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P2-1-61 海馬由来コリン作動性神経刺激ペプチド前駆体ノックアウトマウス海馬におけるHSP90-betaの減少 Suppression of HSP90-beta in the hippocampus of hippocampal cholinergic neurostimulating peptide precursor KO miceite ○金森哲子1, 加藤大輔1, 水野将行1, 及川伸二2, 小鹿幸生1, 松川則之1 ○Tetsuko Kanamori1, Daisuke Kato1, Masayuki Mizuno1, Shinji Oikawa2, Kosei Ojika1, Noriyuki Matsukawa1 名古屋市立大学大学院 医学研究科 神経内科学1, 三重大・医・環境分子医学2 Div. of Dev. Neurol. Neurosci., Grad. Sch. of Med. Sci., Nagoya City Univ., Nagoya, Japan1, Dept. Env. Mol. Med., Mie Univ., Mie, Japan2 Hippocampal cholinergic neurostimulating peptide (HCNP), which induces acetylcholine synthesis, was purified from the soluble fraction of young adult rat hippocampus. This peptide also aligns at N-terminal region of 21kD precursor protein (HCNPpp: HCNP precursor protein) composed of 186 amino acids. Recently, we generated conditional knockout (KO) mice mediated with Cre-transgenic mice driven by CaMKII promoter. In this study, we performed proteomic analysis proteins extracted from hippocampus of homozygous conditional (Cre/+, fHCNPpp/fHCNPpp) and /or wild-type littermate at 12-weeks-old. As results, ten spots of proteins in 2D-DIGE image showed more than 1.1 folds quantitative alternations as absolute logarism value, between those mice. Of two spots, decreasing in HCNPpp (Cre/+, fHCNPpp/fHCNPpp), contained specific 9-amino acids of C-terminal domain of heat shock protein (HSP)-90 beta, and another spot, also decreasing in HCNPpp KO, contained specific 11-amino acids of N-terminal domain of alpha-crystaline B chain (CRYAB) in TOF/TOF/MS analysis. Western blotting also demonstrated the decrease of both HSP90-beta and CRYAB in hippocampus of HCNPpp KO (Cre/+, fHCNPpp/fHCNPpp) compared with wild-type littermate. Recent reports have showed that both HSP90-beta and CRYAB, heat shock proteins, may play a crucial role in folding and/or activating substrate proteins, suggesting molecular chaperons. Interestingly, previous literatures reported that the suppression of chaperons might be involved in pathogenesis of neurodegenative disease via the dysfunction of proteolytic process.	P2-1-62 星状形ナノパターンでの培養によるアストロサイトの分化誘導 Differentiation of astrocyte induced just by culturing on the star-like shaped nano pattern ○武田直也1, 裏田亜里沙1, 井上貴文1, 中村秀樹1 ○Naoya Takeda1, Arisa Urata1, Takafumi Inoue1, Hideki Nakamura1 早稲田大学 先進理工学部 生命医科学科1 Dept of Life Sci and Med Biosci, School of Adv Sci and Eng, Waseda University, Tokyo1 Manipulating differentiation of astrocyte was examined just by culturing on the star-like shaped nano patterns. The electron beam lithography and a polymer surface of the positive resist as the mask material, which have been originally developed for semiconductor processing, were directly applied to create this cellular patterning system in nano resolution. Rat primary astrocyte cultured in vitro usually extends in protoplasmic or radial shape on a flat surface. However, when cultured on the 30 micrometer square pattern overlapping with crossing lines of 100 nanometer width and 200 micrometer long, astrocyte adhered on the square pattern and extended along the line patterns with elongating processes like star shape. By connecting a number of these square-crossing line pattern set to form grid design, the star-like shaped astrocyte also formed cellular network along the pattern. Lucifer Yellow which was microinjected into a single cell laterally diffused to the adjacent cell, showing formation of gap junction in the astrocyte networks. Fluorescence immunostaining analyses quantitative RT-PCR experiments showed that culturing on the nano patterns increased GLT-1 expression, which is normally upregulated in star-shaped astrocyte in vivo or differentiated one by dbcAMP in vitro, in comparison with culturing on a flat glass surface. Thus, it was suggested that just changing cellular shape along the interface topology and/or mechanical stress accompanying with the shape change worked to regulate differentiation and function of astrocyte without applying biosignaling factors.
P2-1-63 Serotonin transporterの取り込み機能におけるMARCKSL1の関与 Involvement of MARCKSL1 in serotonin transporter up take function ○石川淑子1, 松崎伸介1,2,3, 小山佳久2, 遠山正彌1,4, 片山泰一1 ○Toshiko Ishikawa1, Shinsuke Matsuzaki1,2,3, Yoshihisa Koyama2, Masaya Tohyama1,4, Taiichi Katayama1 大阪大学大学院 連合小児発達学研究科 分子生物遺伝学1, 大阪大学大学院医学系研究科神経機能形態学講座2, 大阪大学医学部子どものこころの分子統御機構研究センター3, 大阪府立病院機構4 Dept. Molecular Brain Sci. United Graduate School of Child Development, Osaka Univ. School of Medicine, Osaka, Japan1, Dept. of Anatomy and Neuroscience, Graduate School of Medicine, Osaka Univ., Osaka, Japan2, Molecular Research Center for Children's Mental Development, Osaka Univ., Osaka, Japan3, Osaka Prefectural Hospital Organization, Osaka, Japan4 Autism is a neurodevelopmental disorder characterized by lack of social interaction and communication, obsessiveness, and repetitive behavior. Although it is reported that the prevalence of autism is about 1 to 2 per 1000 people in Japan, the number has been increased, recently. However, no treatment and prevention, even diagnosis for autism has been established, because little is known about the pathogenesis of autism. Recently, it is reported that the risk of autism is associated with density of serotonin transporter (SERT) at brain. So far, we have identified MARCKSL1 as an interactor with SERT. Then, we examine involvement of MARCKSL1 in SERT function. Here, we report about the change of intracellular localization of SERT by MARCKSL1 deletion mutants. Furthermore, we show that serotonin uptake ability as a SERT function is down-regulated by several MARCKSL1 deletion mutants.	P2-1-64 大脳新皮質第5層における微細秩序構造の3次元解析 Three dimensional analysis of the ordered micro-organization in neocortical layer 5 ○木曽かおり1, 黒川裕2, 濱裕2, 宮脇敦史2, 細谷俊彦1 ○Kaori Kiso1, Hiroshi Kurokawa2, Hiroshi Hama2, Atsushi Miyawaki2, Toshihiko Hosoya1 理研BSI局所神経回路1, 理研BSI細胞機能探索2 Lab. for Loacal Neuronal Circuit, BSI, RIKEN, Saitama, Japan1, Lab. for Cell Function Dynamics, BSI, RIKEN, Saitama, Japan2 The organization of functional subtypes of neurons shapes the basic structure of neocortical circuits. Our previous analysis revealed that in mouse layer 5, subcerebral projection neurons (SCPNs), which are a major pyramidal neuron subtype, are aligned radially to form columnar organization. These columnar units of SCPNs are located periodically in the tangential orientation in layer 5. Under specific visual stimulation, SCPNs in a single unit exhibited strongly correlated c-Fos expression, suggesting related functions. These observations suggest that layer 5 has a periodically-repeated functional micro-organization of SCPNs. To further understand the structure of this micro-organization, we are performing three-dimensional analyses. We label SCPNs with various makers, and make whole-brain samples transparent using the Scale method. Three-dimensional positions of SCPNs are obtained by two-photon microscopy and subjected to statistical analyses. We first labelled SCPNs by EGFP expression in the Crym-EGFP transgenic mice, and confirmed the columnar and periodic organization. However, in the Crym-EGFP mice, EGFP was expressed only in limited cortical areas. In addition, even in cortical areas where EGFP was expressed in SCPNs, typically only 50-80% of SCPNs expressed EGFPs. To investigate the micro-organization in wider cortical areas and with higher efficiency, we performed analyses of SCPNs labelled by retrograde-tracing. SCPNs were retrogradely-labelled by injection of tracers into the pons, and the whole brain data was obtained and analyzed by methods optimized for retrograde labeling. Our data indicated that retrogradely-labelled SCPNs showed strong columnar and periodic organization. Detailed analyses of the three-dimensional organization will be presented.

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