Poster

Poster 21 Mood Disorders 3 ポスター 21 気分障害3

P21-1 An approach from familial psychiatric patients in the Ryukyu Islands: Disease modeling based on induced pluripotent stem cell and high-impact variant 沖縄県の精神疾患多発家系を用いた双極性障害・統合失調症細胞モデルの開発：疾患iPS細胞とゲノム解析を融合したアプローチ Takamatsu Gakuya（高松岳矢）^1,2，李俊錫¹，原田綾乃¹，要匡³，柳久美子³，原（宮内）央子⁴，馬目陽子⁴，早川朋子⁵，近藤毅²，岡野ジェイムス洋尚⁴，松下正之¹ ¹Department of Molecular & Cellular Physiology, Graduate School of Medicine, University of the Ryukyus ²Department of Neuropsychiatry, Graduate School of Medicine, University of the Ryukyus ³Department of Genome Medicine, National Center for Child Health and Development ⁴Division of Regenerative Medicine, Jikei University School of Medicine ⁵Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University Bipolar disorder and schizophrenia are common and complex psychiatric diseases characterized by high genetic heterogeneity. Therefore, it would be a rational approach to expand pathological findings in high-risk pedigrees (i.e. genetically recognized subtype with high penetrance variants) to general cases. Patient-derived induced pluripotent stem cells (iPSCs) with high-impact variants could be useful disease cell models of psychiatric diseases. However, while successful for simply inherited traits such as Alzheimer's disease with pathogenic APP mutations, finding the subtype with high penetrance variants remains a challenge in bipolar disorder and schizophrenia. Although DISC1 mutation or 22q11.2 deletion in schizophrenia have been established, high-risk pedigree-based study has not reached its potential. Here, to develop novel iPSC disease models of bipolar disorder or schizophrenia, we searched for families with a high prevalence in the Ryukyu Islands and performed genome sequencing and iPSCs generation. We found (1) a large family with multiple bipolar disorder and recurrent depressive disorder and (2) a family with an early-onset schizophrenia monozygotic twin from first cousin marriage in an isolated island. Exome analysis of these families indicated potential candidate variants including rare-frequency variants in the world or Japan population. iPSCs derived from affected and unaffected individuals in each family were generated and differentiated into neurons. We are now investigating the relation between genomic variants and neuronal phenotype. Our works are expected to establish novel cellular and molecular models of bipolar disorder and schizophrenia.		P21-2 Modeling de novo mutations found in bipolar disorder patients in mice by CRISPR/Cas9 system 双極性障害患者デノボ変異モデルマウスの作製 Nakamura Takumi（中村匠）^1,2，中島一夫²，坪井貴司¹，加藤忠史² ¹Department of Life Science, Graduate School of Arts and Science, The University of Tokyo, Tokyo, Japan ²Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science, Saitame, Japan. Bipolar disorder is a common neuropsychiatric disorder with around 1% life time prevalence. Although various studies have reported that genetic factors are important for the development of bipolar disorder, the pathogenesis of bipolar disorder is still unclear. Recently, a trio-based exome sequencing study for bipolar disorder suggested possible relationship of de novo mutations and bipolar disorder. In the present study, we focused on gene disrupting de novo mutations found in EHD1, MACF1 and KMT2C. These three genes have extreme scores in two indices, Residual Variation Intolerance Score (RVIS) which means an intolerance to protein altering mutation, and probability of Loss-of-Function intolerance (pLi) which indicates an intolerance to loss-of-function mutation. In order to study the effect of the mutations in vivo, we generated mutant mice of Ehd1, Macf1 and Kmt2c by CRISPR/Cas9 system. We validated cleavage activity of each single guide RNAs which cleave each locus by Surveyor assay. We injected each designed CRISPR/Cas9 set into fertilized eggs of mice and got mutant mice. In order to investigate off-target loss of function mutations by CRISPR/Cas9, we performed exome sequence using genome DNA derived from the mutant mouse lines. We did not find any off-target insertion / deletions in these mutant mice. The present study enabled us to analyze the functional impacts of de novo mutations found in bipolar disorder patients in vivo. We are going to perform behavioral tests including intellicage analysis and wheel running activity test. This study will shed light on the pathophysiological mechanisms of bipolar disorder from the viewpoint of the relationship between bipolar disorder and de novo mutations.

P21-3 The potential causal relationship between anti-NMDAR antibody and mood disorders 抗NMDA受容体抗体と気分障害の潜在的因果性 Kawai Hiroki（河合弘樹），酒本真次，岸本真希子，岡久祐子，高木学，山田了士 The Department of Neuropsychiatry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, University of Okayama Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is the autoimmune encephalitis suggested by Dalmau et al. in 2007. This is considered to be closely related to NMDA receptor system. These potential links with glutamate hypothesis of depression and there may be a room for improvement in non-monoamine-based approach for mood disorders. Several studies investigated the relationship between schizophrenia and anti-NMDAR encephalitis, however few studies have focused on the others. We comprehensively tested the existence of anti-NMDAR antibody in the serum and/or cerebrospinal fluid (CSF) of 57 patients initially diagnosed with mood disorders by cell-based-assay (CBA). This study was approved by the Okayama University ethics committee and patients gave informed consent prior to the study. We identified that four patients (7%) had anti-NMDAR antibody. Three of four patients mainly manifested manic symptoms, the other manifested depressive symptoms. One patients had an ovarian teratoma. The other one had also AQP4 antibody in the serum. All four patients were treated with first-line immunotherapy including steroid pulse, plasma exchange and intravenous immunoglobulin (IVIg) and one of four patients was additionally treated with second-line immunotherapy (rituximab). Antipsychotics were not effective to these four patients. Our date suggested that anti-NMDAR antibody exists in the patients initially diagnosed as mood disorders and the improvement of psychiatric symptoms depended on the diminishment of anti-NMDAR antibody.		P21-4 An antidepressant-induced protocadherin Arcadlin/protocadherin-8 regulates dendritic spine density in hippocampal neurons 抗うつ治療によって誘導されるプロトカドヘリンArcadlinが海馬神経細胞スパイン密度に及ぼす影響 Takeuchi Chiaki（竹内千尭）¹，秦侑希¹，石川美帆¹，水田菜々乃¹，長谷川沙紀¹，山本かえで¹，澤野俊憲¹，中谷仁¹，富永恵子²，山形要人³，田中秀和¹ ¹Gr, Sch, Life Sciences, Ritsumeikan Univ, ²Department of Frontier Biosciences, Osaka University ³Synaptic Plasticity Project, Tokyo Metropolitan institute Medical Sciences Sophisticated neural network undergoes remodeling in response to neural activity. Cell adhesion molecules that link pre- and post-synaptic membranes are responsible not only for the establishment of neural circuitry but also for the modulation of the synaptic strength. Among various classes of synaptic cell adhesion proteins, a non-clustered type protocadherin, Arcadlin/protocadherin-8, is unique in that it is induced dramatically in response to neural activity. Although the primary structure of Arcadlin implies its cell adhesion activity, the hemophilic adhesivity in fact is relatively weak, and rather weakens the hemophilic adhesivity of classical cadherins. Furthermore, Arcadlin reduces spine density in cultured hippocampal neurons. We have previously shown that hippocampal dentate granule and CA1 pyramidal cells in Arcadlin ^-/-mice shows a higher spine density than wild type. Here, we investigated the effect of overexpressed Arcadlin by adopting Arcadlin inducing protocols either transiently and chronically. C57BL/6 mice were exposed to either electroconvulsive seizure or daily fluoxetine（25 mg/kg body weight i.p.）for 18 days, followed by the visualization of dendritic morphology of hippocampal neurons with Lucifer Yellow. Electroconvulsive seizure as well as chronic fluoxetine decreased the spine density. The results are consistent with the hypothesis that the Arcadlin level is negatively correlated with spine density. To further confirm this hypothesis we are going to analyze the spine density of Arcadlin ^-/-mice in response to electroconvulsive seizure and chronic fluoxetine.

P21-5 The significance of complete distributional analysis of the 5-HT3A receptor セロトニン3型受容体発現分布全容解明の重要性 Koyama Yoshihisa（小山佳久），近藤誠，島田昌一 Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, Osaka, JAPAN セロトニンは重要な中枢神経伝達物質である。セロトニン産生細胞は脳幹に存在するが、その軸索投射は脳内全域に及ぶ。セロトニン伝達系は知覚、睡眠及び認識など多岐にわたる脳機能を制御しており、その多様性はセロトニン受容体の種類に依存している。セロトニン3型受容体（5-HT3R）は唯一のイオンチャネル型受容体であり、その速い神経応答によって、嘔吐反射や痛覚反応に関わっている。受容体の中でも特徴的な5-HT3Rの脳内局在情報は、セロトニン伝達系の機能多様性を調べる上で非常に有用である。しかしながら、これまで中枢における5-HT3R発現の全容把握は不十分なままであった。中枢神経系における5-HT3Rの局在を詳細に解析するため、我々は5-HT3R-gleen fluorescent protein（GFP）トランスジェニックマウスを用いた。GFP抗体による蛍光免疫染色法を行った結果、嗅球から仙髄における5-HT3R発現の全容を把握することができた。5-HT3ARは、ソマトスタチンもしくはカルレチニン陽性GABA作動性抑制ニューロンに発現しており、特に嗅球、大脳皮質、海馬及び扁桃体で強い発現が観察された。橋、延髄及び脊髄においては、部分的に発現を確認できた。一方で、視床、視床下部及び中脳での発現は弱く、小脳における発現は全く観察されなかった。一方で、前庭神経遠心路起始核、疑核や下唾液核など新たな発現領域の同定に成功した。我々の構築した5-HT3R発現分布図は、中枢神経系におけるセロトニン受容体の新たな機能の発見に貢献できると考える。		P21-6 Identification and functional characterization of novel 5-HTTLPR alleles in SLC6A4 gene SLC6A4における新規セロトニントランスポーター遺伝子多型（5-HTTLPR）の同定とその機能解析 Ikegame Tempei（池亀天平）¹，文東美紀²，浅井竜朗¹，菅原裕子³，近藤健治⁴，池田匡志⁴，吉川茜¹，西村文親¹，河村代志也⁵，垣内千尋¹，佐々木司⁶，岩田仲生⁴，加藤忠史⁷，笠井清登¹，岩本和也² ¹Department of Neuropsychiatry, Graduate School of Medicine, The University of Tokyo ²Department of Molecular Brain Science, Kumamoto University ³Department of Neuropsychiatry, Graduate School of Medical Sciences, Kumamoto University ⁴Department of Psychiatry, Fujita Health University School of Medicine ⁵Department of Psychiatry, Shonan Kamakura General Hospital ⁶Laboratory of Health Education, Graduate School of Education, The University of Tokyo ⁷Laboratory for Molecular Dynamics of Mental Disorders, RIKEN Center for Brain Science Serotonin transporter (5-HTT) is a key molecule to regulate the serotonergic neurotransmission and has been implicated in the pathophysiology of psychiatric disorders. SLC6A4, which encodes 5-HTT, has the functional polymorphic region (5-HTTLPR) in its promoter region. 5-HTTLPR commonly consists of two major groups: the short (S) and the long (L) alleles, each of which has 14 or 16 tandem repeats of 20-23 bp highly homologous units, with S allele exhibiting lower transcriptional efficiency of SLC6A4 gene compare to the L allele. Although the major S and L alleles have been extensively studied, much less is known about the rare 5-HTTLPR allelic variants.In this study, we performed a detailed analysis of 5-HTTLPR genotypes in healthy controls (CT, n = 488) and patients with major psychosis including schizophrenia (SZ, n = 440) and bipolar disorder (BD, n = 450). As a result of this analysis, we found significantly higher frequency of S allele in patients with major psychosis (Fisher’s exact test, p < 0.05). Furthermore, we identified several novel rare 5-HTTLPR allelic variants. One of novel allelic variants consists of 28 tandem repeats (named XL28 according to the previous literature), the longest 5-HTTLPR allelic variant that has not been ever reported, contains 8 times continuous repeats of ζ-η units. This novel variant was found in unrelated two patients with BD. Although we couldn’t find these novel allelic variants in an independent sample set of CT (n = 1,006) and BD (n = 481), a luciferase reporter assay confirmed its lower promoter activity compared to S allele. These observations indicate that low-activity 5-HTTLPR alleles could increase the risk for developing psychiatric disorders.