Establishment of isogenic iPSCs from an individual with SCN1A mutation mosaicism as a model for investigating neurocognitive impairment in Dravet syndrome.

Dravet syndrome (DS) is a severe childhood epilepsy typically caused by de novo dominant mutations in SCN1A. Although patients with DS frequently have neurocognitive abnormalities, the precise neural mechanisms responsible for their expression have not been elucidated. There are wide phenotypic differences among individuals with SCN1A mutations, suggesting that factors other than the SCN1A mutation modify the phenotype. Therefore, a well-controlled cellular model system is required to improve our understanding of the mechanisms underlying DS. Here we generated induced pluripotent stem cell (iPSC) lines from an individual with SCN1A mutation mosaicism, and separately cloned iPSC lines both with and without the SCN1A mutation. These clones theoretically have the same genetic backgrounds, except for the SCN1A gene, and should serve as an ideal pair for investigating the pathophysiology caused by SCN1A mutations. Quantitative reverse transcription-PCR and western blot analysis revealed higher tyrosine hydroxylase mRNA and protein expression levels in mutant neurons than in wild-type neurons. Moreover, dopamine concentrations in media collected from mutant neural cultures were higher than those from wild-type neural cultures. Our findings suggest that SCN1A mutation leads to changes in the dopamine system that may contribute to the behavioral abnormalities in DS.

Mislocalization of syntaxin-1 and impaired neurite growth observed in a human iPSC model for STXBP1-related epileptic encephalopathy.

Syntaxin-binding protein 1 (STXBP1) is essential for synaptic vesicle exocytosis. Mutations of its encoding gene, STXBP1, are among the most frequent genetic causes of epileptic encephalopathies. However, the precise pathophysiology of STXBP1 haploinsufficiency has not been elucidated. Using patient-derived induced pluripotent stem cells (iPSCs), we aimed to establish a neuronal model for STXBP1 haploinsufficiency and determine the pathophysiologic basis for STXBP1 encephalopathy. We generated iPSC lines from a patient with Ohtahara syndrome (OS) harboring a heterozygous nonsense mutation of STXBP1 (c.1099C>T; p.R367X) and performed neuronal differentiation. Both STXBP1 messenger RNA (mRNA) and STXBP1 protein expression levels of OS-derived neurons were approximately 50% lower than that of control-derived neurons, suggesting that OS-derived neurons are a suitable model for elucidating the pathophysiology of STXBP1 haploinsufficiency. Through Western blot and immunocytochemistry assays, we found that OS-derived neurons show reduced levels and mislocalization of syntaxin-1, a component of soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. In addition, OS-derived neurons have impaired neurite outgrowth. In conclusion, this model enables us to investigate the neurobiology of STXBP1 encephalopathy throughout the stages of neurodevelopment. Reduced expression of STXBP1 leads to changes in the expression and localization of syntaxin-1 that may contribute to the devastating phenotype of STXBP1 encephalopathy.

Low-grade intraventricular hemorrhage disrupts cerebellar white matter in preterm infants: evidence from diffusion tensor imaging.

INTRODUCTION: Recent diffusion tensor imaging (DTI) studies have demonstrated that leakage of hemosiderin into cerebrospinal fluid (CSF), which is caused by high-grade intraventricular hemorrhage (IVH), can affect cerebellar development in preterm born infants. However, a direct effect of low-grade IVH on cerebellar development is unknown. Thus, we evaluated the cerebellar and cerebral white matter (WM) of preterm infants with low-grade IVH. METHODS: Using DTI tractography performed at term-equivalent age, we analyzed 42 infants who were born less than 30 weeks gestational age (GA) at birth (22 with low-grade IVH, 20 without). These infants were divided into two birth groups depending on GA, and we then compared the presence and absence of IVH which was diagnosed by cerebral ultrasound (CUS) within 10 days after birth or conventional magnetic resonance imaging (MRI) at term-equivalent age in each group. Fractional anisotropy (FA) and apparent diffusion coefficient (ADC) at the superior cerebellar peduncle (SCP), middle cerebellar peduncle (MCP), motor tract, and sensory tract were measured. RESULTS: In the SCP, preterm born infants with IVH had lower FA values compared with infants without IVH. In particular, younger preterm birth with IVH had lower FA values in the SCP and motor tract and higher ADC values in the MCP. CONCLUSION: Low-grade IVH impaired cerebellar and cerebral WM, especially in the SCP. Moreover, younger preterm infants exhibited greater disruptions to cerebellar WM and the motor tract than infants of older preterm birth.

Longitudinal change in white matter in preterm infants without magnetic resonance imaging abnormalities: Assessment of serial diffusion tensor imaging and their relationship to neurodevelopmental outcomes.

PURPOSE: We used diffusion tensor imaging (DTI) to evaluate longitudinal changes in fractional anisotropy (FA) of white matter tracts in preterm infants without abnormal magnetic resonance imaging (MRI) findings. Imaging was conducted at term equivalent age (TEA) and 1year of corrected age. Furthermore, we assessed correlations between FA and neurodevelopmental outcomes at 3years of corrected age to investigate brain prematurity of preterm infants without MRI abnormalities. METHODS: Preterm infants underwent serial MRI at TEA and 1year of corrected age. Of these, 13 infants entered a retrospective study, undergoing neurodevelopmental assessment at 3years of corrected age. These infants were divided into two groups depending on gestational age (GA): <26weeks and ⩾26weeks. DTI-based tractography was performed to obtain the FA of the motor tract, sensory tract, superior cerebellar peduncle, middle cerebellar peduncle, and corpus callosum. FA was compared between two groups, and correlations between FA and neurodevelopmental outcomes were assessed. RESULTS: FA of the splenium at TEA was significantly different between the two groups divided according to GA. However, this difference was no longer observed at 1year of corrected age. There was no correlation between FA of the splenium at TEA and neurodevelopmental assessment scores at 3years of corrected age. CONCLUSIONS: At TEA, FA of the splenium was lower in younger GA infants without MRI abnormalities, but this may not affect subsequent neurodevelopmental outcomes.

Luteolin attenuates interleukin-6-mediated astrogliosis in human iPSC-derived neural aggregates: A candidate preventive substance for maternal immune activation-induced abnormalities.

Maternal infection during pregnancy increases the risk of neurodevelopmental conditions such as autism spectrum disorders and schizophrenia in offspring. Several previous animal studies have indicated that maternal immune activation (MIA), rather than a specific pathogen, alters fetal brain development. Among them, prenatal exposure to interleukin-6 (IL-6) has been associated with behavioral and neuropathological abnormalities, though such findings remain to be elucidated in humans. We developed a human cell-based model of MIA by exposing human induced pluripotent stem cells (hiPSCs)-derived neural aggregates to IL-6 and investigated whether luteolin-a naturally occurring flavonoid found in edible plants-could prevent MIA-induced abnormalities. We generated neural aggregates from hiPSCs using the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) method, following which aggregates were cultured in suspension. We then exposed the aggregates to IL-6 (100ng/ml) for 24h at day 51. Transient IL-6 exposure significantly increased the area ratio of astrocytes (GFAP-positive area ratio) and decreased the area ratio of early-born neurons (TBR1-positive or CTIP2-positive area ratio) relative to controls. In addition, western blot analysis revealed that levels of phosphorylated STAT3 were significantly elevated in IL-6-exposed neural aggregates. Luteolin treatment inhibited STAT3 phosphorylation and counteracted IL-6-mediated increases of GFAP-positive cells and reductions of TBR1-positive and CTIP2-positive cells. Our observations suggest that the flavonoid luteolin may attenuate or prevent MIA-induced neural abnormalities. As we observed increased apoptosis at high concentrations of luteolin, further studies are required to determine the optimal intake dosage and duration for pregnant women.

Effect of a single inhalation of laninamivir octanoate in children with influenza.

OBJECTIVE: The purpose of this study was to compare the efficiency and safety of a new neuraminidase inhibitor, laninamivir octanoate (LO), with zanamivir (ZN) in pediatric patients with influenza. METHODS: One hundred twelve pediatric patients ≤ 15 years, diagnosed with a rapid diagnostic test as having influenza from January to May 2011, were randomly assigned to the LO group or the ZN group, and their parents were asked to complete a questionnaire during the recovery at home. The LO group was instructed to inhale LO once (20 or 40 mg depending on age), and the ZN group was instructed to inhale ZN (20 mg) twice daily for 5 days. RESULTS: The LO group (n = 55) and the ZN group (n = 57) were well balanced. Finally, 44 patients in the LO group and 41 patients in the ZN group could be evaluated. Median times to fever resolution after initial treatment were 36 hours in the LO group and 37 hours in the ZN group. No differences were observed between the 2 groups with respect to the frequencies of asthmatic symptoms, pneumonia, gastrointestinal symptoms, or abnormal behaviors. Six younger children could not inhale LO well for technical reasons. CONCLUSIONS: Our data suggest that the efficiency and safety of LO are the same as those of ZN in pediatric patients with influenza but that LO may be more convenient than ZN because it requires only a single inhalation. However, younger patients may not inhale LO efficiently.

The incidence and direct medical cost of hospitalization due to rotavirus gastroenteritis in Kyoto, Japan, as estimated from a retrospective hospital study.

Anticipating imminent licensure of rotavirus vaccine for use in Japan, we estimated the incidence of rotavirus hospitalization and calculated the direct medical cost associated with rotavirus hospitalization in a hospital that provided virtually exclusive pediatric beds to the local community adjacent to the northern outskirts of metropolitan Kyoto, Japan. For a 2 year period between September, 2008 and August, 2010, there were 103 hospitalizations due to acute gastroenteritis among children less than 5 years of age. Stool specimens from 77 (75%) of the 103 hospitalized patients were tested for rotavirus antigen, and 46 (60%) were positive. The proportion of rotavirus positives was 65% in the peak-season months (January-June) and 17% in the off-season months (July-December). By extrapolating the test results to those patients with acute gastroenteritis who were not tested, 13 additional cases were estimated to be rotavirus positive. Assuming that all patients with rotavirus gastroenteritis less than 5 years of age in the catchment (5532 according to the 2005 census) were admitted to this hospital, the annual incidence of rotavirus hospitalization was estimated to be 4.1 (testing-unadjusted)-5.3 (adjusted) per 1000 child-years. Thus, it was estimated that one child in 48 or one child in 37 born in this area would be hospitalized due to rotavirus gastroenteritis by the age of 5 years. The incidence of rotavirus hospitalization was similar to the rate in Ise city (4.9 per 1000 child-years), also in central Japan, and lower than the rate in Honjo city in northern Japan (13 per 1000 child-years). Nevertheless, the burden of rotavirus hospitalization was substantial, and the total direct medical cost was estimated to be 6.6 billion Japanese Yen (US$ 57 million). While economic analysis and comparisons with alternative preventive procedures may be necessary, this study provides the policymakers and pediatricians with further evidence that is necessary to decide whether to introduce rotavirus vaccines into the routine childhood immunization schedule in Japan.