Epilepsy is a common neurological disease affecting 0.5–1% of the general population that occurs in various conditions, in which recurrent ‘epileptic seizures‘ are induced. Currently, 20–30% of patients have seizures that are not well controlled by available treatments. Dravet syndrome (DS) is one of these intractable epilepsies, and is characterized by infantile-onset seizures and severe intellectual disability Citation[1]. The disease was named after Charlotte Dravet who first described this condition in 1978. Although rare (the incidence is one in 20,000–40,000), DS has been a focus point in epilepsy research since a robust relationship between the clinical phenotype and genetic cause has been established.
In DS, initial seizures occur in the form of generalized or unilateral convulsive seizures that are frequently associated with fever or simply an elevation in body temperature (e.g., hot-water bathing) Citation[1]. The seizures tend to be prolonged, frequently evolving into status epilepticus, particularly when no therapeutic intervention is made. Other types of seizures, including focal, absence and myoclonic, appear subsequently. Although initially normal, psychomotor development stagnates after seizure onset, and it is often accompanied by hyperactive behaviors and autistic traits. Generalized convulsions and severe cognitive decline persist over the course of patients‘ lives. Ataxia also progresses with age and leads to gait disturbance. Unexpected death at a young age is another critical issue, and 10–20% of the afflicted children die prematurely Citation[2].
Currently, treating DS remains a challenge. Although stiripentol, topiramate and potassium bromide may show efficacy for suppressing seizures, many patients remain epileptic and suffer developmental stagnation Citation[3]. However, despite its progressive nature, DS may be a curable disease. Psychomotor milestones are normal before seizure onset; some patients who attained good initial seizure control showed excellent seizure and intellectual outcomes Citation[4]; and neuronal death was not accelerated in the autopsy of brain specimens of adult patients Citation[5]. These findings strongly imply that early interventions with some new and improved treatments could significantly improve the prognosis of DS.
Genetic defects in SCN1A, which encodes the α1-subunit of the voltage-gated sodium channel NaV1.1, were revealed as a primary cause of DS in 2001 Citation[6]. This has significantly accelerated research into this condition, more specifically by using murine DS models, as well as our understanding of DS pathogenesis Citation[7,8]. It is currently known that many DS mutations in SCN1A cause a significant deterioration in NaV1.1 function (i.e., producing a nonfunctional channel) Citation[9]; haploinsufficiency of this channel may be the primary mechanism Citation[10]. A functional decline in the GABAergic interneurons, where NaV1.1 is primarily expressed in the mouse neocortex and hippocampus, may cause the major symptoms of DS Citation[7,8]; and a functional decline in Purkinje neurons, where NaV1.1 is also strongly expressed, may cause ataxia Citation[11].
Recent studies have revealed that forebrain GABAergic interneuron-specific knockout of SCN1A, which was accomplished by crossing floxed Scn1a mice and Dlx1/2 enhancer-Cre mice, may be sufficient to induce major features of DS – increased seizure susceptibility, high temperature sensitivity of seizures, premature death and autistic traits Citation[12–14]. These features were also observed in mice with global SCN1A defects. These findings indicate that defective GABAergic inhibition in the forebrain may be the key factor in the pathogenesis of DS.
Even so, some researchers observed electrophysiological findings suggesting that in neuronal hyperexcitability Citation[15], not as a result of defective inhibition but by itself, may also underlie epileptogenesis in DS. In both murine models and human patients, the actual underlying pathogenesis may be much more complex, and extensive and systematic approaches must be employed to elucidate the full pathogenesis: determining the functional alteration specific to each neuronal subtype (e.g., glutamatergic neurons, and parvalbumin-, calretinin- and somatostatin-positive subtypes of GABAergic neurons) and each brain region (various cortical and subcortical regions) in both murine and human cells. Accordingly, as a first step to establish a research platform using human cells, we recently generated patient-derived induced pluripotent stem cells (iPSCs) from a DS patient and identified a functional decline in their differentiated GABAergic neurons Citation[16].
In this experiment, the donor patient exhibited a nuclear phenotype of DS and harbored a nonsense mutation in SCN1A. Two lines of iPSCs were established from the patient‘s skin fibroblasts, and an iPSC line, 201B7, which was generated from a healthy female, was used as a control Citation[17]. iPSC-derived neurons were highly heterogeneous regarding neuronal subtypes and their maturities – most were immature. Thus, we generated a reporter lentivirus that selectively detects live SCN1A-expressing neurons. The majority of the reporter-positive neurons also showed GABA immunostaining (i.e., they were GABAergic neurons). After further selection of mature neurons by their morphology and electrophysiological properties, functional capability of these neurons was examined by comparing the depolarizing current evoked-firing properties between the patient and the control neurons. Although no differences were observed for small currents, patient neurons exhibited impaired firing patterns when the currents intensified: a lower firing rate, stronger attenuation in firing amplitude and more frequent occurrence of depolarization block was observed in the patient compared with the control neurons. These data indicated that a limited output capacity in response to intense input stimulation is present in the patient-derived neurons expressing SCN1A.
Our findings suggest that the ‘defective inhibition‘ pathogenesis that had been indicated in the forebrain of the murine DS models is also present in human DS patients Citation[7,8]. Combined with the currently available data on the efficacy of antiepileptic drugs for DS, enhancing GABAergic inhibition will also be an essential part of the treatment of this disease in the future Citation[3]. However, the present study, using patient-derived neurons, has confirmed only one aspect of the complex pathogenesis underlying DS, and the findings are not immediately beneficial for developing new treatments. Considering the intractable nature of DS even under multiple treatments that enhance GABAergic inhibition and the different suggested aspects of its pathogenesis (e.g., increased neuronal hyperexcitability), more comprehensive approaches are crucial. For this purpose, the future direction of iPSC-based DS research should establish methods that allow differentiation of cells into each specific subtype of mature neurons. Then, the functional and other cell biological properties of each type of neurons should be carefully examined. Such studies will help to identify critical pathogenic mechanisms, narrow down the therapeutic focus more specifically and promote the development of new treatments based on a comprehensive understanding of the pathophysiology involved.
Although our study primarily focused on elucidation of human DS pathogenesis, regenerative techniques using patient-derived iPSCs may have the potential to treat DS in future. Recently, Baraban et al. have reported that, in the murine kainic acid model of epilepsy, transplantation of GABAergic interneuron progenitors, which were obtained from the embryonic mouse medial ganglionic eminence, into the hippocampus ameliorated seizures, as well as behavioral deficits, significantly Citation[18]. Other lines of evidence also indicate the efficacy of GABAergic interneurons for suppressing epileptogenesis. Furthermore, recent genome-editing technologies using transcription activator-like effector nucleases (TALENs) Citation[19], clustered regularly interspaced short palindromic repeat (CRISPR)/Cas-based RNA-guided DNA endonucleases or helper-dependent adenoviral vectors Citation[20], hold the potential to repair gene mutations in patient-derived iPSCs. Therefore, if GABAergic interneuron progenitors could be induced from mutation-corrected iPSCs, transplantation of those induced progenitors into brain regions that are critical for the epileptogenesis of the original patients would be an exciting possibility for the future treatment of genetic epilepsies.
Disease modeling using patient-derived iPSCs has just begun, particularly in the field of epilepsy. iPSC technologies are rapidly advancing and will exponentially enhance the value and the utility of this model. This new research platform will significantly expand the possibilities for future research directions and therapeutics of human DS.
Financial & competing interests disclosure
N Higurashi was supported by the Japan Society for the Promotion of Science, the Japan Foundation for Pediatric Research, the Clinical Research Promotion Foundation and Kaibara Morikazu Medical Science Promotion Foundation. H Okano was supported by the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japan Science and Technology Agency. S Hirose was supported by the Japan Society for the Promotion of Science, the Japan Science and Technology Agency, the Ministry of Health, Labor and Welfare, the Mitsubishi Foundation and Fukuoka University. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
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References
- Dravet C , BureauM, OguniH, FukuyamaY, CokarO. Severe myoclonic epilepsy in infancy (Dravet syndrome). In: Epileptic Syndromes in Infancy, Childhood and Adolescence. RogerJ, BureauM, DravetC, GentonP, TassinariCA, WolfPS(Eds) . John Libbey Eurotext, Montrouge, France, 89–113 (2005).
- Sakauchi M , OguniH, KatoIet al. Retrospective multiinstitutional study of the prevalence of early death in Dravet syndrome. Epilepsia52(6), 1144–1149 (2011).
- Chiron C , DulacO. The pharmacologic treatment of Dravet syndrome. Epilepsia52(Suppl. 2), 72–75 (2011).
- Akiyama M , KobayashiK, YoshinagaH, OhtsukaY. A long-term follow-up study of Dravet syndrome up to adulthood. Epilepsia51(6), 1043–1052 (2010).
- Catarino CB , LiuJY, LiagkourasIet al. Dravet syndrome as epileptic encephalopathy: evidence from long-term course and neuropathology. Brain134(Pt 10), 2982–3010 (2011).
- Claes L , Del-FaveroJ, CeulemansB, LagaeL, Van BroeckhovenC, De JongheP. De novo mutations in the sodium-channel gene SCN1A cause severe myoclonic epilepsy of infancy. Am. J. Hum. Genet.68(6), 1327–1332 (2001).
- Yu FH , MantegazzaM, WestenbroekREet al. Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy. Nat. Neurosci.9(9), 1142–1149 (2006).
- Ogiwara I , MiyamotoH, MoritaNet al. NaV1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: a circuit basis for epileptic seizures in mice carrying an Scn1a gene mutation. J. Neurosci.27(22), 5903–5914 (2007).
- Lossin C , RhodesTH, DesaiRRet al. Epilepsy-associated dysfunction in the voltage-gated neuronal sodium channel SCN1A. J. Neurosci.23(36), 11289–11295 (2003).
- Bechi G , ScalmaniP, SchiavonE, RusconiR, FranceschettiS, MantegazzaM. Pure haploinsufficiency for Dravet syndrome Na(V)1.1 (SCN1A) sodium channel truncating mutations. Epilepsia53(1), 87–100 (2012).
- Kalume F , YuFH, WestenbroekRE, ScheuerT, CatterallWA. Reduced sodium current in Purkinje neurons from NaV1.1 mutant mice: implications for ataxia in severe myoclonic epilepsy in infancy. J. Neurosci.27(41), 11065–11074 (2007).
- Cheah CS , YuFH, WestenbroekREet al. Specific deletion of NaV1.1 sodium channels in inhibitory interneurons causes seizures and premature death in a mouse model of Dravet syndrome. Proc. Natl Acad. Sci. USA109(36), 14646–14651 (2012).
- Han S , TaiC, WestenbroekREet al. Autistic-like behaviour in Scn1a+/- mice and rescue by enhanced GABA-mediated neurotransmission. Nature489(7416), 385–390 (2012).
- Kalume F , WestenbroekRE, CheahCSet al. Sudden unexpected death in a mouse model of Dravet syndrome. J. Clin. Invest.123(4), 1798–1808 (2013).
- Liu Y , Lopez-SantiagoLF, YuanYet al. Dravet syndrome patient-derived neurons suggest a novel epilepsy mechanism. Ann. Neurol. doi:10.1002/ana.23897 (2013) (Epub ahead of print).
- Higurashi N , UchidaT, ChristophLet al. A human Dravet syndrome model from patient induced pluripotent stem cells. Mol. Brain6(1), 19 (2013).
- Takahashi K , TanabeK, OhnukiMet al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell131(5), 861–872 (2007).
- Hunt RF , GirskisKM, RubensteinJL, Alvarez-BuyllaA, BarabanSC. GABA progenitors grafted into the adult epileptic brain control seizures and abnormal behavior. Nat. Neurosci.16(6), 692–697 (2013).
- Gaj T , GersbachCA, BarbasCF 3rd. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol.31(7), 397–405 (2013).
- Liu GH , SuzukiK, QuJet al. Targeted gene correction of laminopathy-associated LMNA mutations in patient-specific iPSCs. Cell Stem Cell.8(6), 688–694 (2011).