# EPILEPSY AND NEURODEVELOPMENTAL DISEASES

EDITED BY : Eleonora Palma, Eleonora Aronica and Erwin van Vliet PUBLISHED IN : Frontiers in Cellular Neuroscience

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ISSN 1664-8714 ISBN 978-2-88966-165-7 DOI 10.3389/978-2-88966-165-7

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# EPILEPSY AND NEURODEVELOPMENTAL DISEASES

Topic Editors:

Eleonora Palma, Sapienza University of Rome, Italy Eleonora Aronica, Amsterdam University Medical Center, Netherlands Erwin van Vliet, University of Amsterdam, Netherlands

This topic has been realized in collaboration with Dr. Gabriele Ruffolo, Post Doctoral Researcher at the University of Rome (Sapienza) (ORCID ID: 0000-0002-6554-5496).

Citation: Palma, E., Aronica, E., van Vliet, E., eds. (2020). Epilepsy and Neurodevelopmental Diseases. Lausanne: Frontiers Media SA. doi: 10.3389/978-2-88966-165-7

# Table of Contents

### *04 Editorial: Epilepsy and Neurodevelopmental Diseases* Gabriele Ruffolo, Erwin A. Van Vliet, Eleonora Aronica and Eleonora Palma

*06 Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in*  ATP1A3

August A. Allocco, Sheng Chih Jin, Phan Q. Duy, Charuta G. Furey, Xue Zeng, Weilai Dong, Carol Nelson-Williams, Jason K. Karimy, Tyrone DeSpenza, Le T. Hao, Benjamin Reeves, Shozeb Haider, Murat Gunel, Richard P. Lifton and Kristopher T. Kahle

*14 Identification of KCC2 Mutations in Human Epilepsy Suggests Strategies for Therapeutic Transporter Modulation*

Phan Q. Duy, Wyatt B. David and Kristopher T. Kahle

*20 Functional Genomics of Epilepsy and Associated Neurodevelopmental Disorders Using Simple Animal Models: From Genes, Molecules to Brain Networks*

Richard Rosch, Dominic R. W. Burrows, Laura B. Jones, Colin H. Peters, Peter Ruben and Éric Samarut

*29 Transcriptional Regulation of Channelopathies in Genetic and Acquired Epilepsies*

Karen M. J. van Loo and Albert J. Becker

Anna Fassio, Antonio Falace, Alessandro Esposito, Davide Aprile, Renzo Guerrini and Fabio Benfenati

Simon Levinson, Conny H. Tran, Joshua Barry, Brett Viker, Michael S. Levine, Harry V. Vinters, Gary W. Mathern and Carlos Cepeda

### *100 Multimodal Analysis of STRADA Function in Brain Development* Louis T. Dang, Katarzyna M. Glanowska, Philip H. Iffland II, Allan E. Barnes, Marianna Baybis, Yu Liu, Gustavo Patino, Shivanshi Vaid, Alexandra M. Streicher, Whitney E. Parker, Seonhee Kim, Uk Yeol Moon, Frederick E. Henry, Geoffrey G. Murphy, Michael Sutton, Jack M. Parent and Peter B. Crino

# Editorial: Epilepsy and Neurodevelopmental Diseases

Gabriele Ruffolo1,2, Erwin A. Van Vliet 3,4, Eleonora Aronica4,5 and Eleonora Palma1 \*

*1 Department of Physiology and Pharmacology, Istituto Pasteur-Fondazione Cenci Bolognetti, University of Rome Sapienza, Rome, Italy, 2 Istituto di Ricovero e Cura a Carattere Scientifico San Raffaele, Cassino, Italy, 3 Center for Neuroscience, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, Netherlands, 4 Department of (Neuro)Pathology, Amsterdam UMC, University of Amsterdam, Amsterdam, Netherlands, 5 Stichting Epilepsie Instellingen Nederland, Heemstede, Netherlands*

Keywords: GABA, ion channels, brain immaturity, synaptic transmission, neurodevelopment, epilepsy

### **Editorial on the Research Topic**

### **Epilepsy and Neurodevelopmental Diseases**

The association between epilepsy and neurodevelopmental diseases is well-recognized and has gained significant attention in the field of neuroscience in recent years. One of the main reasons for this interest is the need for a better understanding of the events that lead to the development and maturation of the CNS. This is a fundamental and necessary basis for potential breakthrough strategies that could guide novel and more effective disease-modifying therapeutic approaches to neurodevelopmental syndromes that are frequently characterized by severe and drug-resistant epilepsy.

The perspective of such new therapeutic strategies is very promising. At the state-of-theart, patients afflicted by these rare neurodevelopmental disorders mostly rely on "symptomatic" approaches that mitigate seizures and other major symptoms but do not target the underlying biological causes of the disease.

The study of this vast field of research is extremely complex and requires a multidisciplinary approach, from neuropathological to molecular and functional studies since even "simple" triggering events (e.g., a genetic mutation) during critical periods of brain development can lead to widespread effects on brain morphological and functional features.

This issue represents the concept of an integrated, interdisciplinary translational approach to epileptic syndromes of neurodevelopment. For instance, Rosch et al. thoroughly describe invertebrate animal models such as zebrafish and drosophila which can be used as tools to investigate the early stages of neurodevelopment. Even though these "simple" models are far from the complexity of the human brain, they offer superior manageability to mammalian models, allowing researchers to perform single-cell resolution studies on whole-brain imaging, thus tracking the effects of a pathogenic event from the "larval" stages to the mature brain.

Since the mechanisms by which an insult determines pathological events during neurodevelopment are not always univocal, they can be explored from different angles, using different methods and multidisciplinary perspectives in research design. For example, considering their pivotal role in neurotransmission, it is not surprising that many researchers have made efforts to characterize ion channels. Here, two original research articles by Levinson et al. and by Mao et al. followed this path.

The first by Levinson et al. supports the role of metabotropic GABAB receptors (GABABRs) in pediatric focal cortical dysplasia (FCD) and tuberous sclerosis complex (TSC) through an electrophysiological study of patients' brain slices. This research strengthened the hypothesis that there is significant participation of GABABRs in synaptic inhibition in the aforementioned pathologies, highlighting the importance of studying possible therapeutic targets in a "comparative"

### Edited and reviewed by:

*Dirk M. Hermann, University of Duisburg-Essen, Germany*

\*Correspondence: *Eleonora Palma<EMAIL_ADDRESS>
### Specialty section:

*This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience*

Received: *10 July 2020* Accepted: *24 July 2020* Published: *25 September 2020*

### Citation:

*Ruffolo G, Van Vliet EA, Aronica E and Palma E (2020) Editorial: Epilepsy and Neurodevelopmental Diseases. Front. Cell. Neurosci. 14:255. doi: 10.3389/fncel.2020.00255*

**4**

fashion, since the permissive role of GABABRs was observed in FCD and TSC, but not in non-dysplastic tissues.

The second study by Mao et al. reported two novel mutations in the KCTN2 gene, encoding for KNa1.2 subunit of the sodiumdependent voltage gated potassium channel KNa. This protein frequently mutates in Epilepsy of Infancy with Migrating Focal Seizures (EIMFS) and this study deepened the knowledge of disease genetics and physiology, thus facilitating future studies of the mutated channel in cellular models.

The review by van Loo and Becker describes acquired and genetic channelopathies from an original point of view. This review makes clear that the direct malfunction of a channel protein is not the only problem, as many events precede the incorporation of the channel in the cellular membrane, such as regulation of mRNA transcription, micro-RNA interference, and epigenetic mechanisms such as DNA methylation.

In addition, another review by Fattorini et al. tackles the other relevant mechanisms that can modulate neurotransmission: neurotransmitter reuptake. The authors have reviewed recent findings on the localization of GAT-1 to underline the widespread distribution of the transporter in the CNS as an alternative to traditional ideas about its neuron-specific expression. This is an important contribution to a field that is continuously evolving.

The research article by Duy et al. proposed an interesting approach, aiming to restore an optimal function of KCC2 to enhance neuronal chloride extrusion, making neurons less prone to generate epileptic discharges. Such research also raises the question of whether similar strategies may prevent epileptogenesis and cognitive impairment when applied early during development.

The pathologic traits in the cytoarchitecture of the diseased brain are also explored in this issue. Liu et al. performed a proteomics study that described how the expression of Rho GTPases in hippocampal dentate granule cells may support the abnormal migratory activity observed in mesial temporal lobe epilepsy, determining granule cell dispersion. These dysmaturative processes reproduce events, characterizing early developmental stages, with detrimental consequences. These findings clearly show that the dysmaturation is not confined to the early stages of development but it is also a feature of adult mesial temporal lobe epilepsy. Liu et al. explored the impact of an early developmental dysregulation of the mechanistic target of rapamycin (mTOR) pathway, as a result of mutations of STRADA (pseudokinase STE20-related kinase adaptor alpha, an mTOR regulator), leading to a continuous overactivation of mTOR signaling. In particular, they studied the malfunction of STRADA with complementary techniques such as CRISPRedited Strada mouse N2a cells and induced pluripotent stem cells (iPSCs). This study highlighted the novel contribution of this protein to pathologic hallmarks of mTORopathies such as increased cell size, neuronal hyperexcitability, and impaired cortical lamination.

The list of promising targets that require further investigation is much longer, as indicated in papers by Allocco et al. and Fassio et al.. The first research article defined a role for the impairment of Na+/K+ ATPase function in the pathogenesis of congenital hydrocephalus trough whole-exome sequencing, bioinformatics, and computational modeling. Indeed, these authors, by characterizing novel mutations of α3 subunit of Na+/K+ ATPase, have described, for the first time, a link between this protein and congenital hydrocephalus.

In their review, Fassio et al. highlighted the role of autophagy in neurodevelopment and epilepsy as a key process involved in neurogenesis, neuronal polarity, and synaptic function. Notably, these authors shed new light on this physiological mechanism, suggesting that defective autophagy may represent an additional therapeutic target for epileptic neurodevelopmental diseases.

In conclusion, this Research Topic combines different fields of neuroscience that analyze the correlation between epilepsy and neurodevelopment from different points of view. Therefore, as well as making significant contributions to research, this topic highlights the need for an integrated approach—from anatomy and pathology to molecular biology and electrophysiology—to speed-up the progress in this research field.

# AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

**Conflict of Interest:** The authors declare the absence of any commercial or financial relationship that could be construed as a potential conflict of interest

Copyright © 2020 Ruffolo, Van Vliet, Aronica and Palma. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in ATP1A3

August A. Allocco1† , Sheng Chih Jin2,3† , Phan Q. Duy1† , Charuta G. Furey1 , Xue Zeng2,3 , Weilai Dong2 , Carol Nelson-Williams2 , Jason K. Karimy1 , Tyrone DeSpenza1 , Le T. Hao1 , Benjamin Reeves1 , Shozeb Haider4 , Murat Gunel1,2, Richard P. Lifton2,3 and Kristopher T. Kahle1,5,6,7 \*

### Edited by:

Eleonora Palma, Sapienza University of Rome, Italy

### Reviewed by:

Antonio Gambardella, University of Catanzaro, Italy Ozgun Gokce, Ludwig Maximilian University of Munich, Germany

### \*Correspondence:

Kristopher T. Kahle<EMAIL_ADDRESS>
†These authors have contributed equally to this work

### Specialty section:

This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience

Received: 24 June 2019 Accepted: 04 September 2019 Published: 26 September 2019

### Citation:

Allocco AA, Jin SC, Duy PQ, Furey CG, Zeng X, Dong W, Nelson-Williams C, Karimy JK, DeSpenza T, Hao LT, Reeves B, Haider S, Gunel M, Lifton RP and Kahle KT (2019) Recessive Inheritance of Congenital Hydrocephalus With Other Structural Brain Abnormalities Caused by Compound Heterozygous Mutations in ATP1A3. Front. Cell. Neurosci. 13:425. doi: 10.3389/fncel.2019.00425 1 Department of Neurosurgery, School of Medicine, Yale University, New Haven, CT, United States, 2 Department of Genetics, School of Medicine, Yale University, New Haven, CT, United States, 3 Laboratory of Human Genetics and Genomics, The Rockefeller University, New York, NY, United States, 4 Department of Computational Chemistry, University College London School of Pharmacy, London, United Kingdom, 5 Department of Cellular and Molecular Physiology, School of Medicine, Yale University, New Haven, CT, United States, 6 NIH-Yale Centers for Mendelian Genomics, School of Medicine, Yale University, New Haven, CT, United States, 7 Yale Stem Cell Center, School of Medicine, Yale University, New Haven, CT, United States

Background: ATP1A3 encodes the α3 subunit of the Na+/K+ ATPase, a fundamental ion-transporting enzyme. Primarily expressed in neurons, ATP1A3 is mutated in several autosomal dominant neurological diseases. To our knowledge, damaging recessive genotypes in ATP1A3 have never been associated with any human disease. Atp1a3 deficiency in zebrafish results in hydrocephalus; however, no known association exists between ATP1A3 and human congenital hydrocephalus (CH).

Methods: We utilized whole-exome sequencing (WES), bioinformatics, and computational modeling to identify and characterize novel ATP1A3 mutations in a patient with CH. We performed immunohistochemical studies using mouse embryonic brain tissues to characterize Atp1a3 expression during brain development.

Results: We identified two germline mutations in ATP1A3 (p. Arg19Cys and p.Arg463Cys), each of which was inherited from one of the patient's unaffected parents, in a single patient with severe obstructive CH due to aqueductal stenosis, along with open schizencephaly, type 1 Chiari malformation, and dysgenesis of the corpus callosum. Both mutations are predicted to be highly deleterious and impair protein stability. Immunohistochemical studies demonstrate robust Atp1a3 expression in neural stem cells (NSCs), differentiated neurons, and choroid plexus of the mouse embryonic brain.

Conclusion: These data provide the first evidence of a recessive human phenotype associated with mutations in ATP1A3, and implicate impaired Na+/K+ ATPase function in the pathogenesis of CH.

Keywords: congenital hydrocephalus, ATP1A3, Na+/K+ ATPase, whole exome sequencing, genetics

# INTRODUCTION

fncel-13-00425 September 24, 2019 Time: 17:47 # 2

Congenital hydrocephalus (CH) is the most common reason for brain surgery in children and affects 1 in 1,000 newborns (Tully and Dobyns, 2014; Kahle et al., 2016). CH is characterized by ventriculomegaly, defined as dilation of cerebral ventricles, and thought to be secondary to impaired cerebrospinal fluid (CSF) homeostasis. Consequently, CH is treated by lifelong neurosurgical shunting with high complication rates and morbidity. The lack of satisfactory treatments highlights our incomplete understanding of CH pathogenesis (Kahle et al., 2016). There is a need to identify CH disease-causing genes, given that 40% of CH cases is estimated to have a genetic etiology (Haverkamp et al., 1999). Despite significant efforts to identify CH genes, including a recent whole-exome sequencing (WES) (Furey et al., 2018) study, the majority of CH cases remain idiopathic, underscoring the need for continued gene discovery.

ATP1A3 encodes the α3 subunit of the Na+/K+ ATPase, a fundamental enzyme that regulates ion homeostasis by maintaining ionic gradients across the plasma membrane (Clausen et al., 2017). ATP1A3 is highly expressed in neurons of the adult rodent brain (McGrail et al., 1991; Pietrini et al., 1992; Bottger et al., 2011) and mutations in the gene have been implicated in three autosomal dominant Mendelian diseases, including alternating hemiplegia of childhood (AHC) type 2 (Rosewich et al., 2012), CAPOS syndrome (Demos et al., 2014), and Dystonia-12 (Anselm et al., 2009). Knockdown of Atp1a3 in zebrafish (Doganli et al., 2013) results in hydrocephalus; however, no known association exists between ATP1A3 and human CH.

Here, we present the first case of obstructive CH with aqueductal stenosis and other structural brain abnormalities associated with recessive compound heterozygous mutations in ATP1A3.

## MATERIALS AND METHODS

### Patient/Family Information

The patient is a 23-year-old Caucasian female of European descent. CH was diagnosed on prenatal ultrasound at 18 weeks gestation (**Figure 1A**). MR imaging captured immediately after delivery demonstrated marked asymmetric obstructive hydrocephalus secondary to aqueductal stenosis. The patient underwent ventriculoperitoneal shunt placement at birth with three subsequent surgical shunt revisions and was further diagnosed via MRI and computed tomography (**Figure 1B**) with craniosynostosis, open lip schizencephaly, type 1 Chiari malformation, dysgenesis of the corpus collosum, and learning disability. Routine genetic testing (FISH, microarray) was negative. The patient's mother reports two previous miscarriages. The mother reports a medical history of hyperthyroidism while the father reports a history of hypertension and anxiety. There are no known medical problems that run in the family on either the maternal or paternal side. The patient has one phenotypically normal sister. Institutional review board approval was obtained from the Yale University Human Investigative Committee, and all participants provided written informed consent.

# Exome Sequencing and Analysis

To identify potential disease-causing variants in this patient, we performed WES on the affected individual and her parents. Targeted capture was performed using the xGEN Exome Research Panel v1.0 (IDT) followed by DNA sequencing on the Illumina HiSeq 4000 System. Sequence metrics are shown in **Supplementary Table S1**. Sequence reads were mapped to the reference genome (GRCh37) with BWA-MEM and further processed using the GATK Best Practices workflows (McKenna et al., 2010; Van der Auwera et al., 2013; 1000 Genomes Project Consortium, 2015) as previously described (Jin et al., 2017). Single nucleotide variants and small indels were called with GATK HaplotypeCaller and annotated using ANNOVAR (Wang et al., 2010), ExAC (v3), and gnomAD (v2.1.1) (Lek et al., 2016). The MetaSVM algorithm was used to predict deleteriousness of missense variants (Dong et al., 2015).

We filtered recessive variants for rare [minor allele frequency (MAF) ≤ 10−3 across ExAC and gnomAD] homozygous and compound heterozygous variants that exhibited high quality sequence reads. Only loss-of-function (LoF; including nonsense, frameshift, or canonical splice disruptions), D-Mis (MetaSVMdeleterious), and non-frameshift indels were considered potentially damaging to the disease. For dominant variants, we assessed for rare (MAF ≤ 2 × 10−5 ) and damaging variants (LoF or D-Mis). Finally, false positive variants were excluded by in silico visualization followed by Sanger sequencing validation.

### In silico Modeling

The sequence of the human α3 subunit of the Na+/K+ ATPase (ATP1A3) was taken from the Uniprot (P13637). The crystal structures of the Pig Sodium/potassium-transporting ATPase subunit α(PDB id 4RET and 3WGV) exhibit 86% sequence identity with human ATP1A3 (residues 1-1013). The structure of 4RET and 3WGV were used as templates to construct a homology model of the human ATP1A3 using MODELLER (Fiser and Sali, 2003). Conserved Arginine residues are present at the equivalent positions of R19 and R463 in both human and pigs. The spatial orientations of the side chains in the templates from pig were used to model the side chains of R19 and R463. A total of twenty models were built and subjected to restrained energy minimization to relieve any steric clashes between the side chains and the nucleic acid. The stereo-chemical parameters were analyzed using PROCHECK and PROSA (Wiederstein and Sippl, 2007) and the final model was chosen based on the on the basis of the lowest Cα RMSD value after superimposition on the template structure (0.9 Å). This lies within the permitted range for accurate homology model construction for sequence identity in the high range (>80%) (Fiser, 2010).The mutants, R19C and R463C, were constructed and the free energy of change calculated (11G) in silico using the ICM mutagenesis program (Laskowski et al., 1993) 1 .

### Immunohistochemistry

WT mouse embryos of C57/BL6 background were harvested at embryonic day 15.5. Brains were dissected and fixed in 4%

1www.molsoft.com

PFA in PBS overnight at 4◦C then cryoprotected in 30% sucrose in PBS for 48 h. Brains were then mounted in frozen OCT blocks and sectioned by cryostat at 25 µm in the coronal plane. Sections were mounted on microscope slides and stored in −80◦C until use. To begin staining, slides containing brain sections were first thawed at room temperature, then washed in 0.5% PBST. Sections underwent antigen retrieval using citrate buffer, then washed in 0.5% PBST. Sections were blocked in 10% normal goat serum (NGS) in PBST at room temperature for 1 h, then incubated with primary antibodies diluted in 2.5% NGS in PBST at 4◦C overnight. The primary antibodies were rabbit polyclonal anti-Atp1a3 (1:500 dilution) (Pietrini et al., 1992), mouse monoclonal anti-NeuN (1:250, MAB377, Millipore), and mouse monoclonal anti-Sox2 directly conjugated with Dylight 550 (1:250, MA1-014-D550, Invitrogen). Following primary antibody incubation, sections were washed and incubated with Alexa Fluor-conjugated secondary antibodies (1:500 in 2.5% NGS in PBST) for 1 h at room temperature. Following the final wash, slides were coverslipped with Prolong Gold Antifade mounting medium. For negative control, the primary antibody solution (containing anti-Atp1a3) was blocked with the immunizing peptide sequence (gdkkddsspkksc) (Pietrini et al., 1992). Images were acquired using the Zeiss LSM 880 confocal microscope or the Aperio digital scanner microscope. All experiments were done in accordance with the regulations set forth by the Yale University animal care and use committee.

## RESULTS

Compound heterozygous mutations in ATP1A3 were identified in the affected individual. A maternally inherited single nucleotide G to A variation was identified at cDNA NM\_152296 position 55 in exon 2 (gnomAD MAF = 6.4 × 10−5 ), corresponding to the amino acid substitution p. Arg19Cys in the α3 subunit. A paternally inherited single nucleotide G to A variation was identified at cDNA position 1387 in exon 11 (gnomAD MAF = 4.5 × 10−4 ), corresponding to the amino acid substitution p.Arg463Cys in the α3 subunit (**Figures 1C,D**). ATP1A3 is very intolerant to both loss-of-function mutations (pLI = 1) and missense variants (mis\_z = 6.33) per gnomAD. No homozygous loss-of-function mutations or homozygous damaging missense mutations in ATP1A3 have been reported in gnomAD. Both variants are predicted deleterious per MetaSVM. CADD scores for the p.Arg19Cys and p.Arg463Cys variants were 34 and 24.8, respectively. Conservation of positively charged amino acid residues (arginine and lysine) was observed across species (**Figure 1F**).

In silico modeling of p.Arg19Cys and p.Arg463Cys mutations shows disruptive effects on protein stability. The side chain of arg-19 forms interactions with the backbone atoms of Ser-207 and Leu-267. This interaction is lost when Arg-19 is mutated to a cysteine with a corresponding energy penalty (11G = 2.3 kcal/mole, **Figures 1E,F**). Similarly, the side chain of Arg-463 makes strong hydrogen bond interactions with the side chain of Glu-486 and His-413. These strong interactions are lost when Arg-463 is mutated to a Cys (11G = 3.2 kcal/mole, **Figures 1E,F**).

The association of ATP1A3 mutations with CH and multiple other significant structural brain abnormalities implicate ATP1A3 in human brain development. Thus, we performed immunofluorescence studies in the embryonic mouse brain to characterize Atp1a3 expression. We stained histological brain sections collected from embryonic day 15.5 (E15.5) with an antibody against Atp1a3 (Pietrini et al., 1992) together with Sox2 [a marker of neural stem cells (NSCs)] (Ellis et al., 2004) and NeuN (a marker of differentiated neurons) (Mullen et al., 1992). Overall, Atp1a3 exhibits diffused and cytoplasmic expression throughout all cortical layers of the embryonic mouse brain (**Figure 2**). Co-localization studies showed that Atp1a3 is expressed in differentiated neurons at the cortical plate (CP) and in the NSCs at the ventricular zone (VZ) lining the lateral ventricles (**Figures 2E–N**). We also detected Atp1a3 expression in choroid plexus epithelial cells (**Figures 2O,P**). Furthermore, brain sections depicting the VZ and choroid plexus incubated with the Atp1a3 antibody blocked with the immunizing peptide showed minimal immuno-reactivity (**Figures 2Q,R**), demonstrating antibody specificity.

### DISCUSSION

Atp1a3 expression is exclusive to neurons and highly expressed in inhibitory interneurons (Richards et al., 2007), where it plays a crucial role in electrophysiological functions including ion gradient maintenance (Arystarkhova et al., 2019), after hyperpolarization (Picton et al., 2017), and suppression of burst firing (Vaillend et al., 2002). Mice harboring loss of function mutations in ATP1A3 exhibit increased seizure activity and neuronal hyperexcitability (Clapcote et al., 2009; Hunanyan et al., 2015) suggesting that ATP1A3 is crucial to maintaining normal neurological function. Human mutations in ATP1A3 have previously been discovered in a wide range of autosomal dominant neurological disorders (Heinzen et al., 2014), including encephalopathy with cerebellar ataxia (Sabouraud et al., 2019), AHC (Galaz-Montoya et al., 2019), rapid-onset dystonia Parkinsonism (ADP) (Anselm et al., 2009), cerebellar ataxia (Sabouraud et al., 2019), early-onset epilepsy (Ishihara et al., 2019), and autism spectrum disorder (Torres et al., 2018). Importantly, the majority of the mutations identified in AHC and RDP cluster in exons 8, 14, 17, and 18 (Rosewich et al., 2014), which are distinct from our variants (exon 2 and 11). To our knowledge, damaging recessive genotypes in ATP1A3 have never been associated with any human disease.

We identified two germline mutations in ATP1A3 (p. Arg19Cys and p.Arg463Cys), each of which was inherited from one of the patient's unaffected parents, in a single patient with severe obstructive CH due to aqueductal stenosis, along with open schizencephaly, type 1 Chiari malformation, and dysgenesis of the corpus callosum. Both mutations are predicted to be highly deleterious and impair protein stability. Consistent with our results, morpholino knockdown of Atp1a3 causes ventriculomegaly in zebrafish (Doganli et al., 2013),

SVZ – subventricular zone, VZ – ventricular zone. Dotted boxes in panel (E) shows the cortical plate and ventricular zone. (I–K) High magnification expression of Atp1a3 in NeuN+ cells in the CP. (L–N) Expression of Atp1a3 in Sox2+ cells in the ventricular zone. (O,P) High-magnification expression of Atp1a3 in the choroid plexus. (Q) Brain sections incubated with Atp1a3 primary antibody alone or (R) Atp1a3 primary antibody blocked with immunizing peptide.

recapitulating the hydrocephalus phenotype in our patient. Together, these data provide evidence for a recessive inheritance of CH with aqueductal stenosis caused by ATP1A3 compound heterozygous mutations.

Extending from previous work demonstrating expression of Atp1a3 in mature neurons (McGrail et al., 1991; Pietrini et al., 1992; Bottger et al., 2011), we found Atp1a3 to also be expressed in NSCs, differentiated neurons, and choroid plexus epithelial cells of the mouse embryonic brain. Our findings suggest two separate but not necessarily mutually exclusive mechanisms whereby ATP1A3 compound heterozygous mutations may cause ventriculomegaly. First, hydrocephalus is

classically thought to be a disorder of failed CSF homeostasis secondary to obstructed flow, increased secretion, or decreased absorption by the arachnoid villi (Kahle et al., 2016). The ion pump Na+/K+-ATPase is known to regulate CSF secretion in the choroid by maintaining an osmotic gradient of Na+ that drives the movement of water into the cerebral ventricles (Masuzawa et al., 1984; Fisone et al., 1995; Speake et al., 2001). The Na+/K+- ATPase is composed of three subunits: α, β, and γ (Holm et al., 2016). The catalytic activity of Na+/K+-ATPase has been attributed to the α subunit, which binds ATP, Na+, and K+. Thus, α subunit dysfunction due to p. Arg19Cys and p.Arg463Cys mutations described in this study can impair CSF homeostasis and thus drive the development of hydrocephalus.

Second, our finding of Atp1a3 expression in embryonic NSCs suggests a novel role of ATP1A3 in regulating neural development. Thus, mutations that disrupt ATPA13 function may impair NSC regulation and suggest dysregulation of neural development, rather than failed CSF homeostasis, to be the primary pathogenic driver of human CH. Indeed, multiple lines of evidence from animals (Huang et al., 2007; Lessard et al., 2007; Carter et al., 2012; Roy et al., 2019) and humans (Guerra et al., 2015; Rodriguez and Guerra, 2017; Furey et al., 2018) suggest abnormal NSC development to be a primary driver of CH pathogenesis. This potential impact on the general process of neural development may not only underpin ventriculomegaly but also other developmental anomalies (type 1 Chiari malformation, corpus callosum dysgenesis, and intellectual disability) observed in our patient. In addition, the notable lack of dystonic, epileptic, or motor deficits in this patient suggests that the spectrum of ATP1A3 linked diseases may be broader than previously acknowledged.

In sum, our findings provide the first association of human CH with recessive mutations in ATP1A3, setting the stage for future studies to better understand the role of ATP1A3 in brain development and the pathogenesis of human CH.

### DATA AVAILABILITY STATEMENT

The datasets generated for this study can be found in the dbGAP: phs000744.

### REFERENCES

### ETHICS STATEMENT

The studies involving human participants were reviewed and approved by Yale University Human Investigative Committee. The patients/participants provided their written informed consent to participate in this study.

### AUTHOR CONTRIBUTIONS

AA designed the study, collected and analyzed the data, and wrote the manuscript. SJ and PD collected and analyzed the data, and wrote the manuscript. CF, CW, JK, TD, LH, and BR collected the data. XZ and WD collected and analyzed the data. SH analyzed the data. MG designed the study. RL designed the study and analyzed the data. KK designed the study, analyzed the data, and wrote the manuscript.

# FUNDING

SJ was supported by the James Hudson Brown-Alexander Brown Coxe Postdoctoral Fellowship, an American Heart Association Postdoctoral Fellowship, and the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number K99HL143036. PD was supported by the NIH Medical Scientist Training Program Grant T32GM007205. KK was supported by the NIH 1RO1NS109358-01, the Hydrocephalus Association, and the Rudi Schulte Research Institute.

### ACKNOWLEDGMENTS

The authors would like to thank the family who participated in this study.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2019.00425/full#supplementary-material

(RDP) in the adult mouse brain. J. Comp. Neurol. 519, 376–404. doi: 10.1002/ cne.22524

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Allocco, Jin, Duy, Furey, Zeng, Dong, Nelson-Williams, Karimy, DeSpenza, Hao, Reeves, Haider, Gunel, Lifton and Kahle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Identification of KCC2 Mutations in Human Epilepsy Suggests Strategies for Therapeutic Transporter Modulation

Phan Q. Duy 1,2 , Wyatt B. David1 and Kristopher T. Kahle1,3,4,5,6 \*

1Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, United States, 2Medical Scientist Training Program, Yale University School of Medicine, New Haven, CT, United States, 3Department of Genetics, Yale University School of Medicine, New Haven, CT, United States, 4Departments of Pediatrics and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, CT, United States, 5Yale-Rockefeller NIH Centers for Mendelian Genomics, Yale University, New Haven, CT, United States, 6Yale Stem Cell Center, Yale School of Medicine, New Haven, CT, United States

Epilepsy is a common neurological disorder characterized by recurrent and unprovoked seizures thought to arise from impaired balance between neuronal excitation and inhibition. Our understanding of the neurophysiological mechanisms that render the brain epileptogenic remains incomplete, reflected by the lack of satisfactory treatments that can effectively prevent epileptic seizures without significant drug-related adverse effects. Type 2 K+-Cl− cotransporter (KCC2), encoded by SLC12A5, is important for chloride homeostasis and neuronal excitability. KCC2 dysfunction attenuates Cl− extrusion and impairs GABAergic inhibition, and can lead to neuronal hyperexcitability. Converging lines of evidence from human genetics have secured the link between KCC2 dysfunction and the development of epilepsy. Here, we review KCC2 mutations in human epilepsy and discuss potential therapeutic strategies based on the functional impact of these mutations. We suggest that a strategy of augmenting KCC2 activity by antagonizing its critical inhibitory phosphorylation sites may be a particularly efficacious method of facilitating Cl− extrusion and restoring GABA inhibition to treat medication-refractory epilepsy and other seizure disorders.

### Edited by:

Eleonora Aronica, University Medical Center Amsterdam, Netherlands

### Reviewed by:

Francesco Ferrini, University of Turin, Italy Quentin Chevy, Cold Spring Harbor Laboratory, United States

### \*Correspondence:

Kristopher T. Kahle<EMAIL_ADDRESS>
Received: 12 July 2019 Accepted: 01 November 2019 Published: 15 November 2019

### Citation:

Duy PQ, David WB and Kahle KT (2019) Identification of KCC2 Mutations in Human Epilepsy Suggests Strategies for Therapeutic Transporter Modulation. Front. Cell. Neurosci. 13:515. doi: 10.3389/fncel.2019.00515 Keywords: KCC2, SLC125A, epilepsy, seizure, neuronal excitability, neurodevelopment

# INTRODUCTION

A seizure is a transient increase in the brain's electrical activity that may be triggered by a variety of factors, including medications (Chen et al., 2016), metabolic alterations (Imad et al., 2015), and infections (Zoons et al., 2008). When seizures arise spontaneously, they are considered to be epileptic. Epilepsy is the most common serious brain disorder worldwide (World Health Organization, 2019) characterized by recurrent and unprovoked seizures that can cause loss of consciousness and/or abnormal motor behavior depending on the afflicted brain region (Stafstrom and Carmant, 2015). The disorder affects 0.5% of the general population (Sander and Shorvon, 1996) and is associated with increased rates of mortality (Zieli´nski, 1974), cognitive impairment (Aldenkamp, 2006), and psychosocial dysfunction (Pershad and Siddiqui, 1992) at an annual cost to the US economy of \$12 billion (Begley et al., 2000).

Epilepsy is classically thought to arise from an imbalance between neuronal excitation and inhibition, leading to a hyperexcitable state that is prone to seizure activity. Antiepileptic drugs (AEDs) are the mainstay therapy for epilepsy that aim to restore this balance in neuronal excitability by either suppressing excitatory neurotransmission or augmenting inhibition. Despite decades of medical research and development of novel thirdgeneration AEDs, a third to a half of epilepsy patients on medications continue to have seizures (medication refractory epilepsy; Kwan and Brodie, 2000; Shorvon and Luciano, 2007; Cascino, 2008). Furthermore, AEDs often exert significant drug-related adverse effects, including dizziness, nausea, fatigue, depression, learning and memory impairments, and ataxia (Perucca and Meador, 2005). The lack of a truly satisfactory AED reflects our incomplete understanding of epileptogenesis, the set of pathogenic alterations that render neuronal networks hyperexcitable and thus vulnerable to pathological seizure activity. There is an urgent clinical need for novel insights into cellular and molecular mechanisms of epileptogenesis in order to develop more efficacious AEDs that can achieve seizure freedom with minimal or no side effects.

Human genetic studies have associated mutations in the neuron-specific type 2 K+/Cl− cotransporter KCC2 with the development of epilepsy (Kahle et al., 2014; Puskarjov et al., 2014; Stödberg et al., 2015; Saitsu et al., 2016; Saito et al., 2017; Till et al., 2019). Preclinical studies suggest that modulation of KCC2 activity by targeting critical regulatory domains may be exploited to suppress seizure activity (Moore et al., 2018), highlighting the key role of KCC2 in the regulation of neuronal excitability in physiological and epileptogenic states. In this article, we review the KCC2 mutations that are associated with the development of epilepsy in humans. We also discuss the therapeutic ramifications of these findings and postulate that KCC2 may be a potentially powerful therapeutic target for the development of novel AEDs to treat refractory epilepsy.

# Type 2 K+-Cl− COTRANSPORTER (KCC2) IN CHLORIDE HOMEOSTASIS AND SYNAPTIC INHIBITION

Neuronal excitability describes the propensity of a postsynaptic neuron to generate an action potential, a rapid rise and fall in membrane potential that occurs when the neuron reaches a threshold level of membrane depolarization. Consequently, neuronal excitability is governed by a dynamic balance between excitatory and inhibitory inputs. Excitatory inputs are depolarizing and thus raise the neuronal membrane potential towards the threshold, whereas inhibitory inputs are hyperpolarizing and lower the potential away from threshold. In the central nervous system, neuronal inhibition occurs primarily via activation of ligand-gated γ-aminobutyric acid (GABA) type A receptors (GABAARs) that are highly permeable to Cl−, and to a lesser extent, HCO3 − (Kaila and Voipio, 1987). Ligand binding to GABAARs on the postsynaptic neuron opens a central pore to trigger a hyperpolarizing Cl−

influx that lowers the probability of action potential being generated by neuron. The strength of synaptic inhibition is thus dependent on a low intra-neuronal concentration of Cl−, which provides the basis for an electrochemical gradient that permits passive movement of Cl− through the plasma membrane upon GABAAR activation.

The electroneutral K+/Cl− cotransporter KCC2 (encoded by SLC12A5) is a key determinant of Cl− homeostasis in neurons of the central nervous system (Kahle and Delpire, 2016; Moore et al., 2017). Under normal physiological settings, KCC2 uses the outwardly directed K+ gradient generated by the N+-K+ ATPase pump to extrude Cl− against its electrochemical gradient from neuronal cells in humans and thus maintains low intra-neuronal Cl− concentrations required for hyperpolarizing GABAergic currents. In a likely over-simplified but useful scheme, intra-neuronal Cl− concentrations are high during early brain development secondary to low KCC2 activity (Li et al., 2002; Stein et al., 2004) and high influx of Cl− via Na+-K+-Cl− cotransporter 1 (NKCC1; Plotkin et al., 1997; Yamada et al., 2004) in young neurons, leading to membrane depolarization following ligand binding to GABAAR (Ben-Ari et al., 1989). As the brain matures, NKCC1 activity is downregulated whereas KCC2 activity is upregulated (Plotkin et al., 1997; Stein et al., 2004), leading to hyperpolarizing GABAergic responses, though recent data shows that the expression changes of these molecules vary within the heterogeneous neuronal populations within the brain (Sedmak et al., 2016). Although other KCC isoforms exist, KCC2 is unique in that its expression is primarily localized to central nervous system neurons (Williams et al., 1999; Payne et al., 2003) and it remains constitutively active even under isotonic conditions (Khirug et al., 2005; Mercado et al., 2006). Importantly, at least in the setting of normal neurophysiology, KCC2 is able to remove extra Cl− introduced by GABAergic neurotransmission and thus recover low intracellular Cl− levels in neurons (Kaila et al., 2014; Doyon et al., 2016). These properties indicate that KCC2 is a major extruder of Cl− in mature neurons that establishes the inwardly directed Cl− electrochemical gradient across the plasma membrane necessary for the emergence and maintenance of inhibitory hyperpolarizing responses upon activation of GABAARs.

### KCC2 MUTATIONS AND HUMAN EPILEPSY

The importance of KCC2 in maintaining the strength of synaptic inhibition highlights its potential involvement in epilepsy, a disorder of neuronal hyperexcitability that has been thought to arise from failed neuronal inhibition. Preclinical studies in multiple organisms show that genetic KCC2 deficiency results in diminished Cl− extrusion, neuronal hyperexcitability, and epileptic seizures (Hübner et al., 2001; Hekmat-Scafe et al., 2006; Tanis et al., 2009). Accordingly, downregulation of KCC2 levels is observed in human idiopathic epilepsy (Huberfeld et al., 2007). Recent studies have now demonstrated the presence of KCC2 mutations in human epilepsy patients, providing strong evidence for the role

TABLE 1 | KCC2 (SLC12A5) mutations in human epilepsy.

AA, amino acid; AD, autosomal dominant; AR, autosomal recessive; EIMFS, epilepsy of infancy with migrating focal seizures; IGE, idiopathic generalized epilepsy; NT, nucleotide.

of KCC2 in seizure disorders. All of the KCC2 mutations discovered in human epilepsy thus far are summarized in **Table 1**.

### Idiopathic Generalized Epilepsy 14 (OMIM# 616685, Autosomal Dominant)

Kahle et al. (2014) used a targeted DNA-sequencing approach to screen the cytoplasmic C-terminal region of SLC12A5 which is an important regulatory region of transporter function. They identified two different heterozygous missense variants in SLC12A5 (R952H,<PHONE_NUMBER> and R1049C,<PHONE_NUMBER>) that were enriched among individuals of French Canadian origin with idiopathic generalized epilepsy-14 (EIG14; 616685) compared to controls. Both variants exhibited reduced Cl− extrusion capacity, although unlike the R952H variant, the R1049C variant exhibited normal surface expression with decreased intrinsic cotransporter activity. Both variants also showed decreased phosphorylation of the serine 940 (S940) residue (Kahle et al., 2014), which normally promotes KCC2 activity (Lee et al., 2011). The overall effect impaired the function of KCC2. The variants were inherited from an unaffected parent in several cases, consistent with incomplete penetrance, consistent with other large genomic studies of human idiopathic generalized epilepsy (Mefford et al., 2011). Puskarjov et al. (2014) reported the R952H mutation in an Australian family with early childhood onset of febrile seizures. Segregation of the variant in this kindred was difficult because of uncertain phenotyping, but there was some evidence of incomplete penetrance. Electrophysiological and biochemical assays suggest that the R952H variant exhibits impaired Cl− extrusion likely due to reduced surface expression. Overexpression of this variant in KCC2-deficient mouse cortical neurons failed to rescue defects in dendritic spine development, suggesting a potential role of the R952H variant in formation and maturation of cortical dendritic spines. Puskarjov et al. (2014) suggested that the decrease in KCC2-dependent hyperpolarizing inhibition would promote seizures, and that decreased dendritic spine formation could lead to desynchronization of overall excitability. Importantly, the function of KCC2 in the dendritic spine does not depend on transporter function but rather involves interactions between KCC2 and other proteins (Llano et al., 2015). The most recent study has identified a new missense KCC2 variant, V473I, that causes IGE in a Hungarian patient who is heterozygous for the mutation (Till et al., 2019).

### Early Infantile Epileptic Encephalopathy 34 (OMIM# 616645, Autosomal Recessive)

The strongest genetic evidence for KCC2 dysfunction in epilepsy is demonstrated by studies of patients in families with a severe infantile epilepsy syndrome termed epilepsy of infancy with migrating focal seizures (EIMFS; Stödberg et al., 2015; Saitsu et al., 2016; Saito et al., 2017). To date, nine probands with monogenetic KCC2-related EIMFS have been reported. By whole-exome sequencing of two unrelated families, Stödberg et al. (2015) discovered that affected children with EIMFS harbored biallelic SLC12A5 loss-of-function mutations. Two affected children from a consanguineous family harbored the homozygous mutation L311H that localizes to an extracellular loop. The L426P (localizing to a transmembrane domain) and G551D (localizing to an intracellular loop) variants were found as compound heterozygous mutations in two affected children from another family. All of the KCC2 mutations identified by Stödberg et al. (2015) resulted in diminished Cl− extrusion and reduced cell surface expression. Follow-up studies identified eight additional recessive KCC2 mutations that cause EIMFS (Saitsu et al., 2016; Saito et al., 2017), including the E50\_Q93del variant that causes skipping of exon 3 and the S749del variant that causes an amino acid deletion (Saitsu et al., 2016). Functional characterization of some of these EIFM-causing KCC2 mutations (E50\_Q93del, A191V, S323P, M415V) suggested that they attenuated neuronal Cl− extrusion without altering cell surface expression and distribution (Saitsu et al., 2016). It is important to note that all of the homozygous or compound heterozygous EIMFS patients inherited KCC2 mutations from unaffected heterozygous parents, in contrast to the R952H and R1049 variants that were sufficient to cause epilepsy disorders in heterozygous individuals (Kahle et al., 2014; Puskarjov et al., 2014). The reasons underlying these phenotypic differences are not understood, although some have hypothesized that mutations located in different KCC2 domains exert different effects on functional activity that underlie variations in phenotypic manifestations (Kahle et al., 2016a).

# AUGMENTING KCC2 ACTIVITY TO RESTORE SYNAPTIC INHIBITION AS A THERAPEUTIC AVENUE FOR EPILEPSY

The presence of KCC2 mutations in human epilepsy indicates that accumulation of intracellular Cl− secondary to KCC2 dysfunction may be responsible for driving neuronal hyperexcitability underlying the development of epilepsy syndromes. The association between loss-of-function KCC2 mutations and epilepsy also suggests that augmenting KCC2 activity to enhance Cl− extrusion may confer the opposite effect of rendering neuronal cells more resistant to seizures, representing a potentially powerful therapeutic avenue for idiopathic epilepsy. Indeed, high levels of neural activity due to seizures may promote the intracellular accumulation of Cl− that exceeds the normal Cl− extrusion capacity of KCC2, leading to GABAergic depolarizing currents and loss of synaptic inhibition that underlie epileptogenesis (Ellender et al., 2014; Magloire et al., 2019). Augmenting KCC2 function could theoretically extrude excessive Cl− and restore neuronal inhibition in hyperexcitable states.

Targeting critical phosphorylation sites of KCC2 regulation is one promising strategy to enhance KCC2 function for therapeutic benefit. KCC2 activity is bidirectionally regulated at key phosphorylation sites: S940 phosphorylation increases KCC2 function (Lee et al., 2011), whereas phosphorylation of T906 and T1007 inhibits its function (Rinehart et al., 2009). Dephosphorylation of T906 and T1007 upregulates Cl− extrusion from neurons (Friedel et al., 2015; Titz et al., 2015; Heubl et al., 2017). To test the hypothesis that increasing KCC2 activity is anticonvulsant in vivo, Moore et al. (2018) generated knock-in mice in which threonines 906 and 1007 were substituted for alanines (KCC2-T906A/T1007A) to genetically prevent phosphodependent inactivation, resulting in higher basal neuronal Cl− extrusion (Moore et al., 2018). Strikingly, KCC2- T906A/T1007A mice exhibited profound resistance to chemoconvulsant-induced seizures without altered basal neuronal excitability. These findings suggest that modulation of KCC2 phosphorylation sites may be leveraged to strengthen synaptic inhibition for therapeutic benefit in epilepsy syndromes. For clinical translation, KCC2 function could be enhanced by inhibition of the upstream with no lysine (WNK) and Ste20-related proline-alanine kinase (WNK-SPAK) cascade that normally inhibits KCC2 function by promoting T906 and T1007 phosphorylation (Kahle et al., 2005, 2014, 2016b; Friedel et al., 2015; Kahle and Delpire, 2016; Heubl et al., 2017). Indeed, small molecule inhibitors of the WNK-SPAK pathway are being developed (Yamada et al., 2016). In addition to targeting the WNK/SPAK pathway, other pharmacological strategies to enhance KCC2 activity are also being investigated. A large screening of molecules to identify putative KCC2 agonists was first proposed by Gagnon et al. (2013). Another screening was recently carried out to detect small molecules capable of enhancing KCC2 expression levels (Tang et al., 2019). The ability to augment KCC2 function via different pharmacological approaches will enable flexibility in the selection of the ideal KCC2 modulator that is tailored to the underlying epileptogenic process.

Although efforts to identify KCC2 modulators reflect its promise as a druggable target in epilepsy, there remain several caveats. First, it is unclear which pharmacological approach or combination of approaches to enhance KCC2 activity would rescue the defects in expression and function due to KCC2 mutations observed in epilepsy syndromes. Second, it remains uncertain whether there may be unintended adverse effects that arise from increased KCC2 activity. Indeed, a major shortcoming of current AEDs is not only their inability to prevent seizures in a large population of patients but also their association with drug-related side effects such as cognitive disturbance (Park and Kwon, 2008). Potentiating KCC2 activity does not alter basal neuronal excitability (Moore et al., 2018), and a previously identified KCC2 activator (CLP257) produces analgesia without motor side effects often seen with other analgesics (Gagnon et al., 2013). While these preliminary findings suggest that enhancing KCC2 may be a safe therapeutic avenue to prevent seizures without altering the function of healthy neurons, more systematic studies are still needed to fully characterize potential side effects of the approach before clinical translation.

### CONCLUSION

Epilepsy is a common brain disorder characterized by recurrent and unprovoked seizures thought to be caused by neuronal hyperexcitability. A third to half of epilepsy patients continue to have seizures despite medications (Kwan and Brodie, 2000; Shorvon and Luciano, 2007; Cascino, 2008), underscoring the clinical need for the identification of novel therapeutic targets. KCC2 functions as a major Cl− extruder in mature neurons to establish an inwardlydirected electrochemical gradient of Cl− necessary for the maintenance of fast synaptic inhibition. In the settings of diminished KCC2 activity secondary to risk factor and causal mutations in human epilepsy patients, intracellular Cl− concentrations accumulate, leading to impaired hyperpolarizing responses that render neurons hyperexcitable. In contrast, increasing KCC2 function by overexpression or modulation of key phosphorylation sites confers an anticonvulsant effect. Altogether, the presence of KCC2 mutations in epilepsy coupled with preclinical proof-of-principle for KCC2 as a therapeutic target motivates a rich stream of future studies to further investigate the mechanistic roles of KCC2 in epileptogenesis and how manipulation of KCC2 activity can be leveraged pharmacologically for therapeutic benefit in epilepsy syndromes and conditions of hyperexcitation.

### AUTHOR CONTRIBUTIONS

PD, WD and KK reviewed the literature and wrote the manuscript.

### REFERENCES

### FUNDING

This work was supported by 1R01NS109358-01 (to KK), 1R01NS111029-01A1 (to KK), the Simons Foundation (to KK), the March of Dimes Foundation (to KK), and National Institutes of Health (NIH) Medical Scientist Program Training Grant T32GM007205 (to PD).

### ACKNOWLEDGMENTS

We thank all members of the Kahle lab for their help and support.

**Conflict of Interest**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Duy, David and Kahle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Functional Genomics of Epilepsy and Associated Neurodevelopmental Disorders Using Simple Animal Models: From Genes, Molecules to Brain Networks

Richard Rosch1,2,3, Dominic R. W. Burrows1 , Laura B. Jones4 , Colin H. Peters4 , Peter Ruben4 and Éric Samarut5,6 \*

1 MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, United Kingdom, 2 Department of Paediatric Neurology, Great Ormond Street Hospital, NHS Foundation Trust, London, United Kingdom, 3 Department of Bioengineering, University of Pennsylvania, Philadelphia, PA, United States, 4 Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, BC, Canada, 5 Department of Neurosciences, Research Center of the University of Montreal Hospital Center (CRCHUM), Université de Montréal, Montreal, QC, Canada, 6 Modelis Inc., Montreal, QC, Canada

### Edited by:

Eleonora Aronica, University Medical Center Amsterdam, Netherlands

### Reviewed by:

Michel Joseph Roux, INSERM U964 Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), France Dilja Krueger-Burg, University Medical Center Göttingen, Germany

> \*Correspondence: Éric Samarut<EMAIL_ADDRESS>
### Specialty section:

This article was submitted to Cellular Neuropathology, a section of the journal Frontiers in Cellular Neuroscience

Received: 20 September 2019 Accepted: 02 December 2019 Published: 13 December 2019

### Citation:

Rosch R, Burrows DRW, Jones LB, Peters CH, Ruben P and Samarut É (2019) Functional Genomics of Epilepsy and Associated Neurodevelopmental Disorders Using Simple Animal Models: From Genes, Molecules to Brain Networks. Front. Cell. Neurosci. 13:556. doi: 10.3389/fncel.2019.00556 The genetic diagnosis of patients with seizure disorders has been improved significantly by the development of affordable next-generation sequencing technologies. Indeed, in the last 20 years, dozens of causative genes and thousands of associated variants have been described and, for many patients, are now considered responsible for their disease. However, the functional consequences of these mutations are often not studied in vivo, despite such studies being central to understanding pathogenic mechanisms and identifying novel therapeutic avenues. One main roadblock to functionally characterizing pathogenic mutations is generating and characterizing in vivo mammalian models carrying clinically relevant variants in specific genes identified in patients. Although the emergence of new mutagenesis techniques facilitates the production of rodent mutants, the fact that early development occurs internally hampers the investigation of gene function during neurodevelopment. In this context, functional genomics studies using simple animal models such as flies or fish are advantageous since they open a dynamic window of investigation throughout embryonic development. In this review, we will summarize how the use of simple animal models can fill the gap between genetic diagnosis and functional and phenotypic correlates of gene function in vivo. In particular, we will discuss how these simple animals offer the possibility to study gene function at multiple scales, from molecular function (i.e., ion channel activity), to cellular circuit and brain network dynamics. As a result, simple model systems offer alternative avenues of investigation to model aspects of the disease phenotype not currently possible in rodents, which can help to unravel the pathogenic substratum in vivo.

Keywords: epilepsy, neurodevelopmental disorder, brain disorder, zebrafish, Drosophila

# FROM PHENOTYPE TO GENOTYPE: THE ERA OF GENETICS IN THE FIELD OF NEURODEVELOPMENTAL DISORDERS

Investigating the genetic basis of childhood epilepsy and neurodevelopmental disorders over the last two decades has revealed the unexpected role of a number of key genes in guiding normal brain development and emergent brain dynamics (Myers and Mefford, 2015). Facilitated by the increasing affordability of genomic technologies, genes affecting synaptic function have been identified as causative in a diverse range of epilepsy syndromes and other neurodevelopmental disorders such that many are now considered "synaptopathies" (Grant, 2012). Interestingly, many non-synaptic genes have also been identified as risk factors in various neurodevelopmental disorders. For these genes, the underlying pathogenic mechanisms are puzzling as they are not necessarily known to regulate synaptic activity directly. Taken together, a better understanding of the functional consequences of the wide spectrum of neurodevelopmental genetic mutations is required, for which in vivo systems are particularly useful.

## Severe de novo Mutations and Genomic Alterations in Neurodevelopmental Disorders

Genetic insights have been particularly transformative in our understanding of some of the most severe disorders of neurodevelopment, known now as developmental and epileptic encephalopathies (DEEs) (Scheffer et al., 2016). DEEs usually occur as isolated cases in families, yet in a large proportion of cases, causative de novo mutations in single genes can now be identified from clinical genetic diagnostics (Epi et al., 2013; Oates et al., 2018). The most common genes and their functional effects are illustrated in **Figure 1**. Affordable technology that allows the identification of even small structural genomic alterations [i.e., copy number variations (CNVs)] was key to investigating the genetic basis for common neurodevelopmental disorders. Beginning with transformative studies of people living with autistic spectrum disorders (ASD) (Sebat et al., 2007; Pinto et al., 2010), we have come to understand that individually rare CNVs account for a significant proportion of the incidence not only of autism, but intellectual disability (Cooper et al., 2011), "idiopathic" generalized epilepsies (Mefford et al., 2010; Addis et al., 2016) and schizophrenia (Stefansson et al., 2008; Marshall et al., 2017), particularly where there is overlap between these conditions. Interestingly, genes identified from genome-wide association studies of particular disorders often overlap across disorder categories (Fromer et al., 2014; International League Against Epilepsy Consortium on Complex Epilepsies, 2014; Schizophrenia Working Group of the Psychiatric Genomics Consortium, 2014; Turner et al., 2017), suggesting that many of the genes have a broad neurodevelopmental role that may result in a range of recognizable syndromes or phenotypes.

# Genotype-Phenotype Correlations

The increasing ability to make genetic diagnoses at the level of individual patients carries the promise of allowing the development of targeted therapies informed by underlying pathomechanisms. However, with increasing diagnosis, the phenotypic spectrum widens and linking genotypes to phenotypes is becoming more and more challenging. Indeed, mutations in the same gene (or even identical same genetic mutations) may cause very different phenotypes in different patients. For example, up to 50% of patients with de novo mutations in known epilepsy genes do not have seizures (Deciphering Developmental Disorders, 2017). Another example is the spectrum of epileptic phenotypes caused by different mutations affecting the same GABA receptor gene (GABRA1). This spectrum ranges from some of the most severe developmental DEEs of infancy, to juvenile onset treatable generalized epilepsy syndromes (Johannesen et al., 2016). Some variability in the mapping between affected genes and phenotype can be explained by differences in specific mutations' effects (even at the level of the same gene) on protein function (Ben-Shalom et al., 2017). This genotype-phenotype relation may be addressed in the future by increasing efforts to investigate the functional effects of individual genes as well as individual mutations in vivo through translational research (Scheffer et al., 2016). This gene has been described as both an epilepsy, and an autism gene but also emerges in a range of other neurodevelopmental contexts suggesting potentially shared mechanisms. Understanding the contribution of the genetic alteration to the various phenotypes is essential to now attempt and translate these broad insights into novel, targeted therapies.

# FROM GENE MUTATION TO MOLECULAR DYSFUNCTION (TEMPORAL MICROSCALE)

In the light of the difficulties in relating newly diagnosed genetic variants with their underlying functional consequences, and because of the unclear correlation between phenotype and genotype we described above, there is a need for in vivo models to explore the functional effects of specific genetic alterations. In particular, epilepsy has been studied using multiple model organisms, most traditionally rodents (Seyfried and Glaser, 1985; Yu et al., 2006). Although rat and mouse models have been foundational to the field, recent research has expanded to include non-mammalian models such as round worms, zebrafish, and fruit flies (Baraban, 2007; Cunliffe et al., 2015).

Drosophila melanogaster has become an increasingly popular model organism in epilepsy research due to its small size, short generation time, and the relative ease of stock maintenance and mutant isolation (Bier, 2005; Song and Tanouye, 2008; Cunliffe et al., 2015). These factors, in addition to the large percentage of conserved human disease genes in Drosophila (Fortini et al., 2000; Rubin et al., 2000; Bier, 2005), result in it being an extremely cost-effective model system for epilepsy research (Baraban, 2007; Song and Tanouye, 2008; Cunliffe et al., 2015).

Developments in genome editing technology have facilitated the introduction of human disease-causing mutations into the corresponding genes of Drosophila, resulting in the improved ability to characterize gene-phenotype relationships as well as to perform high-throughput in vivo drug testing (Stilwell et al., 2006). These techniques have advanced the identification of disease-specific epilepsy treatments (Griffin et al., 2018). One example is the study of Dravet syndrome (DS), a severe form of infant-onset febrile epilepsy that is often co-morbid with other developmental disorders. DS patients are typically pharmacoresistant (Chiron, 2011; Dravet, 2011; Griffin et al., 2018), with many common antiepileptic drugs even aggravating their seizures (Guerrini et al., 1998; Chiron, 2011; Nissenkorn et al., 2019). Thus, there exists a demand for increased therapeutic treatment options, an issue that is further complicated by the multitude of different DS-causing mutations (Meng et al., 2015; Schutte et al., 2016). Electrophysiology research has revealed that these mutations exhibit considerable variation in their channel characteristics ("channotype"), ranging from gain-offunction to loss-of-function effects (Escayg and Goldin, 2010; Meng et al., 2015; Peters et al., 2016). Elucidating the molecular variations behind this functional heterogeneity can inform drug selection for preliminary pharmacological testing, which can in turn provide in vivo validation for electrophysiology results.

Combining these two techniques can therefore be a powerful approach to better understanding and treating DS, serving as informative steps on the pathway to clinical drug testing.

A common mutation target for generating DS models is the Drosophila para gene, encoding the voltage-gated sodium channels and corresponding to the human SCN1A gene in which many DS-causing mutations have been identified (Dravet, 2011). The Drosophila para gene is edited using CRISPR-cas9 to reproduce specific human DS causing mutations in SCN1A, whilst also introducing a marker mutation (e.g., eye color). In these flies, transient seizure-like behaviors [falling into their backs or sides and beginning to twitch their legs and wings, sometimes accompanied by abdominal curling (Sun et al., 2012; Schutte et al., 2016; Griffin et al., 2018)] can be induced by hyperthermia (**Supplementary Video S1**). Once a model organism line is validated, potential drug therapies can be assessed by mixing the therapeutic target of interest into liquified cornmeal food and allowing the flies to feed on it before subsequent seizure assays (Sun et al., 2012). Thus, modeling epilepsy with Drosophila enables researchers to use a simple model to shed light on the functional characterization of genetic data and to perform largescale screenings of antiepileptic drug candidates, providing a cost-effective form of preclinical testing.

Danio rerio (zebrafish) is another model appropriate for linking genotype to phenotype in patient models of neurodevelopmental disorders. A major reason for this is the phylogenetic proximity of this vertebrate model, the high homology of the zebrafish and human genomes (∼80% homology), and the ease of genetic manipulation in the zebrafish. In fact, genetic modification in zebrafish is highly efficient, allowing comprehensive in vivo studies even of neurodevelopmental disorders with complex genetic backgrounds. One example is a recent study in which 132 schizophrenia risk variants were generated using CRISPR-Cas9 (Thyme et al., 2019). A wide array of zebrafish models have also been developed across epilepsy genes, and genes associated with broader neurodevelopmental phenotypes [e.g., scn1lab (Baraban et al., 2013), gabrg2 (Liao et al., 2019), GABRA1 (Samarut et al., 2018), mecp2 (Pietri et al., 2013), grin2a/b (Thyme et al., 2019), and others]. Importantly, despite different brain anatomy and physiology to mammalian counterparts, various zebrafish models exhibit phenotypes analogous to corresponding rodent models and clinical phenotypes. In particular a zebrafish model of DS carrying a loss-of-function mutation in the scn1lab gene, exhibits spontaneous electrographic abnormalities reminiscent of seizures (Baraban et al., 2013). Such recordings, obtained from field electrodes placed in the midbrain of agar immobilized larval zebrafish, appear as brief, small amplitude inter-ictal like events and prolonged, multi-spike ictal-like discharges which are qualitatively comparable to epileptiform discharges in patients and mammalian epilepsy models. Furthermore, GABRA1−/− and gabrg2−/− larvae, both modeling mutations reported in common epilepsy syndromes (Wallace et al., 2001; Johannesen et al., 2016), exhibit reflexive seizure-like events in response to light stimulation, reported as convulsive motor abnormalities and abnormal brain synchrony (**Supplementary Video S2**; Samarut et al., 2018; Liao et al., 2019).

It is, however, important to note that zebrafish lack a cortex and therefore qualitative homologies between zebrafish and human epilepsy (a putative cortical pathology) may be more useful for broad functional characterizations of genetic epilepsies, while specific seizure subtypes may be better modeled by more complex model organisms systems. Nonetheless, given that common anti-epileptic drugs correct electrographic, and motor abnormalities in these zebrafish models, the underlying neuropathology is likely to be conserved in genetic models (Baraban et al., 2013; Samarut et al., 2018; Liao et al., 2019). Finally, zebrafish larvae are also highly amenable to high-throughput behavioral drug screens, which have already identified novel drugs for the treatment of DS, thus closing the loop from fish tank to bedside (Griffin et al., 2018).

Therefore the larval zebrafish can provide realistic models of a wide array of neurodevelopmental disorders which may open alternative avenues for investigation at scales not possible in its mammalian counterparts making them complementary models.

## FROM GENE MUTATION TO BRAIN NETWORK PERTURBATION (TEMPORAL MACROSCALE)

As discussed above, in order to translate an improved genetic understanding of neurodevelopmental disorders into novel therapies, a detailed understanding of pathomechanisms is required. Simple animal models can enable the characterization of the functional consequences of genetic mutations at multiple different scales, ranging from single-cell behavior to whole-brain dynamics, and allow this translation much more rapidly and at times more comprehensively than in mammalian models.

The utility of simple model systems for bridging this gap is particularly evident in epilepsy. Given that seizures are an emergent property of microcircuits, understanding the effect of specific genetic mutations at a network level is necessary to explain the emergence of clinical phenotypes. At this juncture, the larval zebrafish is a particularly appealing model for studying brain network dynamics due to its amenability to whole brain imaging at single-cell resolution, allowing identification of abnormal dynamics at multiple scales (Ahrens et al., 2013). The larval fish at 7 days-post-fertilization has a small, simple brain (100,000 cells, <1 mm3 ) but is capable of a variety of complex behaviors whose brain dynamics can be monitored accurately (**Figure 2**). Various genetic lines of pigment deficient larval zebrafish have been developed which enable unrestricted optical access into the developing brain (Antinucci and Hindges, 2016). Furthermore, the development of various transgenic reporters of cellular activity, such as GCaMP and RGECO enable the imaging of calcium dynamics in single cells and whole brain networks during behavior, using fluorescence microscopy (**Figure 2**; Walker et al., 2013; Wolf et al., 2017; Chen et al., 2018). In fact, various transgenic lines have also been developed to monitor specifically GABA (Marvin et al., 2019) or glutamate (Marvin et al., 2013) signaling in vivo. Given that these cellular reporters have been utilized to characterize neuronal function across brain scales [from synapses to cell populations and brain

agarose, allowing in vivo imaging using fluorescence microscopy (shown here is a two-photon microscopy setup). Depending on the experimental paradigm, behavioral output can further be tracked using recordings of tail movements in tail free set ups. This allows e.g., linking of convulsive movements and brain hypersynchrony to identify epileptic seizures in the zebrafish.

networks (Walker et al., 2013; Boulanger-Weill et al., 2017; Betzel, 2018)], functional imaging of zebrafish genetic models may provide a unique window into the multi-scale functional consequences of upstream channel abnormalities. Thus, it may provide an explanatory bridge between gene mutation and whole brain clinical phenotypes.

While at present, the majority of zebrafish functional imaging studies have characterized acute, induced seizures using the GABA-A antagonist pentelynetetrazole (PTZ), a variety of useful network features have been identified which may provide insight into future genetic models. Multiple studies have reported increased functional connectivity across local and distributed brain regions during seizure events (**Figure 2A**; Diaz Verdugo et al., 2019; Liu and Baraban, 2019), in accordance with reports of increased phase locking in EEG recordings during seizures (Meisel et al., 2012).

Importantly, single cell-level information can be harnessed from functional imaging data to explain seizure network dynamics. For example, zebrafish imaging suggests that seizures emerge as cellular ensembles, which are composed of more spatially distant cells than pre-seizure (Diaz Verdugo et al., 2019; Liu and Baraban, 2019). Furthermore, the role of cell subtypes in the emergence of network abnormalities may be probed with the application of double transgenic larval zebrafish, expressing calcium reporters and specific cellular subtype reporters (Lyons et al., 2003; Xi et al., 2011; Shimizu et al., 2015). Such approaches have demonstrated that astrocytes facilitate widespread neuronal synchrony during generalized seizures, thereby enabling seizure state transitions (Diaz Verdugo et al., 2019). Identifying critical cell subpopulations in this way in genetic epilepsies has the potential to identify novel treatment targets in patients. Interestingly, model-based approaches which are widely used to explain network phenomena in EEG data can also be applied to calcium imaging data to test causal mechanisms underlying network features of seizures (Rosch et al., 2018). Such approaches have shown that acute seizures are caused by parameter changes in local excitation-inhibition balance, and alterations in timescales of excitatory and inhibitory connectivity. In this way the cellular mechanisms underlying observed network features in functional imaging data can be uncovered, to provide a

conceptual bridge to explaining EEG phenomena during seizures, such as hypersynchrony and transitions between network states. As more genetic lines become available (Baraban et al., 2013; Samarut et al., 2018; Swaminathan et al., 2018; Liao et al., 2019), such imaging approaches may be harnessed to link gene mutation with network perturbation.

Flies have also become convenient models to perform neuronal cell recording in the adult brain (ex vivo) that can be studies in the context of genetic and/or pharmacological manipulations (Gu and O'dowd, 2007; Roemmich et al., 2018). As an example, electrophysiological studies carried out in adult flies that were genetically modified to mimic DS were pioneers in showing the link between pathological missense mutations and disturbances of sodium ion current activity at the receptor level (Sun et al., 2012; Schutte et al., 2014). These cellular experimentations in a genuine in vivo context are very advantageous in order to unravel the basic cellular mechanisms of brain circuit function and malfunction. They can also be suitable for evaluating the mechanism of action of candidate therapies against neurodevelopmental disorders that were first identified through behavioral assays.

Remarkably, the larval zebrafish is also a suitable model for longitudinal studies of neural development. Indeed, the transparency of the embryo allows one to follow in vivo organogenesis, in particular the observation of central nervous system structures with a single-cell resolution. Moreover, there is a large repertoire of available transgenic lines expressing fluorescent reporters in different neural cell populations such as post-mitotic neurons (huc/elavl3+) (Park et al., 2000), GABAergic interneurons (dlx5/6+) (Zerucha et al., 2000), glutamatergic neurons (vglut2a+) (Kimura et al., 2006) or oligodendrocytes (olig2+) (Shin et al., 2003). This is of a particular interest in the context of neurodevelopmental disorders for which one can expect defects in brain wiring to occur during early neurodevelopment. In this context, the accessibility of the zebrafish embryo from the earliest stages of development is an advantage compared to mammals in which the embryos develops in utero. The use of larval zebrafish has proven particularly useful in modeling several human neurological disorders with a developmental component such as ASD or epileptic encephalopathy. For example, using specific transgenic lines identifying excitatory versus inhibitory neuronal networks, Hoffman et al. (2016) revealed a specific deficit of GABAergic neuronal population networks in the forebrain of zebrafish larvae mutant for CNTNAP2, an ASD-related gene. Another zebrafish model of ASD (shank3b−/−) displays a reduction in the overall brain neuronal content as revealed by a transgenic line expressing a fluorescent protein in post-mitotic neurons (Liu et al., 2018). Interestingly, the development of these neuronal populations can be followed over time and the defects can therefore be monitored throughout neurodevelopment. More recently, two genetic models of epilepsy [idiopathic generalized epilepsy: GABRA1 −/− (Samarut et al., 2018), and focal epilepsy: depdc5−/− (Swaminathan et al., 2018)] have been generated. These models depict relevant phenotypes to the human disorders, but more interestingly, they demonstrated impaired GABAergic synaptic network branching in the mutant larval brains identifying a potential pathomechanism. In this way, zebrafish genetic models can be harnessed to further understand the developmental component of these diseases and in so doing, at least partially, account for the pathogenicity of the mutations tested.

As a result, simple models like flies and zebrafish appear to be an amenable model to (i) mimic human genetic condition associated with neurological disorders, (ii) investigate the consequence at the neuronal network activity level through in vivo calcium imaging, and (iii) unravel neurodevelopmental defects associated with the disorder.

## CONCLUSION: SIMPLE MODELS AS GENETIC AVATARS OF HUMAN PATIENTS FOR A SYSTEMIC APPROACH (FROM GENE TO PHYSIOLOGY)

In the current context of fast-evolving accessibility to genetic diagnosis, more and more genetic basis of neurological disorders are being unraveled. They opened the door to a new challenges that is the translation of this genetic data into functional read-out. Can we predict the functional consequence of a specific mutation in a particular gene? What are the effects of a specific mutation on the activity of the protein? at the level of the neuronal network? At the scale of whole neurodevelopment? These puzzling questions necessitate the use of fast and complementary in vivo approaches. In this review, we are discussing how simple animal models can be employed to bridge the gap between genetic diagnosis and functional studies. Considering the fast development of mutagenesis techniques that now allow to mimic a specific genetic mutation in these simple models, plus their relative lowcost of housing as well as their fast generation time, they represent a model of choice to study neurodevelopmental disorders in an integrated fashion and at multiple scales. Thanks to their versatility, these simple animal models can unravel the basic pathomechanims of gene mutations and therefore open new avenues for therapy development. As mentioned previously, they are also convenient for standardize procedures, in particular for high-throughput screening of small molecules. Interestingly, they are also very convenient for genetic manipulation. Indeed, by microinjecting molecular tools in the one-cell stage embryo, it is possible to either knockdown [with morpholinos or CRISPRi (Long et al., 2015)], knockout [by CRISPR/Cas9 genome editing (Hwang et al., 2013)] or overexpress [by CRISPRa (Long et al., 2015) or by injecting in vitro transcribed messenger RNAs or transposon plasmids for transgenesis] the expression of a gene of interest. As a result, these simple animal models can also serve as tools to test genetic-derived therapeutic strategies through restorative functional assays after modulating the expression of candidate genes.

### AUTHOR CONTRIBUTIONS

RR, DB, LJ, PR, and ÉS wrote the manuscript. CP performed the Drosophila recording. ÉS performed the fish recording.

### FUNDING

fncel-13-00556 December 12, 2019 Time: 14:58 # 7

This work is supported by the Rare Disease Model and Mechanism Network, the Rare Disease Foundation and Dravet Canada. RR is supported by a Sir Henry Wellcome Fellowship (Wellcome Trust, United Kingdom; Grant Number 209164/Z/17/Z). DB is supported by the Medical Research Council and the Sackler Institute for Translational Neurodevelopment.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fncel. 2019.00556/full#supplementary-material

# REFERENCES

VIDEO S1 | Temperature-induced seizures in Drosophila with a mutation in the Para gene, analogous to SCN1A. To introduce the human A1273V mutation into the corresponding location on Drosophila's para gene (A1356V), we used the Drosophila CRISPR-Cas9 system developed by a set of labs at Brown and University of Wisconsin https://flycrispr.org/about/ (citation). We designed 2 RNA ides and a donor vector with mutated template DNA for homology-directed repair as well as insertion of a DsRed eye reporter in the subsequent intron. Embryo injections and genetic crosses were performed by the company BestGene, Inc. (Chino Hills, CA, United States).

VIDEO S2 | Light-induced seizures in zebrafish with a mutation in the GABRA1 gene. As described previously in Samarut et al. (2018). Infrared camera recording of 5, 5 wpf GABRA1 +/+(top, n = 4), ±(middle, n = 4), and −/− (bottom, n = 4) embryos maintained in a 12-well plate, using a DanioVision recording chamber. Light was switched on about 5 s after the video started (see icon on the top left corner). Immediately after light turning on, the −/− juveniles underwent a first phase of tonic-like seizures lasting few seconds and during wish they convulsed heavily and lost their posture. After a short pause, they underwent a second clonic-like phase that lasted up to 1 min and during which they swam quickly in circle (the "whirlpool" phenotype).

transitions to generalized seizures. Nat. Commun. 10:3830. doi: 10.1038/ s41467-019-11739-z

**Conflict of Interest:** ÉS is a co-founder of Modelis Inc.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Rosch, Burrows, Jones, Peters, Ruben and Samarut. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Transcriptional Regulation of Channelopathies in Genetic and Acquired Epilepsies

Karen M. J. van Loo\* and Albert J. Becker

Department of Neuropathology, Section for Translational Epilepsy Research, University of Bonn Medical Center, Bonn, Germany

Epilepsy is a common neurological disorder characterized by recurrent uncontrolled seizures and has an idiopathic "genetic" etiology or a symptomatic "acquired" component. Genetic studies have revealed that many epilepsy susceptibility genes encode ion channels, including voltage-gated sodium, potassium and calcium channels. The high prevalence of ion channels in epilepsy pathogenesis led to the causative concept of "ion channelopathies," which can be elicited by specific mutations in the coding or promoter regions of genes in genetic epilepsies. Intriguingly, expression changes of the same ion channel genes by augmentation of specific transcription factors (TFs) early after an insult can underlie acquired epilepsies. In this study, we review how the transcriptional regulation of ion channels in both genetic and acquired epilepsies can be controlled, and compare these epilepsy "ion channelopathies" with other neurodevelopmental disorders.

### Edited by:

Eleonora Aronica, University Medical Center Amsterdam, Netherlands

### Reviewed by:

Hee Jung Chung, University of Illinois at Urbana-Champaign, United States Darrin Brager, University of Texas at Austin, United States

### \*Correspondence:

Karen M. J. van Loo<EMAIL_ADDRESS>
Received: 12 September 2019 Accepted: 23 December 2019 Published: 14 January 2020

### Citation:

van Loo KMJ and Becker AJ (2020) Transcriptional Regulation of Channelopathies in Genetic and Acquired Epilepsies. Front. Cell. Neurosci. 13:587. doi: 10.3389/fncel.2019.00587 Keywords: genetic and acquired epilepsies, ion channels, channelopathies, transcriptional regulation, neurodevelopmental disorders

### INTRODUCTION

Epilepsy is a severe chronic brain disorder characterized by recurrent seizure activity due to aberrant neuronal network activity (Fisher et al., 2014; Fisher, 2015). Despite many years of research, the underlying mechanisms that orchestrate seizure activity are still not fully understood. This is also reflected in the fact that treatment strategies for epilepsy with antiepileptic drugs (AEDs) are insufficient in about one-third of epilepsy patients (Kwan and Sander, 2004). This relatively high level of pharmacoresistance, together with the often severe side effects of AEDs, asks for a better understanding of its etiology and pathogenesis (Löscher et al., 2013).

Nowadays, it is generally accepted that both genetic as well as environmental factors, such as head trauma, brain tumors, brain infection, stroke, autoimmune diseases, status epilepticus (SE) and hippocampal sclerosis (Engel, 1996; Bien and Elger, 2007; Bien et al., 2007; Liu et al., 2016; Pitkänen et al., 2016; Vezzani et al., 2016) can play a role in the etiopathogenesis of epilepsy. Epilepsies with such a causal injury to the central nervous system (CNS) are called acquired or symptomatic epilepsies, whereas those lacking a clear predisposing cause, are called idiopathic or genetic epilepsies (Shorvon, 2011).

In the last decades, enormous progress has been made in the discovery of epilepsy genes, resulting in a current list of approximately 1,000 epilepsy-associated genes (reviewed by Wang et al., 2017). Since many of the genes annotated on this list are ion channels, the theory was born that epilepsy is a disease of ''ion channelopathies'' (Wallace et al., 1998; Reid et al., 2009). Ion channels are pore-forming membrane proteins involved in maintaining ion homeostasis and the generation and propagation of neuronal action potentials. A disturbance in the neuronal ionic flow might result in hyperexcitability, which can form the basis for seizure activity (Raimondo et al., 2015). In general, ion channels can be divided into two main groups, depending on their mode of activation (Brenowitz et al., 2017). Voltage-gated ion channels are activated by changes in membrane potential and ligand-gated ion channels are opened in response to specific ligands binding to the extracellular domain of the ion channel (Alexander et al., 2015a,b).

In this study, we focus on the transcriptional mechanisms involved in channelopathy-induced epilepsy. We review how the expression of ion channel genes can be affected and compare these mechanisms between genetic and acquired epilepsies. In addition, we also summarize how these transcriptional mechanisms can play a role in the etiopathogenesis of other neurodevelopmental disorders.

### VOLTAGE-GATED AND LIGAND-GATED ION CHANNELS IN GENETIC EPILEPSIES

For decades, scientists try to unravel the molecular background of epilepsies. In 1995, the first epilepsy-associated ion channel was identified; a mutation in a strongly conserved amino acid residue in the acetylcholine receptor alpha 4 subunit (CHRNA4) correlated with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE; Steinlein et al., 1995). After this first discovery, many other ion channels were reported to be linked to epilepsy, including genes belonging to the voltagegated sodium, potassium, calcium and hyperpolarizationactivated cyclic nucleotide-gated (HCN) channels. Besides the voltage-gated ion channels, also several ligand-gated ion channel genes were identified as epilepsy-associated genes, including ionotropic glutamate receptors, GABAA receptors and nicotinic acetylcholine receptors (**Table 1**; reviewed by Lerche et al., 2013; Wang et al., 2017; Wei et al., 2017; Oyrer et al., 2018).

Currently, it is thought that genetic epilepsy can be the result of: (i) rare variants with high penetrance (also known as monogenic or ''common-disease-rare-variant model'') or of (ii) common variants with low penetrance (also known as polygenic or ''common-disease-common-variants model''; Reich and Lander, 2001; Gibson, 2012; Saint Pierre and Génin, 2014). Such rare variants (or mutations) can nowadays be identified by deep sequencing approaches (e.g., exome sequencing or whole-genome sequencing; Dunn et al., 2018), whereas for the identification of common variants (also known as single nucleotide polymorphisms, SNPs), genome-wide association studies are indispensable in large cohorts of patients and controls (International League Against Epilepsy Consortium on Complex Epilepsies, 2018). However, common variants are often difficult to link unequivocally to disease, since these variants contribute only minimally and might also require an additional environmental factor for a pathological outcome.

In epilepsy, both rare as well as common variants have been identified in ion channel genes. Mutations in the sodium channel SCN1A, probably the most studied and best-documented epilepsy gene, can cause a spectrum of epilepsy syndromes including Dravet syndrome and genetic (generalized) epilepsy with febrile Seizures Plus (GEFS+; Brunklaus and Zuberi, 2014), whereas a common variant within an intron of the same gene (rs7587026), was found to associate with mesial temporal lobe epilepsy (mTLE; Kasperaviciute et al., 2013).

Mostly, genetic channelopathies are the result of variants within the coding region of the gene. Both missense mutations (mutations causing an amino acid change), as well as nonsense mutations (mutations causing a premature stop codon), can underlie epilepsy pathogenesis by inducing a loss-of-function (LOF) or a gain-of-function (GOF) channelopathy. In addition, also deletions and duplications of (part of) the gene can strongly affect normal channel function (Borlot et al., 2017; Monlong et al., 2018). Since the focus of this review is on the transcriptional regulation of ion channels, listing all genetic variants within the coding regions of ion channel genes is beyond the scope of this article (for reviews, see Deng et al., 2014; Wei et al., 2017; Zhang et al., 2019).

### TRANSCRIPTIONAL REGULATION OF GENETIC ION CHANNELOPATHIES

Genetic epilepsies can also be the result of a genetic variant positioned in the promoter region, a splice site, or a regulatory region of the gene. How can variants outside the coding region induce a channelopathy? For SCN1A for example, a microdeletion in the 5'-promoter region was found in patients with Dravet syndrome (Nakayama et al., 2010), and another heterozygous mutation in the promoter region (h1u-1962 T >G) was identified in a patient with partial epilepsy and febrile seizures (Gao et al., 2017). Functional analysis of this SCN1A h1u-1962 T >G variant revealed a reduction of SCN1A promoter activity by 42.1% compared to the wild-type variant (Gao et al., 2017), explaining the relatively mild phenotypical impairment caused by this non-coding variant when compared with effects caused by SCN1A coding variants that can result in null expression.

A genetic variant can also be located at a splice site, resulting in alternative splicing of the ion channel gene. This process allows a single gene to produce alternative ion channels with different functional characteristics. In particular, for SCN1A many alternative splicing mutations have been identified in epilepsy pathogenesis (Lossin, 2009; Thompson et al., 2011; Carvill et al., 2018; **Table 1**).

If a genetic variant is located within the binding site of an activating or repressing transcription factor (TF) or a repressor, it may result in altered regulation of the transcriptional machinery. For example, four different haplotypes, consisting of 13 SNPs located in the 5' region of the GABRB3 gene were found to segregate with childhood absence epilepsy (CAE; Urak et al., 2006). The GABRB3 gene encodes the β3 subunit of the GABAA receptor which mediates phasic (synaptic) and tonic (perisynaptic) inhibition (Farrant and Nusser, 2005; Hirose, 2014). Functional analysis of these haplotypes revealed a reduced transcriptional activity of the GABRB3-haplotype-2 promoter (overrepresented in CAE) compared to the GABRB3-haplotype-1 promoter

### TABLE 1 | Transcriptional channelopathies implicated in epilepsies.

(Continued)

TABLE 1 | Continued

\*Epilepsy genes based on Online Mendelian Inheritance in Man (OMIM) database, Wang et al. (2017), Wei et al. (2017) and Oyrer et al. (2018). LOF, Loss-of-function; GOF, Gain-offunction; TF, Transcription Factor; miRNA, microRNA.

(overrepresented in controls). The difference in expression could be explained by reduced binding of the TF N-Oct3 to the GABRB3-haplotype-2 promoter, resulting in decreased expression of the GABRB3 gene (Urak et al., 2006). The reduced β3 levels observed in CAE patients might cause a loss of inhibitory properties of the receptor, eventually causing seizure activity.

### ION CHANNELS IN ACQUIRED EPILEPSIES

Acquired epilepsies are epilepsies, which are on the consequence of an environmental factor. These epilepsies can be: (i) completely dependent on environmental factors; or (ii) can be caused by an interaction of environmental factor(s) with a predisposition genome. In the latter case, the presence of common susceptibility variants (e.g., SNPs or CNVs) can lower the threshold of the environmental factor for inducing an epileptic outcome. Most of our current knowledge of acquired epilepsy pathogenesis comes from the use of animal models, in which insults causing TLE can be mimicked in rodents using approaches like traumatic brain injury, kindling, or by applying one of the chemo-convulsants pilocarpine or kainic acid to induce SE (reviewed by Jefferys et al., 2016; Lévesque et al., 2016; Becker, 2018; Nirwan et al., 2018).

## TRANSCRIPTIONAL REGULATION OF ACQUIRED ION CHANNELOPATHIES

Currently, one of the main questions in epilepsy research is how the expression of ion channel genes in acquired epilepsies can be regulated. The transcriptional regulation of ion channels in acquired epilepsy can occur for example via differential expression of transcriptional activators or repressors. After a brain insult, a transient increase of activity-regulated TFs is evident (e.g., Egr-4, Fos, Jun and Arc), which can as a consequence dysregulate the transcriptional machinery of many genes, including ion channel genes (Herdegen et al., 1993; Beer et al., 1998; Herdegen and Leah, 1998; Honkaniemi and Sharp, 1999). To date, several transcriptional mechanisms have already been identified in the context of channelopathies and epilepsy pathogenesis (**Table 1**).

For CaV3.2, we recently performed an in-depth promoter analysis, examining the molecular mechanisms involved in the transcriptional augmentation of this channel early after pilocarpine-induced SE (Becker et al., 2008). Here, we observed a highly-sophisticated mechanism of transcriptional regulation: the increase of CaV3.2 expression was found to be mediated by metal-regulatory transcription factor 1 (MTF1) upon a rise in intracellular zinc ([Zn2+]i); denoted as the Zn2+- MTF1-CaV3.2 cascade (van Loo et al., 2015). A rise in [Zn2+]i , often seen after a transient insult to the brain (Assaf and Chung, 1984; Zhao et al., 2014), can activate MTF1, which then binds to metal-responsive elements within the CaV3.2 promoter region. Consequently, this results in increased CaV3.2 expression, a larger T-type current and increased burst-firing behavior (van Loo et al., 2015). In this way, the Zn2+-MTF1-CaV3.2 cascade can enhance hippocampal network excitability, resulting in seizure activity. Besides the Zn2+-MTF1-CaV3.2 cascade, also other TFs were found to control CaV3.2 expression, including Egr1 and RE1-silencing transcription factor (REST; van Loo et al., 2012). Such a multifactorial regulation by several TFs, thought to be a general phenomenon of ion channel regulation, severely complicates pharmacological intervention.

### EPIGENETIC CONTROL OF ACQUIRED ION CHANNELOPATHIES

The transcription of ion channels in acquired epilepsies can also be regulated at the epigenetic level: both DNA methylation at cytosine residues, as well as changes in histone modifications (e.g., acetylation or methylation) can strongly affect the transcriptional machinery (reviewed by Hauser et al., 2018). Methylation of DNA generally occurs at cytosines within the 5'-cytosine-guanine-3' context (CpG). Gene promoters often contain large clusters of CpGs (referred to as CpG islands), which are mostly hypomethylated and are linked to transcriptional activation. An increase in DNA methylation may cause reduced transcriptional activity due to physical inhibition of TF binding to their cognate DNA binding motif, or by binding of repressor proteins known as methyl-CpG binding domain proteins (MBDs) to the methylated DNA. In the latter case, MBDs can recruit histone deacetylases (HDAC1 and HDAC2) to the methylated DNA, resulting in the silencing of the corresponding gene (Clouaire and Stancheva, 2008). To date, several epilepsychannelopathies have been described to be caused by an epigenetic control mechanism (**Table 1**).

# REGULATION OF ACQUIRED ION CHANNELOPATHIES BY microRNAs

The transcriptional machinery of ion channels in acquired epilepsies can also be influenced by small non-coding RNAs, such as microRNAs (miRNAs). miRNAs are 22 nucleotides noncoding RNAs that can regulate gene expression by associating with the RNA-induced silencing complex (RISC). The RISC complex then uses the miRNA as a template for recognizing the complementary mRNA of the ion channel gene (Ranganathan and Sivasankar, 2014). The main function of miRNAs appears to be the regulation at the post-transcriptional level: either by hindering protein translation or by enhancing mRNA degradation. Nowadays, it is also debated that miRNAs can have a nuclear function by modulating gene expression directly at the transcriptional level (reviewed by Catalanotto et al., 2016). Numerous miRNAs have been identified in relation to epilepsy pathogenesis (reviewed by Reschke and Henshall, 2015; Henshall et al., 2016; Shao and Chen, 2017; Tiwari et al., 2018), and also several ion channels appear to be under control of miRNAs, including Kv1.1, Kv4.2, Kcnk10 and Grin2a (Sosanya et al., 2013; Alsharafi et al., 2016; Gross et al., 2016; Haenisch et al., 2016; Tiwari et al., 2019; **Table 1**).

### TRANSCRIPTIONAL REGULATION OF CHANNELOPATHIES IN NEURODEVELOPMENTAL DISORDERS

To date, it is generally accepted that ion channelopathies are not unique for epilepsy pathogenesis, but have gained considerable attention for the pathogenesis of several neurodevelopmental disorders, including pathology aspects of autism spectrum disorders (ASDs), schizophrenia, bipolar disorder, major depressive disorder and migraine (reviewed by Imbrici et al., 2013; Schmunk and Gargus, 2013; Albury et al., 2017). Seizures have been noted as a comorbidity feature of neurodevelopmental disorders (Hyde and Weinberger, 1997; Canitano, 2007; Mula et al., 2008; Liao et al., 2018; Salpekar and Mula, 2018; Strasser et al., 2018), which overall may point to the emergence of a functionally impaired neuronal network. For many neurodevelopmental disorders, several genetic variations (both rare mutations as well as common variants) in the coding regions of ion channel genes have been identified and reviewed elsewhere (Imbrici et al., 2013; Schmunk and Gargus, 2013; Daghsni et al., 2018; Weiss and Zamponi, 2019). Interestingly, also the mechanisms described above to be involved in the transcriptional regulation of ion channels in epilepsy pathogenesis, have been observed in the regulation of ion channels in other neurodevelopmental diseases. For example, transcriptional regulation by presence of genetic variants within the promoter region was observed for Grin2a and Grin2b and resulted in schizophrenia pathogenesis (Miyatake et al., 2002; Itokawa et al., 2003; Liu et al., 2015); alternative splicing was documented for Gabrb2, Grin2b and Gabra3 and resulted in mental retardation and ASD (Zhao et al., 2009; Endele et al., 2010; O'Roak et al., 2012; Piton et al., 2013); differential expression of TFs was observed for Scn10a, Kcnq1, Cacna1c, and Grin1 in schizophrenia and other psychiatric disorders (Rannals et al., 2016; Billingsley et al., 2018; Page et al., 2018); epigenetic control mechanisms were described for Gabrb2, Gabrb3, Gria, and Chrna7 in ASD, schizophrenia and Rett syndrome (Samaco et al., 2005; Yasui et al., 2011; Kordi-Tamandani et al., 2013; Zong et al., 2017) and regulation by miRNAs was observed for Cacna1c, Cacnb1, Grin2b and NMDAR in schizophrenia and ASD (Kocerha et al., 2009; Guan et al., 2014; Cammaerts et al., 2015; Zhang et al., 2015; Kichukova et al., 2017).

Many of these ''transcriptional channelopathies'' apparently are rather specific, since they are mostly associated with only one individual neurodevelopmental disorder. However, a few examples exist, in which a comparable transcriptional regulatory mechanism has been observed for channelopathies in epilepsy and other neurodevelopmental disorders, hinting at an explanation for the comorbidity seen between the different disorders. One such example is the GABRB3 gene, an important neurodevelopmental gene and besides epilepsy also associated with Angelman syndrome, Rett syndrome and ASD (Tanaka et al., 2012b). Differential expression of GABRB3 in CAE can be caused by genetic variants within the promoter region (Urak et al., 2006). Interestingly, one of these variants is also associated with schizophrenia and heroin dependence (Chen et al., 2014b; Liu et al., 2019), whereas other genetic variants within the same regulatory region are correlated with ASD (Chen et al., 2014a).

Another example of comparable transcriptional control mechanisms in epilepsy and neurodevelopmental disorders was observed for the NMDAR gene Grin2b. The expression of Grin2b was significantly decreased in the kainic acid-induced SE model and correlated with increased DNA methylation levels at specific CpGs located within the Grin2b locus. Additionally, interfering with the DNA methylation levels prior to SE using a DNA-methyltransferase inhibitor, prevented the Grin2b DNA methylation increase after SE and resulted in augmented Grin2b mRNA and protein expression (Parrish et al., 2013).

Such a glutamatergic hypofunction caused by an epigenetic control mechanism in Grin2b in the epilepsy model can also contribute to the pathophysiology of other neurodevelopmental disorders (Coyle et al., 2002; Lau and Zukin, 2007). Recently, it was reported that in a mouse model for schizophrenia, Grin2b expression was also under control of epigenetic control mechanisms. Here, the reduction in Grin2b expression was caused by an increase in H3K27me3 and REST at the Grin2b promoter (Gulchina et al., 2017). We may thus assume that different neurodevelopmental disorders are associated with a channelopathy with similar underlying transcriptional mechanisms.

Although we see comparable transcriptional mechanisms, no large overlap exists between the individual regulatory players in epilepsy pathogenesis and other neurodevelopmental disorders. Of course, this can also be explained by the fact that most specific mechanisms simply have not been analyzed in all neurodevelopmental disorders, or not in an analogous manner, making a direct comparison at the moment impossible. Further studies will reveal whether the altered diseases-associated expression of more proteins is based on a (partly) general underlying transcriptional phenomenon, possibly explaining the comorbidity between epilepsy and other neurodevelopmental disorders.

### REFERENCES

### FUTURE PERSPECTIVES

In this study, we reviewed the mechanisms involved in the transcriptional regulation of channelopathies in genetic and acquired epilepsies. Although a large amount of data exists, our understanding of transcriptional mechanisms governing ion channel expression is far from complete and requires further detailed investigation, not only for epilepsy pathogenesis, but also for other neurodevelopmental disorders. A better understanding of the underlying mechanisms might result in the development of novel drugs and may provide opportunities for betterindividualized treatment strategies.

### AUTHOR CONTRIBUTIONS

Both authors contributed to the writing and editing of the manuscript.

### FUNDING

This work was supported by the Deutsche Forschungsgemeinschaft (SFB 1089: KL, AB), FOR 2715 (AB), BMBF (EraNet DeCipher to AB), the European Union's Seventh Framework Programme (FP7/2007–2013) under grant agreement n◦ 602102 (EPITARGET; AB), Else Kröner-Fresenius-Foundation 'Promotionskolleg NeuroImmunology' (AB) as well as the BONFOR program of the University of Bonn Medical Center.

**Conflict of Interest**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a past co-authorship with one of the authors AB.

Copyright © 2020 van Loo and Becker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Epilepsy of Infancy With Migrating Focal Seizures: Identification of de novo Mutations of the KCNT2 Gene That Exert Inhibitory Effects on the Corresponding Heteromeric KNa1.1/KNa1.2 Potassium Channel

### Edited by:

Eleonora Palma, Sapienza University of Rome, Italy

### Reviewed by:

Amy McTague, University College London, United Kingdom Terence Hébert, McGill University, Canada

### \*Correspondence:

Hua Wang<EMAIL_ADDRESS>Pierre Szepetowski<EMAIL_ADDRESS>Laurent Aniksztejn<EMAIL_ADDRESS>
†These authors have contributed equally to this work

> Received: 11 July 2019 Accepted: 06 January 2020 Published: 24 January 2020

### Citation:

Mao X, Bruneau N, Gao Q, Becq H, Jia Z, Xi H, Shu L, Wang H, Szepetowski P and Aniksztejn L (2020) The Epilepsy of Infancy With Migrating Focal Seizures: Identification of de novo Mutations of the KCNT2 Gene That Exert Inhibitory Effects on the Corresponding Heteromeric KNa1.1/KNa1.2 Potassium Channel. Front. Cell. Neurosci. 14:1. doi: 10.3389/fncel.2020.00001 Xiao Mao1,2† , Nadine Bruneau3† , Quwen Gao4† , Hélène Becq3 , Zhengjun Jia1,2 , Hui Xi 1,2 , Li Shu1,2 , Hua Wang1,2\*, Pierre Szepetowski 3 \* and Laurent Aniksztejn3 \*

1Hunan Provincial Maternal and Child Health Care Hospital, Changsha, China, 2NHC Key Laboratory of Birth Defects Research, Prevention and Treatment, Changsha, China, 3 INSERM, Aix-Marseille University, INMED, UMR1249, Marseille, France, 4Department of Epilepsy, General Hospital of Southern Theater Command, Guangzhou, China

The epilepsy of infancy with migrating focal seizures (EIMFS; previously called Malignant migrating partial seizures of infancy) are early-onset epileptic encephalopathies (EOEE) that associate multifocal ictal discharges and profound psychomotor retardation. EIMFS have a genetic origin and are mostly caused by de novo mutations in the KCNT1 gene, and much more rarely in the KCNT2 gene. KCNT1 and KCNT2 respectively encode the KNa1.1 (Slack) and KNa1.2 (Slick) subunits of the sodium-dependent voltage-gated potassium channel KNa. Functional analyses of the corresponding mutant homomeric channels in vitro suggested gain-of-function effects. Here, we report two novel, de novo truncating mutations of KCNT2: one mutation is frameshift (p.L48Qfs43), is situated in the N-terminal domain, and was found in a patient with EOEE (possibly EIMFS); the other mutation is nonsense (p.K564∗ ), is located in the C-terminal region, and was found in a typical EIMFS patient. Using whole-cell patch-clamp recordings, we have analyzed the functional consequences of those two novel KCNT2 mutations on reconstituted KNa1.2 homomeric and KNa1.1/KNa1.2 heteromeric channels in transfected chinese hamster ovary (CHO) cells. We report that both mutations significantly impacted on KNa function; notably, they decreased the global current density of heteromeric channels by ∼25% (p.K564∗ ) and ∼55% (p.L48Qfs43). Overall our data emphasize the involvement of KCNT2 in EOEE and provide novel insights into the role of heteromeric KNa channel in the severe KCNT2-related epileptic phenotypes. This may have important implications regarding the elaboration of future treatment.

Keywords: epilepsy of infancy with migrating focal seizures, KNa channels, KCNT genes, epilepsy, encephalopathy

### Mao et al. KCNT2 Mutations in Epileptic Encephalopathies

# INTRODUCTION

Channelopathies represent an important cause of neurological disorders (Kumar et al., 2016). Dysfunction of potassium channels has notably been involved in various types of epileptic encephalopathies, including epilepsy of infancy with migrating focal seizures (EIMFS), previously known as malignant migrating partial seizures of infancy. EIMFS are rare, neonatal epilepsies characterized by onset before the age of 6 months, and usually during the first weeks of life, by continuous migrating polymorphous focal seizures with corresponding multifocal ictal electroencephalographic (EEG) discharges associated with progressive deterioration of psychomotor development (Coppola et al., 1995). EIMFS have a genetic origin and can be caused by de novo mutations in the KCNT1 gene encoding the KNa1.1 subunit (Slack or Slo2.2) of KNa channels (Barcia et al., 2012; Ishii et al., 2013; McTague et al., 2013; Rizzo et al., 2016). More recently, two pathogenic mutations in the KCNT2 gene encoding the KNa1.2 subunit (Slick or Slo2.1) have been reported (Gururaj et al., 2017; Ambrosino et al., 2018). KNa channels are voltagegated potassium channels that are activated by an increase of cytoplasmic Na+ concentration.  These subunits co-assemble to form homo or tetraheteromeric KNa channels. Each subunit is composed of six transmembrane segments and of two intracellular N and C terminal domains (**Figure 1**). These two subunits display structural differences notably regarding their distal C-terminal region, their electrophysiological properties, their responses to neuromodulators, and their sensitivities to changes in cell volume (Bhattacharjee et al., 2003; Santi et al., 2006; Kaczmarek, 2013; Tejada et al., 2017). The C terminal part contains consensus sites for Na+ within the RCK2 (regulator of conductance of K+ ) domain and interaction sites for cytoplasmic proteins (e.g., protein kinase C). In KNa1.2 but not KNa1.1, the C-terminus also harbors a binding site for ATP, which function remains elusive (Bhattacharjee et al., 2003; Berg et al., 2007; Kaczmarek, 2013; Garg and Sanguinetti, 2014; Kaczmarek et al., 2016; Gururaj et al., 2017). In heterologous cells, functional analysis of mutant channels associated with EIMFS mostly revealed gain of function effects: potassium current was increased in cells expressing homomeric KNa1.1 channels harboring either of the p.Val271Phe, p.Gly288Ser, p.Arg398Gln, p.Arg428Gln, p.Arg474His, p.Met516Val, p.Lys629Asn, p.Ile760Met, p.Pro924Leu or p.Ala934Thr missense mutations, and in cells expressing homomeric KNa1.2 channels harboring either of the p.Arg190His or p.Arg190Pro missense mutations (Barcia et al., 2012; Rizzo et al., 2016; Villa and Combi, 2016; Ambrosino et al., 2018). A change in channel function has also been described in cells expressing the KNa1.2 subunit harboring the p.Phe240Leu missense mutation: the mutant channel lost its selectivity to K+ ions and gained permissiveness to Na+ ions (Gururaj et al., 2017).

Here, we have used exome sequencing to identify two novel de novo nonsense and frameshift mutations of the KCNT2 gene in two patients with ascertained EIMFS and with EIMFS-like earlyonset epileptic encephalopathies (EOEE), respectively. We have investigated the functional consequences of the two mutations in heterologous cells expressing heteromeric channels and showed that both mutations reduced whole-cell potassium current. Therefore, EIMFS may be caused not only by an increase but also by a decrease in the function of KNa.

# MATERIALS AND METHODS

# Patients

The two patients with KCNT2 mutations were recruited at Hunan Provincial Maternal and Child Health Care Hospital. Leukocyte DNA was extracted from peripheral blood stored in EDTA tubes by the phenol-chloroform method. Clinical information was collected by experienced neurologists. Patients' parents had informed consents and the study was approved by the Ethics Committee of Hunan Provincial Maternal and Child Health Care Hospital.

## Exome Sequencing

Patients' DNAs were analyzed by next-generation sequencing with the whole-exome sequencing (WES) approach. DNA fragments were sequenced on the HiSeq2500 system (Illumina, San Diego, CA, USA) with a mean depth of 100×. A preliminary processing of WES data (data alignment and filter) followed pipelines as previously reported (Wang et al., 2011).

The annotated data by ANNOVAR (version 20160201; Wang et al., 2011) was used for further data analyses. Public databases (1000Genome, ESP6500, ExAC, dbSNP, and gnomAD) were used to filter known variants with minor allele frequencies (MAFs) over 0.001. Bioinformatics software (PolyPhen, SIFT, CADD or Mutation Taster) were used to predict the pathogenicity of single-nucleotide variations (SNVs). The loss-of-function variants (nonsense variants, frameshift variants, and splicing variants) and predicted pathogenic SNVs were retained. ACMG guidelines were finally used to evaluate the pathogenicity of the variants (Richards et al., 2015).

Sanger sequencing was performed on patients' DNA to validate the findings of WES and on parents' DNA to study familial inheritance. Sequencing primers were designed according to the sequences of the detected variants and polymerase chain reaction amplification was carried out for Sanger sequencing.

### Constructs and Site-Directed Mutagenesis

The human KCNT1 cDNA construct (Genecopoeia EX-Y5001- M02, thereafter designated as pKCNT1) was used for the expression of wild-type human KNa1.1 subunit (NM\_020822). Two human KCNT2 cDNA constructs (Genecopoeia EX-Y5628-M61and EX-Y5628-M83, thereafter designated as pKCNT2-ires-GFP and pKCNT2-ires-mCherry, respectively) were used for expression of mutant or wild-type human KNa1.2 subunits (NM\_198503) coupled with internal ribosome

entry site (IRES)-driven independent expression of eGFP (green fluorescent protein) or mcherry protein, respectively.

Mutant KCNT2 constructs were generated from their wild-type counterpart by using QuikChange Lightning Site-Directed Mutagenesis Kit according to the manufacturer's protocol (Agilent Technologies) and the following forward and reverse primers: KCNT2-A1690T: 50 -tctctgctg gtcttggttttaaaatgctgaattctcttctttg and 50 -caaagaagagaattcagc attttaaaaccaagaccagcagaga (KNa1.2K564∗ ); KCNT2-del143- 144: 50 -cttgatctctggttttttatgaaaaataatttgtctttctttaaatgtattttcattcat atag and 50 -ctatatgaatgaaaatacatttaaagaaagacaaattatttttcataaaaaac cagagatcaag(KNa1.2L48Qfs43). KCNT2 sequences from wild-type and mutant constructs were all verified by Sanger sequencing (GATC Biotech).

### Cell Cultures and Transfections

Chinese hamster ovary (CHO) cells were cultured at 37◦C in a humidified atmosphere with 5% CO2 with an F-12 Nutrient Mixture (Life Technologies) supplemented with 10% FBS (Fetal Bovine Serum) and 100 units/ml antibiotics/antimycotics (Life Technologies). These cells were transiently transfected using the Neonr Transfection System (Life Technologies) according to the manufacturer's protocol. Briefly, 105 cells in suspension were transfected with a total amount of 2 µg of DNA. Non-recombinant pcDNA3.1 was added if necessary and concentrations were adjusted to get a total amount of 2 µg of DNA. Electroporation configuration was 1,400 V, 1 pulse, 20 ms. Cells were transiently co-transfected with pKCNT1 and either of pKCNT2-Mutant-ires-GFP or pKCNT2-WT-ires-mCherry or both (see below). Following electroporation, cells were cultured on pre-coated glass coverslips and maintained at 37◦C and 5% CO2 with a complete medium for 2 days before recordings. Combinations of plasmids used in this study are shown in **Table 1**. The eGFP and mCherry fluorescent dyes were used to ascertain the efficacy of transfection assays and select cells for recordings.

### Electrophysiology

CHO cells were perfused at 1–2 ml/min with the following solution (in mM): 135 NaCl, 3.5 KCl, 5 NaHCO3, 0.5 NaH2PO4, 1 MgCl2, 1.5 CaCl2, 10 HEPES, 10 glucose, and pH 7.3 adjusted with NaOH. Whole-cell patch-clamp recordings were performed with microelectrodes (borosilicate glass capillaries GC 150F-15, Harvard apparatus) filled with a solution containing (in mM): 135 KCl, 0.1 CaCl2, 1.1 EGTA, 10 HEPES, 3 Mg2+ATP, 0.3 Na+GTP, 4 phosphocreatine, pH 7.3 adjusted with KOH and a resistance of 4–6 M. In some experiments, ATP and GTP were omitted from the internal pipette solution. Data were sampled at 10 kHz and filtered with a cut-off frequency of 3 kHz using an EPC-10 amplifier (HEKA Electronik). An hyperpolarizing voltage step of 10 mV during 500 ms followed by incremental depolarizing voltage steps command of 10 mV was applied from a holding potential of −90 mV and up to +110 mV in order to analyze current densities and the conductance–voltage

n, number of recorded cells. Values of voltage for half-maximal activation of potassium channel (V1/2) and of slope factor (k) of conductance/voltage relationships are indicated.

(G–V) relationships. Current densities (expressed in pA/pF) were calculated by measuring current amplitude at the end of the voltage step divided by the capacitance (Cm). G values were obtained from peak amplitudes of the slow outward current divided by the driving force for K+ ions with EK ∼−93 mV and normalized to the maximal conductance. Plotted points were fitted with a Boltzmann function: G/Gmax = 1/[1 + exp(V1/2 − Vm)/k] to yield the voltage for half-maximum activation (Vhalf) and the slope factor (k) values. Currents were analyzed using Origin 8.0 software. Analyses were performed after offline leak current subtraction. Membrane potentials were corrected for liquid junction potential (∼5 mV).

### Statistical Analyses

Data are represented as means ± SEM. Two-way ANOVA with Tukey's correction for multiple testing or Kruskal–Wallis test were used to assess statistical significance; ∗ adjusted p < 0.05; ∗∗adjusted p < 0.01; ∗∗∗adjusted p < 0.001.

### RESULTS

### Identification of Two Novel de novo KCNT2 Defects in Patients With Early-Onset Epileptic Encephalopathies