e ポスター

e ポスター 9. 回路網形成 e Poster 9. Circuit formation

2020/9/11　14:00～15:00　オンデマンドB-1 P2-22 ヒトiPS細胞由来神経系細胞から作成した神経回路標本におけるグルタミン酸トランスポーターの役割 The role of human excitatory amino acid transporters (EAATs) in the neural network samples generated from human induced pluripotent stem cell-derived (hiPSC) neural cells 高橋華奈子¹、中條かおり¹、鈴木郁郎²、佐藤薫¹ 1. 国立医薬品食品衛生研究所薬理部神経薬理、2. 東北工業大学大学院工学部電気電子工学科 kanako TAKAHASHI¹, kaori Chujo¹, ikuro Suzuki², kaoru Sato¹ 1. Lab. Neuropharmacol., Div. Pharm., Nat. Inst. Hlth. Sci, 2. Dept. Electronics, Grad. Sch. Engineering, Tohoku Inst. Tech. We have attempted to generate human neural networks on dish using hiPSC-neural cells. This in vitro assay system is expected to improve the predictability of the non-clinical CNS study in drug development. EAATs are responsible for removal of excitatory neurotransmitter L-glutamate (L-Glu), however, little information is available concerning the roles of EAATs in hiPSC-neural network samples. In this study, we investigated the expression profile and the role of EAATs in the hiPSC-neural network sample. XCell neurons (commercially available hiPSC-neurons) were seeded at 3.0×10⁵ cells/cm² and cultured for 2 months. We first compared the expression profiles of neuronal genes (TUBB3) and astrocytic genes (GFAP and AQP4). The expression level of TUBB3 mRNA was decreased while those of GFAP and AQP4 mRNA were greatly increased as the culture period got longer. We also examined the neural network generation. We could record the synchronized burst firing activity using microelectrode array system from 42 DIV. Furthermore, at 63 DIV, presynaptic synapsin1 puncta and postsynaptic PSD95 puncta were closely apposed. We then examined the functions of EAATs at DIV 63. When we exogenously applied 100 µM L-Glu, the concentration of L-Glu in the medium was decreased to almost zero in 1 hr. In the presence of TFB-TBOA (TFB), non-specific EAAT blocker, the decrease in the L-Glu concentration was not observed. Although 100 µM L-Glu had little effects on cell viability measured by MTT reduction, L-Glu caused a significant decrease in the presence of TFB. When AP5, NMDAR antagonist, was co-applied with TFB, the cell damage was significantly suppressed. These results suggest that EAATs surely uptake the exogenous L-Glu in hiPSC-neural network samples and protect neurons from excitotoxicity.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-23 輸送可能なヒトiPS由来の神経細胞プレート Transportable human iPSC-derived neuron plates 塩野入桃子¹、仲山智明¹、腰塚慎之助¹、北澤智文¹、矢本梨恵¹、荒谷知行¹ 1. 株式会社リコー MOMOKO SHIONOIRI¹, TOMOAKI NAKAYAMA¹, SHINNOSUKE KOSHIZUKA¹, TOMOFUMI KITAZAWA¹, RIE YAMOTO¹, TOMOYUKI ARATANI¹ 1. RICOH COMPANY,LTD. Neurotoxicity is a major cause of failures in drug development. Precise prediction of neurotoxicity has been difficult with animal models because of the species difference. To overcome this difficulty human neurons generated from induced pluripotent stem cell (iPSC) have been extensively tested. In vitro assays using multielectrode arrays (MEAs) are well suited for this purpose. When such neuron plates are prepared by the user himself, know-how for culturing the human iPSC -derived neurons is required, and the length of the culture period requires a great deal of labor and cost. On the other hand, if a plate manufacturer produces neuron plates and the user requests a test, there is a risk of leaking information about the drug under development. These problems can be solved if neuron plates can be transported from the plate manufacturer to the user. However, such neuron plates are extremely delicate, there has been no attempt to transport it. In this study, we developed the transportable human iPSC-derived neuron plates by optimizing neuron culture conditions. The developed plates showed normal neuron characteristics even after transport tests intended for domestic shipment, suggesting that it can be transported from the plate manufacturer to the user. This technology is expected to contribute to reduction the cost and risk associated with toxicity tests using human iPSC-derived neuron plates.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-24 同種移植されたマウスES細胞由来視床下部神経は下垂体後葉に軸索投射する Axon-guidance to the posterior pituitary on grafted hypothalamic neurons from mouse ES cells 河田美穂¹、小谷侑¹、亀山俊樹¹、中島昭²、長崎弘¹ 1. 藤田医科大学医学部生理学講座Ⅰ、2. 藤田医科大学医学部生理化学 Miho Kawata¹, Yu Kodani¹, Toshiki Kameyama¹, Akira Nakashima², Hiroshi Nagasaki¹ 1. Dept Physiol, Sch Med, Fujita Health Univ., 2. Dept Physiol Chem, Sch Med, Fujita Health Univ. We have developed a method to differentiate hypothalamic neurons from mouse embryonic stem cells (mESC). These cells have successfully survived for one month when grafted into the supraoptic nucleus (SON) of the hypothalamus of SCID/NOD mice. However, it is unclear whether grafted neurons would survive for a long time and whether it was appropriately integrated into the host neural circuits. To address these issues, we have sampled both brain and pituitary after 3 months from the grafting and analyzed the graft-derived axonal projection. As previously reported, the hypothalamic neurons were prepared from EB5, mESC cell line, by three-dimensional floating culture method. The hypothalamic progenitors were purified using the cell-surface antigens. For differentiation, cells were enzymatically dissociated and cultured on Matrigel-coated cover glass. After day 28, cells demonstrate hypothalamic identities, and were transduced with AAV-CAG-tdTomato for labeling, and then injected to SON of the SCID/NOD mice by stereotaxic procedure. We found a cluster of graft tissue in the SON region, and no neoplastic changes observed. The tdTomato-positive axons were distributed in wide area around the graft, not only in the hypothalamus but also in the specific hypothalamic target regions along rostro-caudal axis such as hippocampus, amygdala and ventral tegmental area. The tdTomato-positive axons were also identified in the neural lobe of pituitary. They were immunopositive for TUJ1 and showed beaded-shape, which suggest they are hormone-releasing neurons. These results suggested that the grafted hypothalamic neurons survived in the host hypothalamus for long-term and extend axons long distances to the target regions. Further investigations are needed to elucidate the axon guidance mechanism for the proper axonal projections of grafted hypothalamic neurons.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-25 Cdk5はαII-spectrinのリン酸化を介して軸索の周期的細胞骨格構造を制御する Cdk5 regulates phosphorylation of αII-spectrin and axonal periodic cytoskeletal structures. 吉村武¹、Rasband Matthew²、片山泰一¹ 1. 大阪大学大学院・連合小児発達学研究科、2. ベイラー医科大学 Takeshi Yoshimura¹, Matthew N. Rasband², Taiichi Katayama¹ 1. United Graduate School of Child Dev, Osaka Univ, 2. Dept of Neurosci, Baylor College of Medicine Axons have a specific cytoskeletal structure lining the cytoplasmic face of the axolemma. Super-resolution microscopy has revealed a remarkable periodic lattice in axons. The spectrin/ankyrin-based cytoskeleton forms a periodic structure with actin filaments organized in rings spaced 190 nm apart. αII-spectrin, βIV-spectrin and Ankyrin-G form the periodic lattice in the axon initial segment (AIS), whereas the distal periodic cytoskeleton consists of αII-spectrin, βII-spectrin and Ankyrin-B. The distal cytoskeleton progressively fills the axon from its distal growing end back towards the cell body, and defines a boundary limiting Ankyrin-G to the remaining proximal axon that will become the AIS. Spectrins are widely expressed and exist as tetramers consisting of two α and two β subunits. βI-βIV spectrins are found in neurons. However, αII-spectrin is the only α spectrin detected in the nervous system, suggesting that αII-spectrin must play important roles in the entire axon. It was reported that human mutations in αII-spectrin cause West syndrome. While αII-spectrin intracellular localization is fairly well understood, the molecular mechanism by which αII-spectrin is regulated remains unclear. Here, we report that Cdk5 phosphorylates αII-spectrin. Inhibition of Cdk5 impaired AIS formation and neuronal polarity. These results suggest that Cdk5 regulates the axonal periodic cytoskeleton through the phosphorylation of αII-spectrin.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-26 脳に高発現するキナーゼLMTK1のアイソフォーム依存的な機能解析 Expression and functions of two LMTK1 isoforms in brains 魏冉¹、西野尋紀¹、佐藤勇太⁴、杉山亜梨華¹、高橋美由紀¹、浅田明子⁴、福田光則³、五十嵐道弘⁴、友村美根子²、久永真市¹ 1. 東京都立大学、2. 明海大学、3. 東北大学、4. 新潟大学 Ran Wei¹, Hironori Nishino¹, Yuta Satoh⁴, Arika Sugiyama¹, Miyuki Takahashi¹, Akiko Asada⁴, Mitsunori Fukuda³, Michihiro Igarashi⁴, Mineko Tomomura², Shin-ichi Hisanaga¹ 1. Tokyo Metropolitan University, 2. Meikai University, 3. Tohoku University, 4. Niigata University Lemur tail kinase 1 (LMTK1) is a membrane-bound Ser/Thr kinase that is highly expressed in neurons. There are mainly two splicing variants of LMTK1 with different membrane binding modes, palmitoylation at N-terminal cysteines for cytosolic LMTK1A, and transmembrane sequences for LMTK1B. We have recently reported that LMTK1A regulates axon outgrowth and spine formation in neurons. In contrast, almost nothings are known for LMTK1B. Here, we investigated the expression, subcellular localization and roles in axon and spine formation of LMTK1B. Similar to LMTK1A, the wild type of LMTK1B was found to localize to Rab11-positive pericentrosomal compartment. The kinase negative form of LMTK1B was found to associate with the tubular form of endoplasmic reticulum (ER), different from the kinase negative form of LMTK1A. Moreover, unlike LMTK1A kinase negative form, LMTK1B kinase negative form did not promote the axon outgrowth and spine formation. These results suggest that while LMTK1A and LMTK1B share a common function in recycling endosomal trafficking at the pericentrosomal compartment, LMTK1B has an additional unique function in vesicle transport in the ER region.