Ion channels: genetics and as a targets for antiepileptic drugs

Dravet syndrome is caused by mutations of the SCN1A gene that encodes voltage-gated sodium channel alpha-1 subunit. SCN1A-knock-in mouse with a disease-relevant nonsense mutation that we generated reproduced the disease phenotypes. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the fi rst postnatal month. Our immunohistochemical studies showed that in wild-type mice Nav1.1 is dominantly expressed in parvalbumin-positive inhibitory interneurons (PV cells), intensely in its axons and moderately in somata, and mostly not observed in pyramidal cells nor other types of interneurons including somatostatin-positive and calretinin-positive cells. These results suggest that Nav1.1 is largely expressed in PV interneurons and plays critical roles in their spike output, and that impaired function of PV cells would be the cellular basis of Dravet syndrome. PV interneurons and Nav1.1 in those cells would be critical targets for antiepileptic drugs. Neurology Asia 2013; 18 (Supplement 1) : 13 – 14 Address correspondence to: Dr Kazuhiro Yamakawa, Laboratory for Neurogenetics, RIKEN Brain Science Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, JAPAN. E-mail: [email protected] SCN1A GENE MUTATIONS IN PATIENTS WITH DRAVET SYNDROME AND RELATED EPILEPSIES Mutations in SCN1A have been reported to be associated with several types of epilepsies including generalized epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome. Approximately 10%-20% of GEFS+ patients show SCN1A mutations, while approximately 80% of Dravet patients show the mutations. Most exclusively GEFS+ mutations are missense, while two-third of Dravet mutations are truncation mutations such as nonsense and frameshift and remained one-third are missense. In most cases, GEFS+ mutations are inherited, while Dravet mutations are de novo. Further we reported microdeletion mutations affecting SCN1A 5’ promoter region but not affecting the coding region. Missense mutations in SCN1A were also found in 70% of patients having intractable childhood epilepsy with generalized tonic-clonic (ICEGTC), an atypical Dravet syndrome that does not show myoclonic seizures. We also reported that SCN1A mutations associates with psychiatric phenotypes in addition to epilepsy. Dravet mutations are mostly sporadic. However, the fact that several SCN1A mutations were found in familial Dravet syndrome cases in that identical missense mutations were observed in family members with Dravet syndrome or ICEGTC as well as in those having idiopathic epilepsy, febrile seizures only, or even nonsymptomatic5, suggests that genetic backgrounds or environmental modifi ers, or more possibly mosaicisms in parents as shown in our previous study8 largely affects the disease phenotype. These observations further indicates that even in families with sporadic Dravet syndrome cases the Dravet syndrome risk for successive children are higher than the general population, and emphasize the importance of prenatal or preimplantation diagnoses. IN VITRO FUNCTIONAL STUDIES ON NAV1.1 MUTANTS A number of biophysical studies on SCN1A mutations have been reported, however some of those suggested increased activities of mutant channels likely to be enhancing neuronal excitability and others suggested decreased, and interpretation of obtained results still remain elusive. I previously proposed that the ultimate functional consequences of these mutations in the brain may be an overall reduction in channel activity, in which milder GEFS+ phenotypes may be explained by the intermediate reduction, complete loss of the channel activity for one allele, that means half amount of normal channel protein or haproinsuffi ciency, in Dravet syndrome, however in vivo studies were still required to prove it. Neurology Asia 2013; 18 (Supplement 1) 14 MOUSE WITH SCN1A-DEFICIENCY SHOWED SEVERE EPILEPTIC SEIZURES We then generated knock-in (KI) mice with an Scn1a nonsense mutation that appeared in three independent Dravet syndrome patients. Both homozygous and heterozygous knock-in mice developed epileptic seizures within the fi rst postnatal month. All homozygotes died before postnatal day 20 and averagely at 16 postnatal days. In contrast, ~25% and ~40% of heterozygotes died at one month and three months after birth respectively, and remained mice survived thereafter. In heterozygous knock-in mice, trains of evoked action potentials in these fast-spiking, inhibitory cells exhibited pronounced spike amplitude decrement late in the burst. A similar study of an Scn1a KO mouse has been reported by another group one year ahead, in which they also showed specifi c physiological dysfunction in GABAergic neurons. NAV1.1 IS DOMINANTLY EXPRESSED IN AXONS AND SOMATA OF PARVALBUMINE-POSITIVE INHIBITORY NEURONS Yu et al. also reported that the Nav1.1 expressionwas restricted to somata of both pyramidal andinhibitory neurons as described in previous otherstudies. In contrast, in our study we showed,by using three independent antibodies and byusing the KI mice as negative controls, thatthe Nav1.1 protein is expressed dominantly inaxons and moderately in somata of parvalbumin-positive inhibitory interneurons (PV cells). In theneocortex, periphery of somata of pyramidal cellsalso gave signals. Most of these signals wouldbe derived from the Nav1.1 located at the axontermini of the basket cells which are clinging ontothe somata of pyramidal cells, and would not bederived from the pyramidal cell itself. Our datasuggest that Nav1.1 is dominantly expressed inPV-positive inhibitory interneurons and playscritical roles in the spike output from these cells,and further suggest that the impaired functionof PV-interneurons caused by SCN1A mutationis the molecular and cellular basis of Dravetsyndrome. Based on our results, we proposethat PV interneurons and Nav1.1 in the cells arecritical targets for antiepileptic drugs of Dravetsyndrome and its related epilepsies.REFERENCES 1. Escayg A, MacDonald BT, Meisler MH, et al.Mutations of SCN1A encoding a neuronal sodiumchannel, in two families with GEFS+2. Nat Genet2000; 24:343-5.2. Sugawara T, Mazaki-Miyazaki E, Ito M, et al.Nav1.1 Mutations Cause Febrile seizures associatedwith afebrile partial seizures. Neurology 2001;57:703-5.3. Claes L, Del-Favero J, Ceulemans B, et al. De novomutations in the sodium-channel gene SCN1A causesevere myoclonic epilepsy of infancy. Am J HumGenet 2001; 68:1327-32.4. Sugawara T, Mazaki-Miyazaki E, Fukushima K, etal. Frequent Mutations of SCN1A Severe MyoclonicEpilepsy in Infancy. Neurology 2002; 58:1122-4.5. Fujiwara T, Sugawara T, Mazaki-Miyazaki E, et al.Mutations of sodium channel alpha type 1 (SCN1A)in intractable childhood epilepsies with frequentgeneralized tonic-clonic seizures. Brain 2003;126:531-46.6. Nakayama T, Ogiwara I, Ito K, et al. Deletions ofSCN1A 5’ genomic region with promoter activityin Dravet syndrome. Hum Mutat [Epub ahead ofprint]7. Osaka H, Ogiwara I, Mazaki E, et al. Patients witha sodium channel alpha 1 gene mutation show widephenotypic variation Epilepsy Res 2007; 75:46-51.8. Morimoto M, Mazaki E, Nishimura A, et al. SCN1AMutation Mosaicism in a Family with SevereMyoclonic Epilepsy in Infancy. Epilepsia 2006;47:1732-6.9. Lossin C, Wang DW, Rhodes TH, Vanoye CG, GeorgeAL Jr. Molecular basis of an inherited epilepsy.Neuron 2002; 34:877-84.10. Sugawara T, Tsurubuchi Y, Fujiwara T, et al. Nav1.1channels with mutations of severe myoclonic epilepsyin infancy display attenuated currents. Epilepsy Res2003; 54:201-7.11. Rhodes TH, Lossin C, Vanoye CG, Wang DW, GeorgeAL Jr. Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy. Proc Natl Acad Sci U S A. 2004; 101:11147-52. 12. Yamakawa K. Epilepsy and sodium channel gene mutations: gain or loss of function? Neuroreport