Early Rescue of Interneuron Disease Trajectory in Developmental Epilepsies

Abstract

Introduction

Over 150 single gene errors lead to seizure phenotypes, and many of these are early in onset, with life-long comorbid neurological phenotypes that carry a vast health care burden, including cognitive impairment, autism, depression, and premature mortality. Genetic and neurobiological evidence points to critical impairment of synaptic inhibition, the key regulator of dynamic balance of cortical network activity, as a shared hallmark of these neuronal synchronization disorders. Genes for pediatric epilepsy identified so far include those encoding transcription factors, signaling molecules, membrane proteins, and secreted proteins, and define a rich spectrum of biological defects in key programs of brain development, ranging from neuronal proliferation and migration to survival, connectivity and excitability. While interneurons are not alone in defining inhibition within a network, they are the target of at least one third of these genes [1].

The ability to perform repairs on the many roads leading to developmental disinhibition is entering an era when neural circuits can be stably modified by drugs that remodel gene expression, insertion of genes, and the transplantation of cells, all justifiable strategies when other effective therapies are not available. Early modification may provide the greatest opportunity to harness the developmental plasticity of neural circuits. The current challenge is to identify in each case the deleterious events and critical periods to intervene before irreversible network defects appear (Figure 1). Once these obstacles are surmounted, the era of clinical management of gene expression in these cortical excitability disorders will have arrived.

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Figure 1

Summary of developmental pathways for early interventions in the developmental epilepsies. Targeted therapies (e.g. hormones, trophic factors, neuromodulatory drugs, genetic therapy, or cell replacement) can modify the trajectory of disease and prevent epileptogenesis by permanently balancing excitatory and inhibitory network signaling. Cellular processes can be therapeutically targeted in developmental epilepsies. These processes include, but are not limited to, neuronal migration, developmental cell death, neuronal differentiation, and synaptogenesis. Modulating these events during a therapeutic window to rescue or repair damaged cellular processes, particularly interneuron defects, can modify the disease trajectory of developmental epilepsies.

Here we review recent studies that attempt to identify critical periods in brain development and how targeting mutant interneurons may alter the trajectory of disease. Since there are nearly one hundred different types of interneurons with their own connectivity, firing properties, and maturational profiles [2,3], an optimistic view amidst the expanding biological complexity is that many will respond to common proliferative, motogenic, and synaptogenic signals, and that therapies appropriately timed to alter their circuit dynamics during this developmental window may be shared to some degree across genes and phenotypes.

Estradiol rescue of Arx mutations in catastrophic epilepsies

Mutations in Aristaless-Related homeobox (ARX) (OMIM 300382) provide a prototypical example of how primary intrinsic interneuron abnormalities lead to a complex developmental epilepsy, X-linked Infantile Spasms Syndrome (ISSX). Defects in this X-linked transcription factor cause a pleiotropic array of neurodevelopmental disorders including infantile spasms, epilepsy, intellectual disability, and autism spectrum disorder [4,5]. Arx encodes a multidomain transcription factor expressed solely in developing and mature interneurons. Individuals with ARX mutations and orthologous mouse models exhibit losses of cortical interneuron subtypes due to de-repression of Arx- dependent growth signals [6–8]. Several allelic mouse models have been developed and display neurological phenotypes such as epilepsy, cognitive and behavioral phenotypes [9,10].

ARX^(GCG)10+7 (c.333_334ins(GCG)₇), a trinucleotide repeat expansion mutation in the first poly-alanine tract domain of ARX, is a common cause of ISSX [11]. Two models of this expansion mutation have been developed, Arx^(GCG)7 [12] and Arx^(GCG)10+7 [13]. In both models, ARX expression is reduced with selective loss of GABAergic and cholinergic interneuron immunoreactivity in the neocortex and striatum [12–14]. These expansion mutations result in loss of ARX binding to the transcriptional co-repressor, Groucho/transducin-like enhancer of Split (Tle1) [15], and lower transcription of the lysine-specific demethylase 5C (KDM5C), a histone demethylase [16]. This suggests that ARX poly-alanine expansion mutations disrupt the transcriptional regulation capacity of ARX and perturb histone methylation patterns in cortical interneurons, contributing to aberrant interneuronal phenotypes seen in patients [17].

ISSX has been a challenging disorder to treat as patients, like those with idiopathic infantile spasms, are poorly responsive to adrenocorticotropic hormone (ACTH) and antiepileptic drugs [18]. In search of alternative therapy, the effects of estrogen, a neuroprotective hormone, were explored. Daily postnatal treatment of Arx^(GCG)10+7 mutant mice for one week with the neurosteroid 17β-estradiol (E2), restored the decreased forebrain interneuron numbers, reduced neonatal spasms, and prevented seizures in adults [19]. E2 treatment was ineffective when administered to adults, indicating a critical, neonatal window in which E2 corrects pathological processes and modifies disease (Figure 2) [19]. E2 acts on ERα and ERβ receptors located on the plasma membrane or in the cytoplasm, which then activate downstream pathways via transcriptional regulation or non-genomic mediated signaling [20]. ERs are expressed in developing interneurons, and stimulation of these complex pathways may increase migration and survival of these cells [21,22]. It is unclear what factors govern the critical neonatal treatment window, but this may be determined by cell-autonomous events of interneurons and mediated by ERs which display a developmentally-regulated expression pattern in the neonatal rodent cortex [23,24]. E2 and related estrogens show neural and glial protective effects in early CNS injury models [20], and activation of ERs may stimulate transcriptional rescue of Arx mutant interneurons either through recovery of Arx- deficient gene expression or other pathways. While E2 has not yet been used clinically in the treatment of ISSX, a clinical trial can be planned once safety concerns have been fully addressed. Future work is needed to understand the mechanism of E2 in the Arx^(GCG)10+7 model and its benefits in other genetic models of epilepsy with similar phenotypes.

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Figure 2

Neonatal 17β-Estradiol (E2) prevents epilepsy and restores GABAergic interneurons in the Arx^(GCG)10+7 model of X-linked Infantile Spasms. (A) Estradiol treatment paradigms used in [19]. Neonatal Arx^(GCG)10+7 mice were treated with E2 from postnatal day (P)3 to 10 (Early treatment) and adult mice were treated from P33 to P40 (Late treatment). (B) Only early E2 treatment effectively prevented seizures in a majority (9 out of 10) of Arx^(GCG)10+7 mutant (Mut) mice as compared to vehicle (Veh) treated controls. Late treatment had no effect on seizure outcome. (C) Early treatment also rescued numbers of cortical GABAergic interneuron subtypes. For example, numbers of Neuropeptide Y (NPY)+ interneurons were rescued in Arx^(GCG)10+7 mutants with E2 treatment (E2 Mut) as compared to controls (Veh Mut).

Calpain inhibitor rescue of Lis1 neuronal migration disorders

Abnormalities in neuronal migration perturb cortical layering patterns, disrupt the neural networks that rely on this cytoarchitecture, and may be tractable to reversal strategies [25]. Gene-linked migration disorders include lissencephaly, subcortical band heterotopia/double cortex syndrome, and focal cortical dysplasias [25,26]. Deletions in the LIS1 gene (OMIM 607432) and a microdeletion that encompasses LIS1 (Miller-Dieker Syndrome) are causes of lissencephaly and often have severe, intractable epilepsy appearing early in life [27]. Lis1 is essential for regulation and localization of cytoplasmic dynein [28], and knockout mouse models have revealed that cortical interneurons deficient in Lis1 exhibit defective migration due to unstable leading neuritic branch processes [29–31]. Lis1 +/− knockout mice display spontaneous seizures [32], cognitive deficits [33], and motor deficits [34]. Based on the finding that Lis1 protein is degraded by calpain proteases, Yamada and colleagues tested the effects of calpain protease inhibitors in normalizing Lis1 expression and rescuing aberrant phenotypes in Lis1 heterozygous knockout mice [34]. Injection of calpain protease inhibitors and shRNA knockdown of calpain normalized Lis1 protein expression in Lis1^+/− brains, rescued migration of cultured Lis1^+/− cerebellar granule neurons, and partially rescued cortical lamination defects in the Lis1^+/− neocortex [34]. In utero treatment with calpain inhibitors also rescued motor deficits in Lis1^+/− mice, however, it has not yet been determined if calpain inhibitors have any effect on cortical interneuron migration, seizures or cognitive phenotypes in the Lis1^+/− mice. Since calpain inhibitors were administered in utero, it is also unknown whether postnatal administration of calpain inhibitors in Lis1 knockout mice can still increase Lis1 expression, stimulate migration, and improve the neurological deficits. Even once the wave of normal interneuron migration is largely complete, data from transplanted newborn wild type interneurons show they can migrate short distances in a mature neocortex [35], and it is possible that calpain inhibitors may improve migration and synaptic integration of Lis1 mutant interneurons in cortical circuits regardless of anomalous laminar patterns.

Transgenic rescue of Doublecortin mutations

Doublecortin (DCX) (OMIM 300121) is located on the X-chromosome and DCX missense mutations are a common cause of X-linked lissencephaly and subcortical band heterotopia, with a high incidence of epilepsy [26,36]. DCX encodes a microtubule-associated protein expressed in migrating neurons and involved in microtubule stabilization [37]. Neuropathological case studies reveal that human DCX mutations result in loss of cortical GABAergic interneurons [17]. Studies in mouse and rat models have shown that Dcx is important for cortical interneuron migration and reduction of Dcx expression results in loss of interneuron subtypes in the neocortex and hippocampus [38,39]. Interestingly, while genetic deletion of Dcx in mouse fails to produce the same pattern of neocortical heterotopia, the hippocampal cortex is dyslaminated, and Dcx knockout mice still exhibit a seizure phenotype [39]. In rat however, knockdown of Dcx in the embryonic brain by shRNA led to aberrant lamination and morphology of cortical neurons [40]. Manent and colleagues demonstrated that postnatal re-expression of Dcx following embryonic Dcx knockdown rescued cortical lamination defects and the efficiency of rescue was developmentally dependent, since early rescue of Dcx expression was more effective in restoring cortical lamination than late postnatal rescue [41]. Knockdown of Dcx in the rat model has not been shown to cause spontaneous seizures, however postnatal re-expression of Dcx rescued the lowered pentylenetetrazol seizure threshold in Dcx knockdown rats [41]. This suggests that aberrant excitability phenotypes due to hypomorphic DCX mutations may be improved by increasing postnatal DCX expression despite the early migration defect during embryonic development.

mTOR inhibition in the “mTORopathies”

Hyperactivation of the mechanistic target of rapamycin (mTOR) signaling complex has been observed in several monogenic developmental epilepsies, and may represent a hub for early rescue. mTOR complexes control multiple cellular processes including autophagy, protein synthesis, actin dynamics, and mitochondrial function [42]. Somatic or inherited mutations in TSC1 (OMIM 605284), TSC2 (OMIM 613254), PTEN (OMIM 601728), STRADα(OMIM 608626), and MTOR (OMIM 601231) cause developmental epilepsies, brain malformation syndromes, and affect cellular development of glia and excitatory and inhibitory neurons in the developing brain [42–44]. TSC1 and TSC2 are potent negative regulators of mTOR, and mutations in these genes result in focal epilepsy and infantile spasms in up to 75 percent of cases [45,46]. Mouse models have defined several disinhibitory mechanisms by which dysfunction of Tsc1 and augmented mTOR signaling generate seizures [42,44]. In particular, conditional deletion of Tsc1 in interneuron progenitors increased mTOR signaling in GABAergic interneurons, leading to loss of interneuron subtypes, lower convulsant-induced seizure threshold, and early mortality [47].

Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, encodes another upstream negative regulator of mTOR. Loss of function mutations in PTEN are associated with brain malformation disorders and lead to a number of morphologic and functional abnormalities in the brain including dysmorphic neurons, macrocephaly, and seizures [42]. GABAergic interneuron subtypes are also affected by Pten knockout. Conditional deletion of Pten from GABAergic interneuron progenitors disrupted the ratios of two cortical interneuron subtypes, parvalbumin+ and somatostatin+ interneurons, and Pten expression was also crucial for cortical interneuron morphology [48]. Transplant of Pten-deficient cortical interneuron progenitors into fetal WT brain did not rescue their mutant cellular phenotypes, suggesting that Pten function during interneuron development is cell-autonomous [48].

Since pathophysiological mTOR upregulation is common amongst these monogenic disorders, it defines a broader therapeutic target, and recent data indicate that inhibitors of mTOR modify the course of disease in both animal models and in human clinical studies. Rapamycin and clinical analogs reduce seizures and improve other associated phenotypes in models of Tsc1, Tsc2, and Pten [42,44]. Rapamycin has been shown to not only reduce seizures once they have become manifest, but also to prevent epileptogenesis in these models [49–51]. Human studies with rapamycin analogs found these mTOR inhibitors reduced seizures and other clinical manifestations of tuberous sclerosis complex due to TSC1/TSC2 mutations [45,52,53], and polyhydramnios, megalencephaly, and symptomatic epilepsy (PMSE) caused by STRADα mutations[54]. Despite their efficacy in seizure reduction, few studies have examined the effects of these inhibitors in Pten or TSC1/TSC2-associated infantile spasms. Vigabatrin, a GABA transaminase inhibitor that elevates GABA levels, has been shown to be effective for infantile epilepsies and has become a first-choice drug for treatment of TSC1/TSC2-associated infantile spasms [45,46]. The opportunity to diagnose these mutations prenatally in humans augurs a clinical trial of early pre-seizure intervention in this disorder.

Tau ablation rescue of ion channelopathy

Mutation of ion channel subunit genes comprises the largest fraction of known epilepsy mutations, and human and experimental evidence indicates that their phenotype is particularly susceptible to epistatic masking, even by subunits among the 400 member voltage-gated channel gene family [55,56]. Recent studies have defined MAPT (Tau), a microtubule stabilizing protein highly expressed in axons, as a particularly strong gene modifier of epilepsy arising from potassium (Kcna1/Kv1.1) [57] and sodium (Scn1a/NaV1.1) [58] channel mutants. Deletion of Tau rescued neural hyperexcitability, megencephaly, and early lethality in Kcna1 knockout mice [57]. Recent work has also shown that loss of Tau in Kcna1 knockout mice corrected the lower threshold for spreading depolarization in the brainstem, a mechanism thought to contribute to sudden, unexpected death in epilepsy (SUDEP) [59]. Despite being a potent phenotypic modifier, little is known about the mechanism of Tau loss in reducing hyperexcitability in models of ion channelopathy, and whether there is a critical developmental window for its therapeutic action.

Conclusion

New monogenic causes of epilepsy found in the clinical exome continue to emerge, and we are only at the beginning of exploring early reversibility in these gene disorders. Interneurons are an important common starting point, but certainly not the sole target to prevent the development of epilepsy. What is critical to realize is that even when gene defects are cell-type specific, both selective and non-selective treatment strategies can be developed, taking advantage of plasticity at this early stage by altering the cellular milieu. A second important take home message is that seizures may be separable from structural circuit abnormalities. For example, some genes produce striking brain malformations such as cortical dyslamination in the double cortex syndrome (DCX), or prominent balloon cells and tubers of tuberous sclerosis (TSC1), however, mouse models show that these structural defects are not essential for epilepsy to occur [26,42]. Thus partial phenotypic rescue, that is preventing seizures, may be possible even in the face of major brain malformations. These studies serve as optimistic reminders that just as many genes lead to epilepsy, there may be multiple ways of therapeutically masking the most deleterious effects and improving the outcomes of patients with developmental epilepsies.

Acknowledgments

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