataya-Kaneda M. Sirolimus relieves seizures and neuropsychiatric symptoms via changes of microglial polarity in tuberous sclerosis complex model mice. Neuropharmacology. 2022;218:109203.
109. Korotkov A, Luinenburg MJ, Romagnolo A, et al. Down-regulation of the brain-specific cell-adhesion molecule contactin-3 in tuberous sclerosis complex during the early postnatal period. J Neurodev Disord. 2022;14:8.
110. Iyer A, Prabowo A, Anink J, Spliet WGM, van Rijen PC, Aronica E. Cell injury and premature neurodegeneration in focal malformations of cortical development. Brain Pathol. 2014;24:1–17.
111. Kovacs GG, Adle-Biassette H, Milenkovic I, Cipriani S, van Scheppingen J, Aronica E. Linking pathways in the developing and aging brain with neurodegeneration. Neuroscience. 2014;269:152–172.
112. Sarnat HB, Flores-Sarnat L. Infantile tauopathies: Hemimegalencephaly; tuberous sclerosis complex; focal cortical dysplasia 2; ganglioglioma. Brain Dev. 2015;37:553–562.
113. Hwang J-HL, Perloff OS, Gaus SE, et al. Tuberous sclerosis complex is associated with a novel human tauopathy. Acta Neuropathol. 2023;145:1–12.
114. Liu AJ, Lusk JB, Ervin J, Burke J, O’Brien R, Wang S-HJ. Tuberous sclerosis complex is a novel, amyloid-independent tauopathy associated with elevated phosphorylated 3R/4R tau aggregation. Acta Neuropathol Commun. 2022;10:27.
115. Liu AJ, Staffaroni AM, Rojas-Martinez JC, et al. Association of cognitive and behavioral features between adults with tuberous sclerosis and frontotemporal dementia. JAMA Neurol. 2020;77:358–366.
116. Chen S, Chen Y, Zhang Y, et al. Iron metabolism and ferroptosis in epilepsy. Front Neurosci. 2020;14:601193.
117. Owens GC, Garcia AJ, Mochizuki AY, et al. Evidence for innate and adaptive immune responses in a cohort of intractable pediatric epilepsy surgery patients. Front Immunol. 2019;10:121.
118. Mühlebner A, van Scheppingen J, de Neef A, et al. Myelin pathology beyond white matter in tuberous sclerosis Complex (TSC) cortical tubers. J Neuropathol Exp Neurol. 2020;79:1054–1064.
119. Figlia G, Gerber D, Suter U. Myelination and mTOR. Glia. 2018;66:693–707.
120. Gruber V-E, Lang J, Endmayr V, et al. Impaired myelin production due to an intrinsic failure of oligodendrocytes in mTORpathies. Neuropathol Appl Neurobiol. 2021;47:812–825.
121. Zonouzi M, Berger D, Jokhi V, Kedaigle A, Lichtman J, Arlotta P. Individual oligodendrocytes show bias for inhibitory axons in the neocortex. Cell Rep. 2019;27:2799–2808.e3.
122. Fang L-P, Zhao N, Caudal LC, et al. Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects social cognitive behavior. Nat Commun. 2022;13:1394.
123. Chen W, Luo B, Gao N, et al. Neddylation stabilizes Nav1.1 to maintain interneuron excitability and prevent seizures in murine epilepsy models. J Clin Invest. 2021;131:e136956.
124. Ichise E, Chiyonobu T, Ishikawa M, et al. Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons. Hum Mol Genet. 2021;30:1337–1348.
125. Powell EM. Interneuron development and epilepsy: Early genetic defects cause long-term consequences in seizures and susceptibility. Epilepsy Curr. 2013;13:172–176.
126. Fu C, Cawthon B, Clinkscales W, Bruce A, Winzenburger P, Ess KC. GABAergic interneuron development and function is modulated by the Tsc1 gene. Cereb Cortex. 2012;22:2111–2119.
127. Ka M, Smith AL, Kim W-Y. MTOR Controls genesis and autophagy of GABAergic interneurons during brain development. Autophagy. 2017;13:1348–1363.
128. Hui KK, Takashima N, Watanabe A, et al. GABARAPs dysfunction by autophagy deficiency in adolescent brain impairs GABA_A receptor trafficking and social behavior. Sci Adv. 2019;5:eaau8237.
129. Alsaqati M, Heine VM, Harwood AJ. Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation. Mol Autism. 2020;11:80.
130. Talos DM, Sun H, Kosaras B, et al. Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia. Ann Neurol. 2012;71:539–551.
131. Aronica E, Boer K, Redeker S, et al. Differential expression patterns of chloride transporters, na+-K+-2Cl–cotransporter and K+-cl–cotransporter, in epilepsy-associated malformations of cortical development. Neuroscience. 2007;145:185–196.
132. Ruffolo G, Cifelli P, Roseti C, et al. A novel GABAergic dysfunction in human dravet syndrome. Epilepsia. 2018;59:2106–2117.
133. Ahtam B, Dehaes M, Sliva DD, et al. Resting-State fMRI networks in children with tuberous sclerosis Complex. J Neuroimaging. 2019;29:750–759.
134. Shrot S, Lawson P, Shlomovitz O, et al. Prediction of tuberous sclerosis-associated neurocognitive disorders and seizures via machine learning of structural magnetic resonance imaging. Neuroradiology. 2022;64:611–620.
135. Cook IA, Wilson AC, Peters JM, et al. EEG Spectral features in sleep of autism Spectrum disorders in children with tuberous sclerosis Complex. J Autism Dev Disord. 2020;50:916–923.
136. Neal A, Bouet R, Lagarde S, et al. Epileptic spasms are associated with increased stereo-electroencephalography derived functional connectivity in tuberous sclerosis complex. Epilepsia. 2022;63:2359–2370.
137. Wang Y, Yuan L, Zhang S, et al. Fast ripples as a biomarker of epileptogenic tuber in tuberous sclerosis Complex patients using stereo-electroencephalograph. Front Hum Neurosci. 2021;15:680295.
138. De Ridder J, Lavanga M, Verhelle B, et al. Prediction of neurodevelopment in infants with tuberous sclerosis Complex using early EEG characteristics. Front Neurol. 2020;11:582891.
139. Davis PE, Kapur K, Filip-Dhima R, et al. Increased electroencephalography connectivity precedes epileptic spasm onset in infants with tuberous sclerosis complex. Epilepsia. 2019;60:1721–1732.
140. Bernardo D, Nariai H, Hussain SA, et al. Visual and semi-automatic non-invasive detection of interictal fast ripples: A potential biomarker of epilepsy in children with tuberous sclerosis complex. Clin Neurophysiol. 2018;129:1458–1466.
141. Wu JY, Goyal M, Peters JM, et al. Scalp EEG spikes predict impending epilepsy in TSC infants: A longitudinal observational study. Epilepsia. 2019;60:2428–2436.
142. Scheper M, Romagnolo A, Besharat ZM, et al. miRNAs and isomiRs: Serum-based biomarkers for the development of intellectual disability and autism Spectrum disorder in tuberous sclerosis Complex. Biomedicines. 2022;10:1838.
143. Pawlik B, Smyczyńska U, Grabia S, et al. mTOR inhibitor treatment in patients with tuberous sclerosis Complex is associated with specific changes in microRNA Serum profile. J Clin Med. 2022;11:3395.
144. Moavero R, Benvenuto A, Emberti Gialloreti L, et al. Early clinical predictors of autism Spectrum disorder in infants with tuberous sclerosis Complex: Results from the EPISTOP study. J Clin Med. 2019;8:788.
145. Moavero R, Marciano S, Pro S, et al. Event-Related potentials in ADHD associated with tuberous sclerosis Complex: A possible biomarker of symptoms severity? Front Neurol. 2020;11:546.
146. Moavero R, Kotulska K, Lagae L, et al. Is autism driven by epilepsy in infants with tuberous sclerosis Complex? Ann Clin Transl Neurol. 2020;7:1371–1381.
147. Rosina E, Battan B, Siracusano M, et al. Disruption of mTOR and MAPK pathways correlates with severity in idiopathic autism. Transl Psychiatry. 2019;9:50.
148. Yang J, Yang X, Tang K. Interneuron development and dysfunction. FEBS J. 2022;289:2318–2336.
149. Powell EM, Campbell DB, Stanwood GD, Davis C, Noebels JL, Levitt P. Genetic disruption of cortical interneuron development causes region- and GABA cell type-specific deficits, epilepsy, and behavioral dysfunction. J Neurosci. 2003;23:622–631.
150. Suleymanova EM. Behavioral comorbidities of epilepsy and neuroinflammation: Evidence from experimental and clinical studies. Epilepsy Behav. 2021;117:107869.
151. Vezzani A, Aronica E, Mazarati A, Pittman QJ. Epilepsy and brain inflammation. Exp Neurol. 2013;244:11–21.
152. Mazarati AM, Lewis ML, Pittman QJ. Neurobehavioral comorbidities of epilepsy: Role of inflammation. Epilepsia. 2017;58(Suppl 3):48–56.
153. Dalva MB, McClelland AC, Kayser MS. Cell adhesion molecules: Signalling functions at the synapse. Nat Rev Neurosci. 2007;8:206–220.
154. Krishnan ML, Commowick O, Jeste SS, et al. Diffusion features of white matter in tuberous sclerosis with tractography. Pediatr Neurol. 2010;42:101–106.
155. Arulrajah S, Ertan G, Jordan L, et al. Magnetic resonance imaging and diffusion-weighted imaging of normal-appearing white matter in children and young adults with tuberous sclerosis complex. Neuroradiology. 2009;51:781–786.
156. Lewis WW, Sahin M, Scherrer B, et al. Impaired language pathways in tuberous sclerosis complex patients with autism spectrum disorders. Cereb Cortex. 2013;23:1526–1532.
157. Simao G, Raybaud C, Chuang S, Go C, Snead OC, Widjaja E. Diffusion tensor imaging of commissural and projection white matter in tuberous sclerosis complex and correlation with tuber load. AJNR Am J Neuroradiol. 2010;31:1273–1277.
158. Kovacs GG, Ghetti B, Goedert M. Classification of diseases with accumulation of tau protein. Neuropathol Appl Neurobiol. 2022;48:e12792.
159. Sarnat H, Flores-Sarnat L, Crino P, Hader W, Bello-Espinosa L. Hemimegalencephaly: Foetal tauopathy with mTOR hyperactivation and neuronal lipidosis. Folia Neuropathol. 2012;50:330–345.
160. Shephard E, McEwen FS, Earnest T, et al. Oscillatory neural network alterations in young people with tuberous sclerosis complex and associations with co-occurring symptoms of autism spectrum disorder and attention-deficit/hyperactivity disorder. Cortex. 2022;146:50–65.
161. Moavero R, Voci A, La Briola F, et al. Sleep disorders and neuropsychiatric disorders in a pediatric sample of tuberous sclerosis complex: A questionnaire-based study. Sleep Med. 2022;89:65–70.
162. Zhang B, Guo D, Han L, Rensing N, Satoh A, Wong M. Hypothalamic orexin and mechanistic target of rapamycin activation mediate sleep dysfunction in a mouse model of tuberous sclerosis complex. Neurobiol Dis. 2020;134:104615.
163. Löscher W, Potschka H, Sisodiya SM, Vezzani A. Drug resistance in epilepsy: Clinical impact, potential mechanisms, and new innovative treatment options. Pharmacol Rev. 2020;72:606–638.
164. Aronica E, Sisodiya SM, Gorter JA. Cerebral expression of drug transporters in epilepsy. Adv Drug Deliv Rev. 2012;64:919–929.
165. Boer K, Troost D, Jansen F, et al. Clinicopathological and immunohistochemical findings in an autopsy case of tuberous sclerosis complex. Neuropathology. 2008;28:577–590.
166. Formisano L, Napolitano F, Rosa R, et al. Mechanisms of resistance to mTOR inhibitors. Crit Rev Oncol Hematol. 2020;147:102886.
167. Amin S, Mallick AA, Edwards H, et al. The metformin in tuberous sclerosis (MiTS) study: A randomised double-blind placebo-controlled trial. EClinicalMedicine. 2021;32:100715.
168. Chiron C, Dumas C, Jambaqué I, Mumford J, Dulac O. Randomized trial comparing vigabatrin and hydrocortisone in infantile spasms due to tuberous sclerosis. Epilepsy Res. 1997;26:389–395.
169. Ounissi M, Rodrigues C, Bienayme H, et al. Proposition of a minimal effective dose of vigabatrin for the treatment of infantile spasms using pediatric and adult pharmacokinetic data. J Clin Pharmacol. 2019;59:177–188.
170. Rodrigues C, Chiron C, Ounissi M, et al. Pharmacokinetic evaluation of vigabatrin dose for the treatment of refractory focal seizures in children using adult and pediatric data. Epilepsy Res. 2019;150:38–45.
171. Thiele EA, Bebin EM, Bhathal H, et al. Add-on cannabidiol treatment for drug-resistant seizures in tuberous sclerosis Complex. JAMA Neurol. 2021;78:285.
172. French JA, Lawson JA, Yapici Z, et al. Adjunctive everolimus therapy for treatment-resistant focal-onset seizures associated with tuberous sclerosis (EXIST-3): A phase 3, randomised, double-blind, placebo-controlled study. Lancet. 2016;388:2153–2163.
173. Franz DN, Lawson JA, Yapici Z, et al. Everolimus dosing recommendations for tuberous sclerosis complex-associated refractory seizures. Epilepsia. 2018;59:1188–1197.
174. Ben-Menachem E. Mechanism of action of vigabatrin: Correcting misperceptions. Acta Neurol Scand Suppl. 2011;124:5–15.
175. Willmore LJ, Abelson MB, Ben-Menachem E, Pellock JM, Shields WD. Vigabatrin: 2008 update. Epilepsia. 2009;50:163–173.
176. Ben-Menachem E. Vigabatrin. In: Levy RH, Mattson RH, Meldrum BS, Perucca E, eds. Antiepileptic drugs. 5th ed. Lippincott Williams & Wilkins; 2002:855–863.
177. Leach JP, Sills GJ, Majid A, et al. Effects of tiagabine and vigabatrin on GABA uptake into primary cultures of rat cortical astrocytes. Seizure. 1996;5:229–234.
178. Yang J, Shen J. Elevated endogenous GABA concentration attenuates glutamate-glutamine cycling between neurons and astroglia. J Neural Transm. 2009;116:291–300.
179. Zhang B, McDaniel SS, Rensing NR, Wong M. Vigabatrin inhibits seizures and mTOR pathway activation in a mouse model of tuberous sclerosis complex. PLoS One. 2013;8:e57445.
180. Rosenberg EC, Tsien RW, Whalley BJ, Devinsky O. Cannabinoids and epilepsy. Neurotherapeutics. 2015;12:747–768.
181. Patra PH, Serafeimidou-Pouliou E, Bazelot M, Whalley BJ, Williams CM, McNeish AJ. Cannabidiol improves survival and behavioural co-morbidities of dravet syndrome in mice. Br J Pharmacol. 2020;177:2779–2792.
182. Hawkins NA, Anderson LL, Gertler TS, Laux L, George AL, Kearney JA. Screening of conventional anticonvulsants in a genetic mouse model of epilepsy. Ann Clin Transl Neurol. 2017;4:326–339.
183. Patra PH, Barker-Haliski M, White HS, et al. Cannabidiol reduces seizures and associated behavioral comorbidities in a range of animal seizure and epilepsy models. Epilepsia. 2019;60:303–314.
184. Perucca E. Cannabinoids in the treatment of epilepsy: Hard evidence at last? J epilepsy Res. 2017;7:61–76.
185. Pertwee RG. The diverse CB₁ and CB₂ receptor pharmacology of three plant cannabinoids: Δ₉-tetrahydrocannabinol, cannabidiol and Δ₉-tetrahydrocannabivarin. Br J Pharmacol. 2008;153:199–215.
186. Gray RA, Whalley BJ. The proposed mechanisms of action of CBD in epilepsy. Epileptic Disord. 2020;22(S1):10–15.
187. Ibeas Bih C, Chen T, Nunn AVW, Bazelot M, Dallas M, Whalley BJ. Molecular targets of cannabidiol in neurological disorders. Neurotherapeutics. 2015;12:699–730.
188. Serra I, Scheldeman C, Bazelot M, et al. Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of tuberous sclerosis Complex. Behav Brain Res. 2019;363:135–144.
189. Ebrahimi-Fakhari D, Agricola KD, Tudor C, Krueger D, Franz DN. Cannabidiol elevates mechanistic target of rapamycin inhibitor levels in patients with tuberous sclerosis Complex. Pediatr Neurol. 2020;105:59–61.
190. Eisen HJ, Tuzcu EM, Dorent R, et al. Everolimus for the prevention of allograft rejection and vasculopathy in cardiac-transplant recipients. N Engl J Med. 2003;349:847–858.
191. Saran U, Foti M, Dufour J-F. Cellular and molecular effects of the mTOR inhibitor everolimus. Clin Sci. 2015;129:895–914.
192. van Rossum HH, Romijn FPHTM, Smit NPM, de Fijter JW, van Pelt J. Everolimus and sirolimus antagonize tacrolimus based calcineurin inhibition via competition for FK-binding protein 12. Biochem Pharmacol. 2009;77:1206–1212.
193. Merino D, San Segundo D, Medina JM, et al. Different in vitro proliferation and cytokine-production inhibition of memory T-cell subsets after calcineurin and mammalian target of rapamycin inhibitors treatment. Immunology. 2016;148:206–215.
194. Raab-Graham KF, Haddick PCG, Jan YN, Jan LY. Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites. Science. 2006;314:144–148.
195. Leitner DF, Kanshin E, Askenazi M, et al. Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia. PLoS One. 2022;17:e0268597.
196. Skardelly M, Glien A, Groba C, et al. The influence of immunosuppressive drugs on neural stem/progenitor cell fate in vitro. Exp Cell Res. 2013;319:3170–3181.
197. Huang X-Y, Hu Q-P, Shi H-Y, Zheng Y-Y, Hu R-R, Guo Q. Everolimus inhibits PI3K/akt/mTOR and NF-kB/IL-6 signaling and protects seizure-induced brain injury in rats. J Chem Neuroanat. 2021;114:101960.
198. Zeng L-H, Xu L, Gutmann DH, Wong M. Rapamycin prevents epilepsy in a mouse model of tuberous sclerosis complex. Ann Neurol. 2008;63:444–453.
199. Tillema J-M, Leach JL, Krueger DA, Franz DN. Everolimus alters white matter diffusion in tuberous sclerosis complex. Neurology. 2012;78:526–531.
200. Carson RP, Van Nielen DL, Winzenburger PA, Ess KC. Neuronal and glia abnormalities in Tsc1-deficient forebrain and partial rescue by rapamycin. Neurobiol Dis. 2012;45:369–380.
201. Meikle L, Pollizzi K, Egnor A, et al. Response of a neuronal model of tuberous sclerosis to mammalian target of rapamycin (mTOR) inhibitors: Effects on mTORC1 and akt signaling lead to improved survival and function. J Neurosci. 2008;28:5422–5432.
202. Magri L, Cominelli M, Cambiaghi M, et al. Timing of mTOR activation affects tuberous sclerosis complex neuropathology in mouse models. Dis Model Mech. 2013;6:1185–1197.
203. Peters JM, Prohl A, Kapur K, et al. Longitudinal effects of everolimus on white matter diffusion in tuberous sclerosis Complex. Pediatr Neurol. 2019;90:24–30.
204. Curatolo P, Bjørnvold M, Dill PE, et al. The role of mTOR inhibitors in the treatment of patients with tuberous sclerosis Complex: Evidence-based and expert opinions. Drugs. 2016;76:551–565.
205. Jambaqué I, Cusmai R, Curatolo P, Cortesi F, Perrot C, Dulac O. Neuropsychological aspects of tuberous sclerosis in relation to epilepsy and MRI findings. Dev Med Child Neurol. 1991;33:698–705.
206. Kingswood JC, Belousova E, Benedik MP, et al. Tuberous SClerosis registry to increAse disease awareness (TOSCA) post-authorisation safety study of everolimus in patients with tuberous sclerosis Complex. Front Neurol. 2021;12:630378.
207. Wiemer-Kruel A, Stiller B, Bast T. Cannabidiol interacts significantly with everolimus—Report of a patient with tuberous sclerosis Complex. Neuropediatrics. 2019;50:400–403.
208. Gilmartin CGS, Dowd Z, Parker APJ, Harijan P. Interaction of cannabidiol with other antiseizure medications: A narrative review. Seizure. 2021;86:189–196.
209. Lamb YN. Ganaxolone: First approval. Drugs. 2022;82:933–940.
210. Carter RB, Wood PL, Wieland S, et al. Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor. J Pharmacol Exp Ther. 1997;280:1284–1295.
211. Nohria V, Giller E. Ganaxolone. Neurotherapeutics. 2007;4:102–105.
212. Lasarge CL, Danzer SC. Mechanisms regulating neuronal excitability and seizure development following mTOR pathway hyperactivation. Front Mol Neurosci. 2014;7:18.
213. Díaz-Caneja CM, State MW, Hagerman RJ, et al. A white paper on a neurodevelopmental framework for drug discovery in autism and other neurodevelopmental disorders. Eur Neuropsychopharmacol. 2021;48:49–88.
214. Iannone AF, De Marco García NV. The emergence of network activity patterns in the somatosensory Cortex—An early window to autism Spectrum disorders. Neuroscience. 2021;466:298–309.
215. Marín O. Interneuron dysfunction in psychiatric disorders. Nat Rev Neurosci. 2012;13:107–120.
216. Bombardieri R, Pinci M, Moavero R, Cerminara C, Curatolo P. Early control of seizures improves long-term outcome in children with tuberous sclerosis complex. Eur J Paediatr Neurol. 2010;14:146–149.
217. Ehninger D. From genes to cognition in tuberous sclerosis: Implications for mTOR inhibitor-based treatment approaches. Neuropharmacology. 2013;68:97–105.
218. Tsai PT, Hull C, Chu Y, et al. Autistic-like behaviour and cerebellar dysfunction in purkinje cell Tsc1 mutant mice. Nature. 2012;488:647–651.
219. Curatolo P, Franz DN, Lawson JA, et al. Adjunctive everolimus for children and adolescents with treatment-refractory seizures associated with tuberous sclerosis complex: Post-hoc analysis of the phase 3 EXIST-3 trial. Lancet Child Adolesc Heal. 2018;2:495–504.
220. Marín O. Developmental timing and critical windows for the treatment of psychiatric disorders. Nat Med. 2016;22:1229–1238.
221. Meredith RM. Sensitive and critical periods during neurotypical and aberrant neurodevelopment: A framework for neurodevelopmental disorders. Neurosci Biobehav Rev. 2015;50:180–188.
222. Loth E, Spooren W, Ham LM, et al. Identification and validation of biomarkers for autism spectrum disorders. Nat Rev Drug Discov. 2016;15:70–73.
223. Krueger DA, Northrup H, Northrup H, et al. Tuberous sclerosis Complex surveillance and management: Recommendations of the 2012 international tuberous sclerosis Complex consensus conference. Pediatr Neurol. 2013;49:255–265.
224. He W, Chen J, Wang Y-Y, et al. Sirolimus improves seizure control in pediatric patients with tuberous sclerosis: A prospective cohort study. Seizure. 2020;79:20–26.
225. Wang X, Ding Y, Zhou Y, et al. Prenatal diagnosis and intervention improve developmental outcomes and epilepsy prognosis in children with tuberous sclerosis complex. Dev Med Child Neurol. 2022;64:1230–1236.

Studies

Conditions

Cannabinoids and active compounds

Related research

Abstract

Untitled section

Teaser

Article notes

Untitled section

Author notes

Introduction

Search strategy

Cellular and molecular mechanisms of TSC associated epileptogenesis

Insights from human pathology and genetic models

mTORC1 signalling and its roles in normal brain development

Morphological and functional alterations in TSC models

Morphological and functional alterations in human brain

Neural circuit dysfunctions and altered neurotransmission

Non-neural mechanisms

Age-dependent pathophysiological mechanisms and GABA signalling

Biomarkers for TSC-related developmental and epileptic encephalopathies

Mechanisms of developmental encephalopathy

Contributing mechanisms in TSC

Inflammation

Extracellular matrix and cell adhesion

Myelin pathology

Early neurodegeneration progress: tauopathy

Abnormal functional connectivity

Mechanism of action of drugs and targeted therapeutic options

Mechanisms of drug resistance

Vigabatrin

Cannabidiol

Everolimus

Ganaxolone

Discussion

Acknowledgements

Contributor Information

Funding

Competing interests

References

Untitled section

References

Additional materials