Iori Ohmori

Significant correlation of the SCN1A mutations and severe myoclonic epilepsy in infancy.

To investigate the possible correlation between genotype and phenotype of epilepsy, we analyzed the voltage-gated sodium channel alpha1-subunit (SCN1A) gene, beta1-subunit (SCN1B) gene, and gamma-aminobutyric acid(A) receptor gamma2-subunit (GABRG2) gene in DNAs from peripheral blood cells of 29 patients with severe myoclonic epilepsy in infancy (SME) and 11 patients with other types of epilepsy. Mutations of the SCN1A gene were detected in 24 of the 29 patients (82.7%) with SME, although none with other types of epilepsy. The mutations included deletion, insertion, missense, and nonsense mutations. We could not find any mutations of the SCN1B and GABRG2 genes in all patients. Our data suggested that the SCN1A mutations were significantly correlated with SME (p<.0001). As we could not find SCN1A mutations in their parents, one of critical causes of SME may be de novo mutation of the SCN1A gene occurred in the course of meiosis in the parents.

A Missense Mutation of the Gene Encoding Voltage-Dependent Sodium Channel (Nav1.1) Confers Susceptibility to Febrile Seizures in Rats

Although febrile seizures (FSs) are the most common convulsive syndrome in infants and childhood, the etiology of FSs has remained unclarified. Several missense mutations of the Nav1.1 channel (SCN1A), which alter channel properties, have been reported in a familial syndrome of GEFS+ (generalized epilepsy with febrile seizures plus). Here, we generated Scn1a-targeted rats carrying a missense mutation (N1417H) in the third pore region of the sodium channel by gene-driven ENU (N-ethyl-N-nitrosourea) mutagenesis. Despite their normal appearance under ordinary circumstances, Scn1a mutant rats exhibited remarkably high susceptibility to hyperthermia-induced seizures, which involve generalized clonic and/or tonic–clonic convulsions with paroxysmal epileptiform discharges. Whole-cell patch-clamp recordings from HEK cells expressing N1417H mutant channels and from hippocampal GABAergic interneurons of N1417H mutant rats revealed a significant shift of the inactivation curve in the hyperpolarizing direction. In addition, clamp recordings clearly showed the reduction in action potential amplitude in the hippocampal interneurons of these rats. These findings suggest that a missense mutation (N1417H) of the Nav1.1 channel confers susceptibility to FS and the impaired biophysical properties of inhibitory GABAergic neurons underlie one of the mechanisms of FS.

A CACNB4 mutation shows that altered Cav2.1 function may be a genetic modifier of severe myoclonic epilepsy in infancy

Mutations of SCN1A, encoding the voltage-gated sodium channel alpha1 subunit, represent the most frequent genetic cause of severe myoclonic epilepsy in infancy (SMEI). The purpose of this study was to determine if mutations in other seizure susceptibility genes are also present and could modify the disease severity. All coding exons of SCN1B, GABRG2, and CACNB4 genes were screened for mutations in 38 SCN1A-mutation-positive SMEI probands. We identified one proband who was heterozygous for a de novo SCN1A nonsense mutation (R568X) and another missense mutation (R468Q) of the CACNB4 gene. The latter mutation was inherited from his father who had a history of febrile seizures. An electrophysiological analysis of heterologous expression system exhibited that R468Q-CACNB4 showed greater Ba(2+) current density compared with the wild-type CACNB4. The greater Ca(v)2.1 currents caused by the R468Q-CACNB4 mutation may increase the neurotransmitter release in the excitatory neurons under the condition of insufficient inhibitory neurons caused primarily by the SCN1A mutation.

Acute encephalopathy in children with Dravet syndrome

Purpose:  The occurrence of acute encephalopathy in children with Dravet syndrome has been reported sporadically. This study clarified the features of acute encephalopathy in children with Dravet syndrome.

Long-term follow-up of childhood epilepsy associated with tuberous sclerosis

Summary: Purpose: To study the clinical and electroencephalographic (EEG) characteristics of patients whose epilepsy is associated with tuberous sclerosis, with special reference to their clinical course.

A protein transduction method using oligo-arginine (3R) for the delivery of transcription factors into cell nuclei

Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer.

Antidepressant-like effect of sildenafil through oxytocin-dependent cyclic AMP response element-binding protein phosphorylation

Oxytocin (OT) levels in plasma increase during sexual response and are significantly lower in patients with depression. A drug for the treatment of sexual dysfunction, sildenafil, enhances the electrically evoked release of OT from the posterior pituitary. In this study, we showed that sildenafil had an antidepressant-like effect through activation of an OT signaling pathway. Application of sildenafil reduced depression-related behavior in male mice. The antidepressant-like effect was blocked by an OT receptor (OTR) antagonist and was absent in OTR knockout (KO) mice. Sildenafil increased the phosphorylation of cAMP response element-binding protein (CREB) in the hippocampus. The OTR antagonist inhibited sildenafil-induced CREB phosphorylation and sildenafil had no effect on CREB phosphorylation in OTR KO mice. These results suggest sildenafil to have an antidepressant-like effect through the activation of OT signaling and to be a promising drug for the treatment of depression.

Combining poly-arginine with the hydrophobic counter-anion 4-(1-pyrenyl)-butyric acid for protein transduction in transdermal delivery.

Topical therapy is the most favored form of treatment for whitening against hyperpigmentation and sunburn because it lends itself to self-administration, patient compliance, and absence of systemic adverse effects. However, transdermal delivery of hydrophilic chemicals is difficult. The main purpose of this study is to develop a delivering system of hydrophilic drugs and proteins across the skin. Hydroquinone (HQ), a well-known tyrosinase inhibitor and antimelanogenesis compound, and enhanced green fluorescent protein (EGFP) were fused with eleven poly-arginine (11R). Both HQ-11R and EGFP-11R were efficiently delivered in B16 cells, a mouse melanoma cell line. HQ-11R was as effective as HQ alone at inhibiting melanin synthesis in B16 cells. EGFP-11R was efficiently delivered into cells of the epidermis with 4-(1-pyrenyl)-butyric acid (PB), a counteranion bearing an aromatic hydrophobic moiety, in vivo, but EGFP alone or EGFP-11R without PB was not. Finally, topical application of HQ-11R with PB significantly inhibited UV irradiation-induced pigmentation in guinea pigs compared with HQ alone. These results suggest that topical therapy using poly-arginine in combination with PB is useful for the delivery of hydrophilic drugs and proteins by the transdermal route.

Nationwide survey (incidence, clinical course, prognosis) of Rasmussen’s encephalitis

PURPOSE Rasmussens encephalitis (RE) is a progressive and catastrophic epileptic disorder caused by chronic localized encephalitis. We performed a nationwide survey of RE to assess the clinical picture, treatment effect, and prognosis of Japanese RE patients. SUBJECTS & METHODS The subjects were 27 patients (male:12; female:15) from 13 medical facilities. All of them satisfied the clinical and neuroimaging criteria for RE, including 14 pathologically proven cases. RESULTS They were divided into the childhood-onset rapidly progressive type (CORP, n=19), and late-onset slowly progressive type (LOSP, n=8). The mean age at epilepsy onset was 4 years and 4 months in CORP, and 16 years in LOSP. The mean period between the onset age of epilepsy and development of frequent seizures was 1 year and 4 months in the former, and 3 years and 4 months in the latter. The immunomodulatory treatment including high-dose steroid (n=14) and high-dose intravenous immunoglobulin therapies (IVIgG, n=12) achieved more than a 50% reduction in the seizure frequency in 5 (36%) and 4 (33%) patients, respectively. Eight and seven patients underwent focal cortical resection and functional hemispherectomy, leading to significant improvement in 5 of the 8 patients and excellent seizure control in all 7 patients, respectively. CONCLUSION Although the high-dose steroid and IVIG therapies may have alleviated the exacerbation of seizures in those with RE, they could not halt the disease progression. Functional hemispherectomy is still the only curative therapy for RE, despite the fact that the early introduction of this procedure remains controversial.

Mutation screen of GABRA1, GABRB2 and GABRG2 genes in Japanese patients with absence seizures

Absence seizures are classified into typical and atypical absences according to clinical and EEG characteristics. Although missense mutations in the GABA(A) receptor gamma2 subunits (GABRG2) gene have recently been detected in two families with typical absence seizures, no study has been carried out to clarify the relationship between atypical absence and GABA(A) receptors. We performed mutation analysis of all the coding exons of GABA(A) receptor alpha1, beta2 and gamma2 subunit (GABRA1, GABRB2 and GABRG2) genes by direct sequencing to clarify whether there was common molecular biological mechanism underlying both typical and atypical absences. We recruited 52 unrelated Japanese patients, thirty-eight with typical absences and 14 with atypical absences. They consisted of 38 with childhood absence epilepsy, three with Lennox-Gastaut syndrome, two with epilepsy with myoclonic-astatic seizures and nine with epilepsy with continuous spike-waves during slow wave sleep. All of the subjects were idiopathic or cryptogenic cases without any organic brain lesions or underlying diseases. We detected five polymorphisms (T156C in GABRA1, C1194T in GABRB2, and C315T, T588C and C1230T in GABRG2), and they are silent mutations. In conclusion, mutations in GABRA1, GABRB2 and GABRG2 do not seem to be a major genetic cause of epilepsy with typical and atypical absences in Japanese subjects.