神経発達障害 Neurodevelopmental Disorders
P3-2-159 出生直後雌ラットへのEthynyl estradiol 曝露が摂食・学習・性行動に与える影響 Effects of neonatal ethynyl estradiol exposure to female rats on feeding, learning and sexual behavior ○小峰千亜希1, 神島愛未1, 小林由紀1, 千本隆志1, 植村英恵1, 吉田緑2, 近藤保彦3, 川口真以子1 ○Chiaki Komine1, Manami Kamishima1, Yuki Kobayashi1, Takashi Senbon1, Hanae Uemura1, Midori Yoshida2, Yasuhiko Kondo3, Maiko Kawaguchi1 明治大院・農学研究科1, 国立医薬品食品衛生研究所2, 帝京科学大・アニマルサイエンス学科3 School of Agriculture, Meiji University, Kanagawa1, National Institute of Health Sciences, Tokyo2, Department of Animal Sciences, Teikyo University of Science,Tokyo3 Objective: Perinatal estrogen causes masculinization and defeminization of the brain in female rats. Since ethynyl estradiol (EE), one of the most widely used ingredients of oral contraceptive pills and cosmetics, mimics estrogenic activity, EE treated in female rat neonates may prevent normal development of the brain. In this study, we examined the effects of neonatal treatment with EE on feeding, learning and sexual behaviors in adult female rats. Method: Female rats were injected with sesame oil (CONT), 0.02 mg/kg EE (LEE), 2 mg/kg EE (HEE), or 20 mg/kg 17β-estradiol (E2) within 24 h after birth. All groups were tested for passive avoidance learning at 8 week-old. Two weeks after the test, they were ovariectomized (OVX) under pentobarbital anesthesia. Between 13 and 15 week, they were subjected to sexual behavior tests. In these tests, the rats were primed with estradiol benzoate (EB) and progesterone. Food intake in each female was also measured during 15-16 week-old. We also prepared another set of the 4 groups, a half of each group injected with EB and the other without the treatment after OVX. Next day, a passive avoidance test was carried out to examine the prompt effect of EB. Result: In the sexual behavior test, HEE and E2 showed a lower percentage of lordosis behavior (a female typical sexual reflex) than CONT. Passive avoidance learning was impaired in gonadally intact LEE. The effect was also confirmed in the second serious in OVX-EB treated LEE, which showed declined tendency in the passive avoidance performance. There was no significant difference in food intakes. These results suggest that neonatal EE in female rats disturbs the normal development of female brain, resulting in impairment of sexual behavior and learning performance.	P3-2-160 生後発達期のセロトニンが成体期のBALB/cマウスの行動に及ぼす影響 Roles of serotonin during the postnatal period in the anxiety, depression, and the spatial learning in adult BALB/c mice ○石川千尋1, 大谷彰子1, 吉川雅朗2, 先崎浩次1, 志賀隆1 ○Chihiro Ishikawa1, Akiko Ohtani1, Masaaki Yoshikawa2, Koji Senzaki1, Takashi Shiga1 筑波大院・医学医療系1, 日本医・機能形態生体構造医2 Fac of Med, Univ of Tsukuba, Tsukuba, Japan1, Div of Anat Sci, Dept of Funct Morphol, Nihon Univ Sch of Med, Tokyo, Japan2 Serotonin (5-hydroxytryptoamine, 5-HT) is known to act as a neurotransmitter in the adulthood and a neurotrophic factor in the brain development. We have previously reported that the oral administration of fluoxetine, selective serotonin reuptake inhibitor (SSRI) during postnatal weeks 1-3 normalized 5-HT turnover rate, dendritic spine and synapse densities, and spatial learning in prenatally stressed C57BL/6J mice (Ishiwata et al., 2005). Interestingly, the same treatment had no effect in no-prenatally stressed mice. These results suggest that adequate amount of 5-HT is important for the proper development of brain and behavior. BALB/c mice is known to have less amount of brain 5-HT and be more anxious compared to other strains such as C57BL/6 and 129x/Sv. Therefore, we hypothesized that fluoxetine has some positive effects in BALB/c mice without stressing prenatally. In the present study, we examined the effects of SSRI on BALB/cCrSlc male mice by oral administration of fluoxetine (5 mg/kg BW/day) during postnatal weeks 1-3. We found that fluoxetine treatment increased the entries of open arms significantly and the tendency to stay longer in open arms in elevated plus maze, suggesting the decrease of anxiety. In addition, fluoxetine treatment significantly improved the spatial learning in Morris water maze, and decreased depression-like behavior such as floating in forced swim test. In these mice, the 5-HT2A-receptor mRNA expression was increased in the frontal cortex. We are currently studying the roles of 5-HT receptors, which mediate the effects of 5-HT, using 5-HT1A-receptor agonist (8-OH-DPAT) and 5-HT2A-receptor antagonist (ketanserin).
P3-2-161 バルプロ酸の妊娠期投与による海馬神経回路の興奮―抑制バランスの破綻のVSD可視化解析 Disruption of the excitatory/inhibitory balance of hippocampal neural activity by prenatal valproic acid application: A voltage-sensitive dye imaging study ○冨永洋子1, 五十嵐勝秀2, 種村健太郎3, 菅野純2, 中島欽一4, 冨永貴志1 ○Yoko Tominaga1, Katsuhide Igarashi2, Kentaro Tanemura3, Jun Kanno2, Kinichi Nakashima4, Takashi Tominaga1 徳島文理大・香川薬・神経科学研1, 国立医薬品食品衛生研・安全性生物試験研究センター・毒性部2, 東北大院・農・動物生殖科学3, 奈良先端大・バイオサイエンス・分子神経分化制御4 Inst Neurosci., Kagawa Sch Pharm Sci, Tokushima Bunri Univ, Sanuki, Japan1, Div Cellular & Molecular Toxicol, NIHS, Tokyo, Japan2, Lab Animal Reproduction, Grad Sch Agr Sci, Tohoku Univ., Sendai, Japan3, Lab Mol Neurosci, NAIST, Nara, Japan4 Valproic acid (VPA) is a first-line therapy for epilepsy. Recently, the FDA has warned that children born from mothers treated with VPA have an increased risk of autism spectrum disorder (ASD)-like cognitive deficiencies. It is widely accepted that disruptions in the excitatory/inhibitory (E/I) balance and alterations in the communication between different brain areas are important factors in the etiology of ASDs. In other words, ASDs are "circuit disorders" that are more likely to be caused by alterations in macroscopic neural circuit functions than in specific microscopic synapses or neurons. To date, a limited number of methods for demonstrating alterations in neuronal circuit activity have been reported. Here, we examined the alteration of neural circuit activity by using single-photon wide-field voltage-sensitive dye (VSD) imaging. Previously, we found that mice born to female mice treated with VPA on embryonic days 12, 13, and 14 showed clear deficits in behavior related to learning and memory function after postnatal day 84. In the hippocampal slice preparation of VPA-treated mice, we searched for deficits in synaptic activation patterns in the perforant, mossy fiber, and Schaffer collateral pathways by using VSD imaging. The neuronal response of VPA-treated mice observed with VSD imaging was not different from that of control mice, but did show a reduced sensitivity to a GABAA receptor inhibitor. We hypothesized that this resulted from reduced inhibitory activation in VPA-treated mice, accompanied by reduced excitatory activation. In other words, the disruption of the E/I balance in the hippocampal circuitry may be the cause of the behavioral deficits.	P3-2-162 自閉症スペクトラム患者共通リスクSNPsを含むゲノム領域は発達期脳においてエンハンサー活性を有する A 5p14.1 gene-poor region encompassing Autism Spectrum Disorder associated SNPs has enhancer activities in the developing brain ○井上-上野由紀子1, 井上高良1 ○Yukiko U Inoue1, Takayoshi Inoue1 国立精神・神経セ神経研疾病6部1 Dept Biochem and Cellular Biol, NCNP, Tokyo1 Our understanding for the genetics of autism spectrum disorder (ASD) has expanded rapidly in the past decade. Whereas rare and de novo mutations in 10-20% of ASD cases have been identified, the involvement of common genetic risk factors in most patients is still under investigation. A recent genome-wide association study (GWAS) indicated a significant association of ASD with the SNPs (rs4307059 and five others), located in a gene-poor region between cadherin10 (CDH10) and cadherin9 (CDH9) on chromosome 5p14.1. One approach to annotate non-protein coding risk regions is thoroughly searching for as yet un-annotated transcripts by means of bioinformatics and tiling arrays, and the transcription of a novel non-coding RNA (moesin pseudogene1 antisense) from the aforementioned ASD risk region was reported last year. Here, as the other approach, we utilized a bacterial artificial chromosome (BAC) enhancer-trapping strategy to scan the gene desert encompassing the ASD associated SNPs for cis-regulatory elements. We chose a human BAC clone covering all the six ASD risk SNPs and modified it by in vitro transposition of Tn1000β-lacZ, followed by in vivo transgenic mouse reporter assay. This assay successfully trapped enhancer activities specific for the developing neocortex and striatum, the brain regions implicated in the clinical conditions of ASD. This is the first employment of human BAC enhancer-trapping to neurodevelopmental disorder related common genetic variants, and the results may lead to the implication of functional variations that alter the activity of brain enhancer in ASD etiology. Moreover, this approach can be applied to any GWAS non-coding SNPs as an efficient tool.
P3-2-163 IL1RAPL1欠損マウスの行動学的解析 Behavioral phenotypes of IL1RAPL1 knockout mice ○安村美里1,2, 吉田知之1,3, 高雄啓三4, 山崎真弥5, 阿部学5, 植村健1,2, 宮川剛4,6, 崎村建司5, 三品昌美1,7 ○Misato Yasumura1,2, Tomoyuki Yoshida1,3, Keizo Takao4, Maya Yamazaki5, Manabu Abe5, Takeshi Uemura1,2, Tsuyoshi Miyakawa4,6, Kenji Sakimura5, Masayoshi Mishina1,7 東京大院・医・薬理・分子神経生物1, 信州大・医・神経生理2, 科学技術振興機構さきがけ3, 生理研・行動様式解析室4, 新潟大・脳研・細胞神経生物5, 藤田保健衛生大・総医研・システム医科学6, 立命館大・総合科学技術研究機構・脳科学7 Dept of Mol Neurobiol & Pharmacol, Grad Sch of Med, Univ of Tokyo, Tokyo, Japan1, Dept of Neurophysiol, Shinsyu Univ, Sch Med, Matsumoto, Japan2, PRESTO, JST, Saitama, Japan3, Sec Behav Patterns, Ctr Genet Anal Behav, NIPS, Okazaki, Japan4, Dept Cell Neurobiol, Brain Res Inst, Niigata Univ, Niigata, Japan5, Div Sys Med Sci, ICMS, Fujita Health Univ, Toyoake, Japan6, Brain Sci Lab, Res Org of Sci & Tech, Ritsumeikan Univ, Kusatsu, Japan7 Precise synaptic connections between nerve cells provide the basis of brain functions. Dysfunction in neuronal communication is implicated in the pathogenesis of many psychiatric and neurologic diseases. Interleukin-1 receptor accessory protein-like 1 (IL1RAPL1) is responsible for X-linked nonsyndromic intellectual disability and is also associated with autism. Recently we found that postsynaptic IL1RAPL1 mediates excitatory synapse formation through transsynaptic interaction with presynaptic protein tyrosine phosphatase δ. To elucidate the behavioral effects of IL1RAPL1 deficiency, we generated IL1RAPL1 knockout mice and subjected the mutant mice to a behavioral test battery. IL1RAPL1 knockout mice showed the decrease of spine density of basal dendrites of cortical layer 2/3 and hippocampal CA1 pyramidal neurons. Reference memory learning in the Barnes maze test, and working memory learning in the eight-arm radial maze test and T-maze forced alternation task were mildly impaired in IL1RAPL1 knockout mice. Moreover, the mutant mice exhibited impairments in fear conditioning test. Impaired behavioral flexibility in reversal tasks, increased social interactions in novel environment and Crawley's three-chamber social interaction test were observed in IL1RAPL1 knockout mice. Locomotor activity, the time spent in the central area of open field, and the percentage of entries into open arm in the elevated plus maze were increased in IL1RAPL1 knockout mice. These results suggest that IL1RAPL1 plays roles in the regulation of learning and memory, social behavior, locomotor activity, and anxiety-like behavior. IL1RAPL1 knockout mice may provide insight into therapeutic approach for neurodevelopmental and psychiatric disorder.	P3-2-164 in vivoおよびin vitro自閉症モデルのシナプスにおける表現型の解析 Molecular analysis of autism related synaptic phenotypes using in vivo and in vitro models ○飯島陽子1, , 飯島崇利1 ○Yoko Hanno-Iijima1, Barbara Biemans2, Takatoshi Iijima1, Peter Scheiffele1 バーゼル大学・バイオセンター1, ロシュ2 Dept. of Cell and Neurobiology, Biozentrum, University of Basel, Basel, Switzerland1, CNS Discovery pRED, F. Hoffmann-La Roche Ltd., Basel, Switzerland2 Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairment in several brain areas, such as reciprocal social interaction skills, communication difficulties, and the presence of stereotyped behaviors and restricted interests. Valproic acid (VPA) is frequently used for the treatment of epilepsy and bipolar disorders. However, when VPA is taken during pregnancy, exposure to VPA in utero dramatically increases the prevalence of ASD. Previous reports suggested that VPA injection into pregnant rodents can recapitulate behavioral phenotypes related to ASD in the offspring such as impairments in social interaction. Therefore, this model may allow for identification of molecular mechanisms and synaptic pathophysiology underlying ASD. We screened for proteins that showed alteration in the offspring of VPA exposed mice, and found some candidate proteins that might be responsible for giving rise to autism related behavior. In addition, we demonstrated that direct VPA exposure of neuronal primary cultures reveals protein alterations that are similar to the in vivo VPA model. Using the in vitro VPA model, we further investigated molecular mechanisms underlying the phenotype triggered by VPA. Based on these findings, we propose a hypothesis for the molecular mechanism underlying synaptic pathophysiology in this ASD model.
P3-2-165 自閉症スペクトラムにおけるヒト声に特異的な脳活動の検出 Voice-specific brain responses in persons with/without autism spectrum disorders ○軍司敦子1, 小林朋佳1, 竹市博臣1, 鈴木浩太1, 山本寿子1, 安村明1, 稲垣真澄1 ○Atsuko Gunji1, Tomoka Kobayashi1, Hiroshige Takeichi1, Kota Suzuki1, Hisako Yamamoto1, Akira Yasumura1, Masumi Inagaki1 (独)国立精神・神経医療研究センター　精神保健研究所1 Dept Developmental Disorders, NIMH, NCNP, Kodaira, Japan1 Background and Purpose: Appropriate assessment tools in each cognitive function are an effective means to the intervention in persons with autism spectrum disorders (ASD). To date only limited research has been conducted on voice perception in social interaction and communication. Because persons with ASD show a different pattern of brain response to human voice (Gervais et al., 2004), the neuropsychological evaluation may be an index to assess the developmental stage of social cognition through auditory signals. We aimed at developing an efficient measurement method for this assessment. Method: 113 typically-developing (TD) persons (9.1 ± 4.4 yrs) and 34 persons with ASD (10.5 ± 4.5 yrs) participated in the study. To make a direct comparison with the fMRI study (Belin et al. 2000), the same exact materials were used: human voice, non-voice, spectrally scrambled voice and spectrally scrambled non-voice sounds as stimuli. A stimulus with duration of 20 seconds contained spectrally scrambled portions (256 segments) at random timing according to an m-sequence for electroencephalography (EEG) measurements. Each of eight stimuli was presented once. EEGs were recorded from eleven scalp electrodes (Fpz, Fz, Cz, Pz, Oz, FC5, T7, CP5, FC6, T8, CP6) referenced to nose tip. An index of component score was defined as (voice - non-voice) - (scrambled voice - scrambled non-voice), to evaluate the voice-specific brain responses. Results: The index showed a peak with correlation delay time of 317.6 ± 31.3 ms in the TD group which was shorter than that in the ASD group (332.9 ± 30.0 ms) (p < .01). Conclusions: A neuropsychological evaluation using m-sequence technique evidenced for perceptual specificity elicited by human voice. This application may contribute to segregation between persons with and without ASD.	P3-2-166 成人高機能自閉症の脳機能的ネットワークの変化 Altered functional brain networks in adults with autism spectrum disorder ○板橋貴史1, 山田貴志2, 神保太樹3, 塩田清二3, 渡部洋実2, 中村元昭2, 山形文2, 金井智恵子4, 鳥居塚和生1, 加藤進昌2,6, 橋本龍一郎5 ○Takashi Itahashi1, Takashi Yamada2, Daiki Jimbo3, Seiji Shioda3, Hiromi Watanabe2, Motoaki Nakamura2, Bun Yamagata2, Chieko Kanai4, Kazuo Toriizuka1, Nobumasa Kato2,6, Ryuichiro Hashimoto5 昭和大学薬学部　創薬分子薬学1, 昭和大学医学部 精神医学2, 昭和大学医学部　顕微解剖学3, 相模女子大学学芸学部　子ども教育学科4, 首都大学東京 人文科学研究科5, 科学技術振興機構 CREST6 Department of Medicinal Chemistry, School of Pharmacy, Showa University, Tokyo, Japan1, Department of Psychiatry, School of Medicine, Showa University, Tokyo, Japan2, Department of Anatomy, School of Medicine, Showa University, Tokyo, Japan3, Department of Education and Child Studies, Faculty of Arts & Sciences, Sagami Women's University, Kanagawa, Japan4, Graduate School of Humanities, Tokyo Metropolitan University, Tokyo, Japan5, CREST, JST, Tokyo, Japan6 Previous findings of reduced functional connectivity in people with autism spectrum disorder (ASD) have been reported in resting-state functional magnetic resonance (rs-fMRI) studies. Typically, network abnormalities have been investigated by seed-based analyses that intend to localize abnormalities rather than to find global properties of networks in ASDs. Network analysis has been used to describe the global and local properties of the large-scale brain networks, and has provided new insights for understanding the brain. In this study, we acquired rs-fMRI data from 46 ASDs (age: 31.11±8.13, female: 7) and 46 normal controls (NCs) (age: 32.02±7.94, female: 7) using a 1.5 Tesla GE Signa system (repetition time: 2000 ms, in-plane resolution: 64×64, slice thickness: 4 mm with a 1-mm slice gap, 27 axial slices, flip angle: 90º, 204 volumes). Using 160 brain regions as nodes, we calculated a correlation matrix for each participant. Applying a range of sparsity thresholds, a binary and undirected graph was generated at each sparsity to explore the properties of the networks. In global metrics, ASDs showed higher global and lower local efficiency compared to NCs, indicating the possibility of increasing randomness. In addition, several nodes showed differences in local metrics (i.e., degree, betweenness, eigenvector centrality, and nodal efficiency). Notably, the dorsal anterior cingulate cortex (dACC) showed decreases in all the metrics. Partial correlation analyses revealed that 3 metrics (i.e., degree, eigenvector and nodal efficiency) of the dACC were negatively correlated with the Autism-Spectrum Quotient score. Our findings suggested that increased randomness and that the dACC might particularly play a key role in the development of autistic symptoms.
P3-2-167 ゲノム編集技術を用いた結節性硬化症モデルマーモセットES細胞の作製 Generation of TSC model marmoset ES cells with genome editing technology ○岸憲幸1,2, 佐久間哲史3, 奥野弥佐子1,2, 山本卓3, 岡野栄之1,2 ○Noriyuki Kishi1,2, Tetsushi Sakuma3, Misako Okuno1,2, Takashi Yamamoto3, Hideyuki Okano1,2 慶應大学・医・生理1, 理研・BSI2, 広島大院・理・数理分子生命3 Dept Physiol, Keio Univ School of Medicine, Tokyo1, RIKEN-Keio Univ. Joint Research Lab, RIKEN BSI, Wako, Japan2, Dept Mathematical and Life Science, Graduate School of Science, Hiroshima Univ, Higashi-Hiroshima, Japan3 Autism is a complex genetic neurodevelopmental disorder that impairs social interaction and communication and causes restricted and repetitive behavior, and is typically diagnosed in early childhood. Due to the wide range of severity of symptoms within patients displaying autistic behavior, this condition is usually classified within autism spectrum disorders (ASDs). Tuberous sclerosis or tuberous sclerosis complex (TSC) is one of ASDs and a multi-system genetic disease that causes non-malignant tumors to grow in the brain and on other vital organs such as the kidney, heart, eye, lung, and skin. Patients with TSC have a very high prevalence of epilepsy, autism, developmental delay, mental retardation, and other neurological and psychiatric problems. TSC is caused by a mutation of either of two genes, TSC1 and TSC2, which encode the proteins harmatin and tuberin respectively and act as tumor suppressors. Several lines of TSC rodent models have been generated and analyzed to investigate how mutated harmatin or tuberin affects normal neural development. However, our understanding of pathogenesis in TSC is limited by the lack of an animal model that fully replicates the pathology of the human disorder. The common marmoset (Callithrix jacchus) is a small New World primate that, because of its size, availability, and unique biological characteristics, has attracted considerable attention as a potentially useful biomedical research animal in fields such as neuroscience and stem cell research. Therefore, to investigate molecular and cellular mechanisms of TSC, we are currently pursuing to generate TSC model marmoset ES cells with transcription activator-like effector nuclease (TALEN) technology, which enables to create specific double-strand break at desired locations in the genome. These approaches will not only reveal pathogenesis of TSC, but also potentially contribute to future therapeutic strategies for TSC.	P3-2-168 結節性硬化症におけるスパイン形成のmTOR非依存的機構 An mTOR-independent mechanism for impaired spinogenesis and behavioral deficits in a rodent model of tuberous sclerosis ○安田新1, 杉浦弘子1, 桂林秀太郎2, 岩崎克典2, 小林敏之3, 樋野興夫3, 山形要人1 ○Shin Yasuda1, Hiroko Sugiura1, Shutaro Katsurabayashi2, Katsunori Iwasaki2, Toshiyuki Kobayashi3, Okio Hino3, Kanato Yamagata1 東京都医学研・神経可塑性1, 福岡大・薬・神経薬理2, 順大・医・腫瘍病理3 Neural Plasticity Project, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan1, Department of Neuropharmacology, Fukuoka University, Pharmaceutical Science, Fukuoka, Japan2, Department of Pathology and Oncology, Juntendo University, School of Medicine, Bunkyo-ku, Tokyo, Japan3 Mutation of tuberous sclerosis complex gene (Tsc2) causes tuberous sclerosis (TSC). The TSC2 protein, also called tuberin, has the GAP activity towards small GTPase Rheb. Tuberin suppresses the mTOR activity by stimulating GTP hydrolysis of Rheb. We analyzed Tsc2+/- rats to examine an involvement of the TSC-Rheb-mTOR pathway in dendritic spinogenesis. Tsc2 mutation abolished spine formation in cultured hippocampal neurons. A similar abnormality was also observed in wild-type neuron when expressed with a GTP-bound form of Rheb. Rapamycin completely blocked the mTOR activity, but it did not restore spine formation in Tsc2+/- neurons, indicating that the mTOR signaling may not be involved in impaired spinogenesis in TSC. To clarify this mTOR-independent mechanism, we performed yeast two-hybrid screening and found a new Rheb-binding protein (RBP). Knockdown of RBP induced the spine formation in Tsc2+/- neurons, whereas overexpression of RBP abolished spine maturation in wild-type neurons. The expression of dominant-negative Rheb reduced the RBP levels in neurons, and increased the spinogenesis in Tsc2+/- neurons. Furthermore, intraventricular infusion of RBP siRNA into the Tsc2+/- rat brains restored impaired contextual fear discrimination. We have generated conditional RBP knockout mice, and are currently investigating whether the genetic downregulation of RBP rescues dendritic spine abnormalities and behavioral deficits in Tsc2+/- mice.
P3-2-169 相互作用蛋白探索とloss-of-function解析による神経発達障害原因遺伝子CDKL5 の機能研究 Functional studies of the CDKL5, a causative gene for neurodevelopmental disorders, by interactome screening and loss-of-function analyses ○奥田耕助1, 渡邉紀1, 小林静香2, 真鍋俊也2, 高雄啓三3, 宮川剛3, 深谷昌弘4, 阪上洋行4, 西岡朋生5, 天野睦紀5, 貝淵弘三5, 水口雅1, 田中輝幸1 ○Kosuke Okuda1, A Watanabe1, S Kobayashi2, T Manabe2, K Takao3, T Miyakawa3, M Fukaya4, H Sakagami4, T Nishioka5, M Amano5, K Kaibuchi5, M Mizuguchi11, T Tanaka1 東京大学大学院 医学系研究科 発達医科学教室1, 東京大学医科学研究所　神経ネットワーク分野2, 自然科学研究機構　生理学研究所　行動・代謝分子解析センター3, 北里大学医学部　解剖学教室4, 名古屋大学医学系研究科　神経情報薬理学講座5 Dept. of Developmental Medical Sciences, Graduate School of Medicine, The Univ. of Tokyo1, Division of Neuronal Network, Dept. of Basic Medical Sciences, The Univ. of Tokyo2, Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences3, Dept. of Anatomy, Kitasato Univ. School of Medicine4, Dept. of Cell Pharmacology, Graduate School of Medicine, Nagoya Univ.5 The Cyclin-dependent kinase-like 5 (CDKL5) gene encodes for a serine-threonine kinase sharing homology to Mitogen-activated kinases (MAPKs) and Cyclin-dependent kinases (CDKs). Recently, mutations in the CDKL5 gene have been identified in the patients with neurodevelopmental disorders associated with epilepsies, such as West syndrome or atypical Rett syndrome, suggesting its critical role in neurodevelopment. However, neither its molecular functions or pathomechanisms caused by its mutations are unknown. Aiming to elucidate these problems, we have taken mainly two strategies, interactome screening and its loss-of-function analysis. For interactome screening, we performed the yeast two-hybrid screening and identified several CDKL5 interacting proteins. Also we are screening the CDKL5 kinase substrates using the affinity column chromatography and shotgun LC-MS/MS. For loss-of-function analysis, we have generated the Cdkl5 knock-out mouse and its phenotypic analyses are ongoing. The combination of interactome screening and loss-of-function analysis suggested us possible its functions and mechanisms of CDKL5 regulating neurodevelopment.	P3-2-170 Pax6のヘテロ変異がマウス超音波発生に及ぼす影響 The effect of Pax6 haploinsufficiency on mouse ultrasonic vocalization ○木村龍一1, 吉崎嘉一1, 杉山拓1, 鈴木淳1,2, 中村龍司1, 大隅典子1 ○Ryuichi Kimura1, Kaichi Yoshizaki1, Taku Sugiyama1, Jun Suzuki1,2, Ryuji Nakamura1, Noriko Osumi1 東北大学大学院　医学系研究科　発生発達神経科学分野1, 東北大学病院耳鼻咽喉・頭頚部外科学教室2 Division of Developmental Neuroscience, Tohoku University, Sendai, Japan1, Department of Otolaryngology, Head and Neck Surgery, Tohoku University School of Medicine2 Autism is neurodevelopmental disorder that is characterized with three core symptoms, i.e., social impairment, repetitive behavior/restricted interest, and communication impairment. A recent large-scale linkage study implicated chromosome 11p12-13 for autism susceptibility loci. We previously reported that PAX6, which resides in chromosome 11p13, was associated with autism and actually Pax6 heterozygous mutant rat displayed some behavioral abnormality related with autism. Ultrasonic vocalization (USV) is considered as rodent vocal communication. Although the number of USV emission was decreased in Pax6 heterozygous mutant rat, the mechanism why Pax6 haploinsufficiency caused abnormal USV is not determined yet. To examine how Pax6 haploinsufficiency contributes to abnormal USV displayed in Pax6 heterozygous mutant rat, we first measured USV activity in Pax6 heterozygous mutant mouse (Sey/+). The number of USV was also decreased in Sey/+ at postnatal day 6 and the extent of the reduction was similar between male and female pups. Because Pax6 was highly expressed in the cerebellum at postnatal day 6, a development of the cerebellum was examined.The density of Purkinje cells characterized by cabindin expression was not affected (51.6±4.1cells/mm in WT and 50.2±2.9cells/mm in Sey/+). Relative intensity of VGluT1 staining, which was used as a marker of parallel fiber projection in the molecular layer of the cerebellum, was similar in both animals, indicating that parallel fiber projection from granule cell layer to Purkinje cells is intact. We are now performing further histological analysis explaining USV deficit caused by Pax6 haploinsufficiency.
P3-2-171 X連鎖性精神遅滞患者における関連遺伝子解析 Mutation analysis revealed molecular etiology of X-linked MR in Japanese ○和賀央子1, 竹下絵里1,3, 中川栄二2, 後藤雄一1,2 ○Chikako Waga1, Eri Takeshita1,3, Eiji Nakagawa2, Yu-ichi Goto1,2 （独）国立精神・神経医療研究センター　神経研究所　疾病研究第二部1, （独）国立精神・神経医療研究センター　病院　小児神経科2, （独）国立精神・神経医療研究センター　病院　臨床検査部3 Department of Mental Retardation and Birth Defect Research, National Institute of Neuroscience, Tokyo, Japan1, Department of Child Neurology, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan.2, Department of Laboratory Medicine, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo, Japan.3 Mental retardation (MR) is characterized by cognitive impairment with an IQ < 70, and has long been recognized as a common and etiologically heterogeneous disorder, affecting 1-3% of the general population. Numerous studies of large families containing males with MR have highlighted the importance of the X-chromosome (X-linked mental retardation: XLMR). To delineate molecular etiology in Japanese XLMR patients, we analyzed the XLMR-related 13 genes (SYP, GRIA3 , RAB39B , ZDHHC15 , ZNF674 , ZNF711 , JARID1C , BRWD3 , HUWE1 , CUL4B , RPL10 , ZDHHC9 and SRPX2 ) in 53 male patients with probable in X-linked inheritance. We identified novel mutations in SYP (5 patients in 2 pedigrees: 3.8%), JARID1C (4 patients in 2 pedigrees: 3.8%) and RPL10 (2 patients in 1 pedigree: 1.9%) genes. We extended our JARID1C analysis in 210 sporadic male and female MR patients and detected 2 additional mutations (4 patients in 2 pedigrees: 0.95%). Next we examined JARID1C expression in patients' lymphoblast cells. Quantitative RT-PCR showed a significant reduction of JARID1C mRNA in one patient who carries a frameshift mutation which was restored by treatment with cycloheximide, a potent inhibitor of nonsense-mediated mRNA decay (NMD). Therefore, mRNAs carrying JARID1C frameshift mutation are likely subject to degradation by NMD. Western blotting analysis showed that JARID1C protein was reduced in all 4 patients carrying 2 frameshift and 2 missence mutations. These results suggest that reduction of JARID1C causes MR in these patients. Our mutation analysis of XLMR genes revealed mutations in 3 genes, all of which are novel in Japanese, thus contributing the delineating of molecular etiology of XLMR in Japanese.	P3-2-172 妊娠期の非晶質ナノシリカ曝露が次世代の情動機能へ及ぼす影響探索 The effects of in utero exposure to amorphous nanosilica particles on neonatal emotional functions ○森下裕貴1, 吉岡靖雄1, 高雄啓三2,3, 吾郷由希夫4, 佐藤宏祐1, 野尻奈央1, 田中智大1, 田熊一敞4, 角田慎一5,6, 松田敏夫4, 宮川剛2,3, 東阪和馬1, 堤康央5,6 ○Yuki Morishita1, Yasuo Yoshioka1, Keizo Takao2,3, Yukio Ago4, Hiroyoshi Satoh1, Nao Nojiri1, Tomohiro Tanaka1, Kazuhiro Takuma4, Shin-ichi Tsunoda5,6, Toshio Matsuda4, Tsuyoshi Miyakawa2,3, Kazuma Higashisaka11, Yasuo Tsutsumi5,6 大阪大院・薬・毒性学1, 藤田保衛大・総医研・システム医2, 生理研・行動様式解析3, 大阪大院・薬・薬物治療学4, 医薬基盤研・バイオ創薬5, 大阪大・MEIセ6 Lab Toxicol Safety Sci, Grad Sch Pharm Sci, Osaka Univ, Osaka, Japan1, Divi Sys Med Sci, Inst Comprehen Med Sci, Fujita Health Univ, Aichi, Japan2, Sec Behav Patterns, Center Genetic Anal Behav, NIPS, Aichi, Japan3, Lab Medicinal Pharmacol, Grad Sch Pharm Sci, Osaka Univ, Osaka, Japan4, Lab Biopharm Res, Nat Inst Biomed Innov, Osaka, Japan5, MEI Center, Osaka Univ, Osaka, Japan6 Nanomaterials (NMs) have already been used in various fields such as cosmetics and foods. However, concerns about the potential risks of NMs have been raised. Therefore, assessing the safety of NMs is important. We had already revealed that amorphous nanosilica particles (nSP) could be distributed to fetal brain and might increase the rate of low birth weight infants, when they were intravenously administrated to pregnant mice. Fetal brain is very susceptible to chemical substances, and low birth weight infants are known to have high risks of contracting neurological disorders. Thus, to assess postnatal effects on emotional and cognitive functions of prenatal exposure to nSP is one of the most important issues in assessing the safety of NMs. In this study, we attempted to identify the hazards of prenatal exposure to nSP on neonatal emotional functions. Pregnant mice were intravenously treated with nSP on gestational day 16 and 17. To evaluate anxiety-like behavior and depression-like behavior, pups performed elevated plus maze test, Porsolt forced swim test, tail suspension test, and wire hang test from 10 weeks old. No difference was found on the score of elevated plus maze test and Porsolt forced swim test between the pups of nSP treated mice (nSP pups) and control mice. On the other hand, nSP pups showed lower activity in Porsolt forced swim test and shorter latency to fall in wire hang test than control mice. These results suggest that prenatal exposure to nSP might induce depression-like behavior of pups. This information would be important for verifying the safety of NMs, because few studies have assessed the postnatal effects of prenatal exposure to NMs on neonatal emotional functions.
P3-2-173 安静時脳機能画像を用いた機械学習による自閉症スペクトラム障害のバイオマーカー開発 A machine-learning-based biomarker for autism spectrum disorder using resting-state fMRI ○八幡憲明1, 森本淳2, 橋本龍一郎3, 柴田和久2,4, 今水寛2, 福田めぐみ2, 川久保友紀5, 桑原斉5, 山田貴志3, 佐々木由香2,4, 加藤進昌3, 渡邊武郎2,4, 笠井清登1, 川人光男2 ○Noriaki Yahata1, Jun Morimoto2, Ryuichiro Hashimoto3, Kazuhisa Shibata2,4, Hiroshi Imamizu2, Megumi Fukuda2, Yuki Kawakubo5, Hitoshi Kuwabara5, Takashi Yamada3, Yuka Sasaki2,4, Nobumasa Kato3, Takeo Watanabe2,42,4, Kiyoto Kasai1, Mitsuo Kawato2 東京大院・医・精神医学1, ATR脳情報通信総合研究所2, 昭和大学附属烏山病院3, ブラウン大学・認知言語心理科学部4, 東京大院・医・こころの発達医学5 Dept Neuropsych, Univ of Tokyo, Tokyo, Japan1, ATR Brain Info Comm Res Lab Group, Kyoto, Japan2, Showa Univ Karasuyama Hosp, Tokyo, Japan3, Dept Cogn Ling & Psychol Sci, Brown Univ, RI, USA4, Dept Child Neuropsych, Univ of Tokyo, Tokyo, Japan5 Autism spectrum disorder (ASD) is a developmental disorder characterized by a triad of impairments in social interactions, communication, and behavioral flexibility. A neural mechanism model of ASD built on previous neuroimaging and postmortem brain studies has centered on disrupted brain networks, suggesting that the pattern of functional connectivity (FC) can be potentially used as a diagnostic biomarker of this disorder. Here, we have developed a novel, machine-learning-based classifier of ASD by utilizing resting-state functional magnetic resonance imaging (rsfMRI). Seventy-four adult patients with ASD and 124 typically developed (TD) individuals were incorporated into a training data set in the present study. Their MRI data were acquired at 3 different sites in Japan and were preprocessed in a standard manner. Then, for each individual, a matrix of 4,278 FCs among 93 anatomically defined regions of interest was calculated. The classifier based on a sparse logistic regression analysis selected 21 of these FCs for a reliable classification of the ASD and TD populations. A leave-one-out justification indicated that the classifier successfully distinguished the two populations with the specificity of 73% and sensitivity of 83% with the diagnostic odds ratio of 13.5. Further, the same classifier was applied to an independent data set that had been acquired at 6 sites in the United States (34 with ASD and 34 TD individuals). The accuracy of the classification was close to 70%. This indicates that our rsfMRI-based classifier was successfully generalized to the external data set. These results collectively demonstrate a high degree of feasibility of the FC-based biomarker for ASD. The extracted pattern of FCs may deepen our understanding of the neural mechanisms of ASD and increase the ratio of successfully identifying therapeutic targets of this disorder.	P3-2-174 変異型セレブロン導入によるミトコンドリア動態の解析 Analysis of mitochondrial dynamics in cereblon overexpressed PC12 cells ○中村智菜1, 若林慧1, 和田丈慶1, 朝日透1,2, 澤村直哉1,2 ○China Nakamura1, Satoru Wakabayashi1, Takeyoshi Wada1, Toru Asahi1,2, Naoya Sawamura1,2 早大院・先進理工1, 早大・科健機構2 Fac. Adv. Sci. Eng., Waseda Univ.1, ASMeW, Waseda Univ.2 A nonsense mutation of the gene cereblon (CRBN) is associated with mild mental retardation (Higgins et al., Neurology 63, 1927-1931, (2004)). Therefore we expect that elucidation of the function of CRBN protein would reveal the molecular mechanism of mental retardation. Because aberrance of mitochondrial dynamics were reported in Alzheimer's disease, Parkinson's disease, and other mental disorders (Wang et al, PNAS 105, 19318-19323 (2008), Wang et al, HMG 21, 1931-1944 (2012)), we hypothesize that similar mitochondrial defects could be observed by expression of mutant CRBN (mCRBN). First, we found that DLP1 and OPA1, protein to regulate mitochondrial fission and fusion, were reduced in mCRBN overexpressed PC12 cells compared to wild type (wCRBN) cells. Next, we homogenized the cells and then fractionated into mitochondrial and cytosol fractions. In mitochondrial fraction from , the levels of DLP1, OPA1, and mCRBN were increased compared to those of wCRBN. We examined the interaction of DLP1 or OPA1 with CRBN using immunoprecipitation, but we could not observe interaction of DLP1 and OPA1 with neither wCRBN nor mCRBN. In conclusion, mitochondrial dynamics may be affected by altered DLP1 and OPA1 expression levels.
P3-2-175 Rett症候群患者由来のiPS細胞から誘導した神経細胞ではアストロサイトの亢進がみられる Astrocytes are increased in neural cells derived from Rett syndrome iPS cells ○安藤友子1,2,5, 赤松和土1, 松本拓也1, 三宅邦夫2, 山口亮1, 岡田洋平1,3, 今泉陽一1, 大山学4, 黒澤尋5, 天谷雅行4, 久保田健夫2, 岡野栄之1 ○Tomoko Ando1,2,5, Wado Akamatsu1, Takuya Matsumoto1, Kunio Miyake2, Ryo Yamaguchi1, Yohei Okada1,3, Yoichi Imaizumi1, Manabu Ohyama4, Hiroshi Kurosawa5, Masayuki Amagai4, Takeo Kubota2, Hideyuki Okano11 慶應義塾大・医・生理学1, 山梨大院・医・環境遺伝2, 慶應義塾大・医・咸臨丸プロジェクト3, 慶應義塾大・医・皮膚科学4, 山梨大院・医工学・人間環境医工学5 Dept. of Physiol., Sch. of Med., Keio Univ., Tokyo, Japan1, Dept. of Epigenetic Med., Univ. of Yamanashi, Yamanashi, Japan2, Kanrinmaru-Progect, Sch. of Med., Keio Univ., Tokyo, Japan3, Dept. of Derm., Sch. of Med., Keio Univ., Tokyo, Japan4, Div. of Med. and Eng. Science, Interdisciplinary Grad. Sch. of Med. and Eng., Univ. of Yamanashi, Yamanashi, Japan5 Rett syndrome (RTT) is a neurodevelopmental disorder caused by lack of methyl CpG binding protein 2 (MECP2). MECP2 is an X-linked gene that is involved in random X-chromosome inactivation (XCI). RTT patients are usually females with mosaic expression of mutant and normal MECP2 based on random XCI. To elucidate the genetic phenomenon and neuro-pathogenesis of RTT, we generated induced pluripotent stem cells (iPSCs) from two patients by using retroviral vector expressing Yamanaka reprogramming four factors (Sox2, Oct4, c-Myc and Klf4). After establishing iPS clones from these patients, we analyzed MECP2 expression in each iPS clone to determine whether paternal or maternal X-chromosome was inactivated. We obtained both MECP2+ due to paterernally inherited mutant MECP2 activation and MECP2- iPS clones due to materernally inherited normal MECP2 activation from both patients, suggesting that the expression of MECP2 in iPS cells determined by XCI was maintained from original single skin fibroblast despite the reprogramming procedure. Next, we differentiated each iPS clone into neural cells via neural stem cells by using neurosphere cultivation. By defining MECP2+ clones as control, we found that neurons derived from MECP2- iPSC clones highly expressed astrocytes related genes. Our results suggested that lack of MECP2 in neural cells in RTT patients changes differentiation property of neural stem cells or viability of neurons.	P3-2-176 Pax6は父親の高齢による自閉症の発症脆弱性を調節する Pax6 modulates predisposition of autism by advanced paternal age in mouse ○吉崎嘉一1, 木村龍一1, 大隅典子1 ○Kaichi Yoshizaki1, Ryuichi Kimura1, Noriko Osumi1 東北大院・医・発生発達1 Div Dev Neurosci, Univ of Tohoku, Sendai1 Most recent studies indicate that prevalence of autism spectrum disorder (ASD) has increased dramatically. Although the risk of autism is associated with several prenatal factors including advanced parental age, the exact reason is unclear. Here we examined how advanced paternal age contributes to pathophysiology for autism by using wild type mice and Pax6 heterozygous mice (Sey/+, C57BL6/J) that show impaired ultrasonic vocalization (USV). Both young (under 8-month-old) and advanced age (over 12-month-old) male Sey/+ mouse were bred with virgin female (10-week-old, C57BL6/J), and obtained offspring were subjected to measure isolation-induced USV for 5 minutes at postnatal day 6. The number of USV in Sey/+ offspring derived from young Sey/+ was significantly decreased by 54.0% compared to that in wild-type offspring. Interestingly the number of USV in wild-type offspring derived from advanced age Sey/+ was comparable decrease to that in Sey/+ offspring. These results suggest that Pax6 is necessary for proper development of USV. It is also implied that advanced paternal age might affect predisposition of ASD in cases where the risk genes are not inherited in offspring.
P3-2-177 Rare De novo Copy number Variations in Japanese Autism Subjects ○Thanseem Ismail1 Dept of Psychiatry and Neurology, Hamamatsu University School of Medicine, Hamamatsu, Japan1 Rare De novo Copy number Variations in Japanese Autism Subjects Ismail Thanseem1, Kazuhiko Nakamura1, Ayyappan Anitha1, Masafumi Ohtsubo2, Shinsei Minoshima2 and Norio Mori1. 1Dept of Psychiatry and Neurology 2Medical Photonics Research Center Hamamatsu University School of Medicine, Hamamatsu, Japan Although autism has a strong genetic basis, consistent picture of a common susceptibility loci is still lacking. Recently, there is paradigm shift away from the "common disease-common variant" hypothesis to a "common disease-rare variant" model for the genetic architecture of autism. Here we tried to identify de novo rare copy number variations (CNVs) in Japanese autism subjects. A total of 108 trio samples were analyzed so far using the Affymetrix Genome-Wide Human SNP Nsp/Sty 6.0 array. PennCNV was used to call the CNVs. An average of 37.21 autosomal CNVs are detected per patient samples, after all the QC corrections. There is no significant difference in the total number/average size of CNVs per sample between patients and their parents. However, the percentage of deletions in the total length of CNVs is 42% in case of patients, while it is only 31.2% in parents. Almost 90% of the CNVs in children are found to be inherited; almost equally from both the parents. After several QC corrections, 124 highly confident de novo CNVs were identified. Among these, 21 are found be rare de novo events. In previous studies, de novo events have consistently shown the greatest genetic effects and were more frequent in ASD probands with only one affected child (the simplex families). Following the logic that CNV deletions should decrease the dosage of affected genes, our results that almost 75% of CNVs affecting genes are deletions, is particularly interesting. The likely morphological consequences of genes hit by rare de novo variants in the present study are now being investigated.	P3-2-178 Maternal immune activation affects the neuronal architectures and cognitive functions of the offspring ○Wai Yu Li1, Yi-Chun Chang1, Li-Jen Lee1,2,3 Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan1, Institute of Brain and Mind Sciences National Taiwan University, Taipei, Taiwan2 Recent studies have suggested a link between maternal immune activation (MIA) and subsequent development of psychiatric disorder such as schizophrenia and autism. Accumulating evidence also indicates MIA may result in cognitive deficits in their offspring. In the present study, we treated the pregnant mice of C57BL/6 background with a single injection of polyinosinic-polycytidylic acid (poly I:C, 20 mg/kg, i.p.) at gestation day 9 as the MIA model. The treatment did not exhibit significant effects on the emotional status and social interaction in the adult male offspring. However, poly I:C offspring demonstrated considerable reduction in locomotor activity and impairments in short-term and spatial memory. Since the medial prefrontal cortex (mPFC) and the dentate gyrus (DG) are implicated in various cognitive functions, we examined the neuronal structures in these regions. In poly I:C offspring, the layer II/III pyramidal mPFC neurons exhibited extensive dendritic arbors with greater segment length yet fewer branches and dendritic spines compared to those in control mice. On the other hand, the DG granule neurons in poly I:C offspring had reduced dendritic complexity, dendritic length and spine density compared to the controls. Together, the structural deficits in mPFC and DG neurons might account for the cognitive defects in poly I:C offspring. Further study is required to elucidate the underlying mechanism of MIA-induced neuronal malformations.

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