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2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P1 症候性⾃閉スペクトラム症ティモシー症候群の新規モデルマウス開発 Establishment of a novel mouse model of Timothy syndrome with syndromic autism spectrum disorder ^○小澤享弘¹, Fumi Hyodo^1,2, Miao Pan^1,2, Shuhei Ueda^1,2, Manabu Abe⁴, Kenji Sakimura⁴, Shin-ichiro Horigane^1,2, Sayaka Takemoto-Kimura^1,2 1.Department of Neuroscience I, Research Institute of Environmental Medicine, Nagoya University, 2.Molecular/Cellular Neuroscience, Nagoya University Graduate School of Medicine, 3.Department of Pathology and Laboratory Medicine, Nagoya University Hospital, 4.Department of Animal Model Development, Brain Research Institute, Niigata University ^○Yukihiro Ozawa¹, Fumi Hyodo^1,2, Miao Pan^1,2, Shuhei Ueda^1,2, Manabu Abe⁴, Kenji Sakimura⁴, Shin-ichiro Horigane^1,2, Sayaka Takemoto-Kimura^1,2 1.Department of Neuroscience I, Research Institute of Environmental Medicine, Nagoya University, 2.Molecular/Cellular Neuroscience, Nagoya University Graduate School of Medicine, 3.Department of Pathology and Laboratory Medicine, Nagoya University Hospital, 4.Department of Animal Model Development, Brain Research Institute, Niigata University Ca²⁺ signaling plays key roles in multiple cellular functions, including neural circuit formation and plasticity. Ca²⁺ signaling dysfunction is linked with psychiatric and neurological disorders, including autism spectrum disorder (ASD). Timothy syndrome (TS) is a rare genetic disorder caused by a single de novo missense mutation G406R in L-type Ca²⁺ channel (Ca_v1.2), causing excessive Ca²⁺ influx into the cells (Splawski et al., 2004, 2005). TS is characterized by ASD, gross motor dysfunction, and QT prolongation, and its pathophysiological mechanism remains largely unknown. TS2-neo, a G406R general knock-in mouse strain has been previously established as a TS mouse model (Bader et al., 2011). Several research groups, including ours, have reported ASD-related behaviors in social and repetitive/restricted behavior domains in this mouse model. Moreover, neurodevelopmental structural abnormalities were also revealed in the TS2-neo mice. These findings significantly advanced our understanding of the pathophysiology of TS. However, it is noteworthy that the expression level of G406R mutant channels is suppressed in this mouse model to avoid mortality before weaning. In this study, we developed a nervous system-specific G406R knock-in mouse to elucidate the outcome of a G406R mutation in the nervous system. The novel TS mouse model, bearing high levels of mutant channels in the nervous system, presented ASD-like behaviors and motor coordination impairment. We also found histological differences in the neocortex of the mouse model. Therefore, we conclude that we successfully developed a novel TS mouse model with construct and face validity, which helps us greatly in better understanding the complex pathophysiology of TS.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P2 1次繊毛に局在するNPY受容体-Y2の特性評価：モデル細胞とマウス脳を用いた繊毛動態およびシグナル解析 Characterization of primary cilia in NPY receptor 2-expressing cells in vitro and in vivo ^○西村宣哉, Yuki Kobayashi, Yumiko Saito 広島大学　統合生命科学研究科 ^○Nobuya Nishimura, Yuki Kobayashi, Yumiko Saito Graduate School of Integrated Sciences for Life, Hiroshima University The primary cilium is a specialized sensory organelle protruding from the plasma membrane. Ciliary dysfunction results in severe human diseases called ciliopathies. It is also becoming evident that a cilium length of the right size is relevant in physiological conditions, as ciliary shortening is observed in several types of obese rodent model. Ciliary membrane is highly enriched in selective receptors such as a small set of GPCRs including feeding-related neuropeptide Y (NPY) receptor Y2. We previously found that NPY caused efficient cilia length shortening with a lower EC50 value in Y2 expressing hTERT-RPE1 (hRPE1) clone cells. The initiation of Y2-mediated cilia shortening depends on two parallel signaling events: Gi/o-Akt activation and Gi/o-JNK activation. In the present study, we performed the transcriptome analysis in Y2-expressing hRPE1 cells in response to NPY to identify the target molecules involved in cilia length regulation. Because both Gi/o and Akt/JNK activation is essential for NPY-elicited the ciliary shortening, RNA-sequencing analysis of Y2 cells were subjected to NPY with combination of Gi/o- , Akt- and JNK-inhibitors. Collectively, we found upregulation of 79 genes and downregulation of 52 genes compared with static control cells. To narrow down the number of candidate genes, we are now cross-referencing our list with other systematic studies. Regarding the in vivo mouse brain, we observed higher levels of ciliary Y2 in hypothalamus and amygdala. Furthermore, Y2 positive cilia length in a specific nucleus of the hypothalamus was found to be significantly shortened in fasted mice compared with fed mice. We believe that our study will lead to a better understanding of the molecular mechanisms underlying ciliary Y2-mediated cilia length control.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P3 中枢性一次繊毛局在型GPCR (MCHR1)を基盤とするトランスクリプトーム解析－繊毛ダイナミクスを制御する新規責任分子の同定 Transcriptome analysis of ciliary-dependent GPCR signaling-Identification a novel regulatory step underlying cilia length control ^○斎藤祐見子¹, Tatsuo Miyamoto¹, Yuko Sekino¹, Tomoaki Shirao¹, Noriko Koganezawa¹, Yuki Kobayashi¹ 1.広島大学統合生命科学研究科, 2.Res Inst Rad Bio Med, Hiroshima University, 3.Grad Sch Pharma Sci, Univ Tokyo, 4.AlzMed, Inc, 5.Grad Sch Med, Gunma University ^○Yumiko Saito¹, Tatsuo Miyamoto¹, Yuko Sekino¹, Tomoaki Shirao¹, Noriko Koganezawa¹, Yuki Kobayashi¹ 1.Grad Sch Int Sci Life, Hiroshima University, 2.Res Inst Rad Bio Med, Hiroshima University, 3.Grad Sch Pharma Sci, Univ Tokyo, 4.AlzMed, Inc, 5.Grad Sch Med, Gunma University The primary cilium is a biomechanical sensor plugged in at the cell surface. It is implicated in the processing of extracellular signals and its absence or misfunctioning lead to a broad variety of serious defects. Ciliary signaling is mediated, in part, by a limited set of GPCRs preferentially enriched in the cilium membrane. This includes melanin-concentrating hormone (MCH) receptor 1 (MCHR1), which plays a role in feeding and mood. In addition to its receptor composition, the length of the cilium is a characteristic parameter that is implicated in its function. We previously found that MCH shorten cilia length via the Gi/o-Akt pathways in both MCHR1-expressing hTERT-RPE1 cells (hRPE1 cells) and hippocampal neurons. However, the detailed step by which MCHR1 activity elicits cilia shortening remains unknown. In this study, we used RNA-seq to identify the target molecules involved in MCHR1-mediated cilia shortening. The analysis of ciliated cells with MCH treatment showed 536 genes changing compared with static control cells. Validation by qRT-PCR and loss-of-function technology identified a scaffolding molecule, PDLIM5 as the most significant key factor for MCHR1-mediated cilia shortening. We further demonstrated the crucial importance of the F-actin network regulator alpha-actinin 1/4 as a downstream target of PDLIM5. In the endogenous MCHR1-expressing hippocampus, transcriptional upregulation of PDLIM5 and actinin 1/4, following the application of MCH, was detected when the MCHR1-positive cilia were shortened. This is the first study to analyze ciliary GPCR-based transcriptome profiles in terms of ciliary dynamics. Our approach also serves as a template for future studies aiming to identify key insights into the signaling role of other ciliary GPCRs such as D2R.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P4 TRPA1チャネルは細胞外のアルカリ状態により活性化が増強する Activation of TRPA1 channel is synergistically potentiated by extracellular alkaline condition ^○柴崎貢志, Akane Egoshi, Miho Kusano, Tomoko Matsumoto 長崎県立大学大学院　人間健康科学研究科 ^○Koji Shibasaki, Akane Egoshi, Miho Kusano, Tomoko Matsumoto Laboratory of Neurochemistry, University of Nagasaki We previously reported that TRPA1 is activated by extracellular alkaline condition which contributes pain sensation (J. Clin. Invest. 2008). It has been reported that TRPA1 is activated by various stimuli such as cold (<17°C), Allyl isothiocyanate (AITC in Wasabi and mustard oil) and mechanical stimulus. All TRP channels have unique properties called as synergistic effects. If we apply two different agonists, thresholds of each agonist can be effectively reduced. Thus, we can observe significant TRP channel activation by combinational application of two different agonists. These backgrounds indicate that TRPA1 activation can be potentiated by weak alkaline condition. In this study, we examined the possibility by an electrophysiological experiment. Two-electrode voltage clamp in Xenopus oocytes is a conventional electrophysiological technique used to artificially control the membrane potential of large cells to study the properties of electrogenic membrane proteins, especially ion channels. We ectopically expressed mouse TRPA1 in Xenopus oocytes, and examined the effects of extracellular alkaline condition on TRPA1 activation by AITC. Although we failed observed TRPA1 activation in weak alkaline condition, AITC-activated TRPA1 currents were significantly potentiated in the alkaline condition compared with those in normal pH (pH7.4). These results indicate that alkaline condition significantly reduces the thresholds for AITC responses. We hypothesize that these properties involve in various physiological responses such as gliotransmitter release.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P5 Quick-Neuron™Plate - MEA 48を用いた抗てんかん薬の神経応答評価 Evaluation neuronal responses of anti-epileptic drugs with Quick-Neuron™ Plate ^○仲山智明, 荒谷知行, 東基記 Biomedical Business Center, Ricoh Futures Business Unit, Rioch Company LTD. ^○Tomoaki Nakayama, 知行荒谷, 基記東 Biomedical Business Center, Ricoh Futures Business Unit, Rioch Company LTD. 【Background】 The significant difference between human and animals is one of the most frequent cause of drug discovery project closures in central nervous system (CNS) field. And, it is also well known that in vitro/ in vivo animal models are often insufficient to predict drug efficacies and toxicities in human. To overcome this challenge, many assay methods with the human induced pluripotent stem cell (iPSC) derived neurons are being reported. Multielectrode array (MEA) is one of the well-known methods to evaluate nerve functions. However, MEA has some challenges. For example, the preparation of the MEA plate with iPSC-derived neurons by the users themselves involves tremendous labor, cost, and risks, including cell culture know-how, a prolonged culture period, and problems such as agglutination and stripping off cells. To solve these challenges, we have developed the Quick-NeuronTM Plate - MEA48, which is the ready-to-use plate. 【Objective】 In this study, we evaluated the effects of anti-epileptic drugs on the iPSC-derived neurons by using Quick-NeuronTM Plate - MEA48. And, we showed if the plate can be used for drug screening in CNS-related disease such as epilepsy. 【Results】 It was shown that the anti-epileptic drugs which act on ion channels reduced the number of spontaneous neuronal firing. This result suggested that Quick-Neuron™ Plate-MEA 48 is suitable for pharmacological evaluation of anti-epileptic drugs. As a future study, we will investigate whether Quick-Neuron™ Plate-MEA 48 can be used in other diseases.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P6 酸化ガレクチン-1によって刺激されたマクロファージにおける神経再生関連因子の発現の詳細な分析 Detailed analysis of the expression of nerve regeneration-related factors in macrophages stimulated by oxidized galectin-1 ^○寺島裕美¹, Ryu-ichiro Hirano¹, Kazunori Sango¹, Toshihiko Kadoya¹ 1.前橋工科大学大学院工学研究科生物工学専攻　タンパク質化学研究室, 2.Diabetic Neuropathy Project, Tokyo Metropolitan Institute of Medical Science ^○Yumi Terashima¹, Ryu-ichiro Hirano¹, Kazunori Sango¹, Toshihiko Kadoya¹ 1.Department of Biotechnology, Maebashi Institute of Technology, 2.Diabetic Neuropathy Project, Tokyo Metropolitan Institute of Medical Science It is well known that macrophages contribute the nerve regeneration after injury, involving Wallerian degeneration and axonal regeneration. One of the roles of macrophages in the regeneration is the secretion of factors involved in the repair of nerve tissue. However, the details of these secretory factors and the time course of expression have not yet been clarified. We identified oxidized galectin-1 (Gal-1/Ox) as an essential factor to promote peripheral nerve regeneration by stimulating macrophages. Galectin-1 (Gal-1) is an animal lectin specific to β-galactoside, showing the lectin activity when all 6 cysteine residues of the molecule are in the reduced state. On the other hand, Gal-1/Ox, in which three sets of S-S bonds are formed, does not show lectin activity but promotes nerve regeneration after injury. In this study, we examined the mRNA expression of nerve regeneration-related factors in U937 derived macrophages after Gal-1/Ox stimulation. Human monocyte derived U937 cells were differentiated into macrophages by PMA stimulation. Cells cultured for 3, 9, 18, 24, and 48 hours after the addition of Gal-1/Ox or Gal-1 were collected and mRNA expression of nerve regeneration-related factors were analyzed using real-time-PCR. Expression changes specific to Gal-1/Ox stimulated cells were observed in the factors of BDNF, GDNF, NT-3, NT-4, FGF-2, HGF and IL-6. Expression of GDNF, NT-4 and FGF-2 increased early and maximally reached at 3 hours after stimulation. Then, the expression of HGF and NT-3 reached the maximum at 9 hours. BDNF and IL-6 were maximally expressed at 48 hours, respectively. It was suggested that peripheral nerve is successfully repaired by the action of these factors, which show different expression time course, in early stage after injury.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P7 腸管神経系におけるタウリンの生理作用の解明 The physiological role of taurine in the enteric nervous system ^○森谷大地, Tatsunori Maekawa, Rei Kawashima, Fumitaka Kawakami, Takafumi Ichikawa 北里大学大学院医療系研究科医科学専攻生体制御生化学研究室 ^○Daichi Moriya, Tatsunori Maekawa, Rei Kawashima, Fumitaka Kawakami, Takafumi Ichikawa Department of Regulation Biochemistry, Graduate School of Medical Sciences, Kitasato University [Background and aim] Taurine is one of the amino acid analogs found in organisms including humans. Since taurine is abundant in the central nervous system and is an important molecule for nerve development and protection, we focused on the role of taurine in the enteric nervous system (ENS). Given that it has been reported that dysfunction of the ENS can be a factor in the development of irritable bowel syndrome, inflammatory bowel disease, depression, and Parkinson’s disease, maintaining homeostasis of the ENS could affect the health of the body. It has been known that taurine is involved in gastrointestinal functions by promoting gastric acid secretion and small intestinal smooth muscle contraction, but the cellular mechanisms have not been elucidated. In this study, to reveal the physiological effects of taurine in the ENS, we performed ex vivo intestinal motility analysis and immunofluorescence staining of enteric plexus using C57BL/6J mice in the presence of taurine. [Results] To examine the expression of taurine synthase and neuronal activation, whole-mount staining using anti-cysteinesulfinic acid decarboxylase (CSAD/CSD) antibody and anti-c-Fos antibody were performed. The results showed that CSAD-positive cells were found in enteric glial cells but not in enteric neurons. Immunostaining using anti-c-Fos antibody showed that low concentrations of taurine promoted neuronal activation, while high concentrations of taurine suppressed neuronal activation. [Conclusion] The present study revealed the expression of taurine synthase in enteric glial cells. Inhibitory effect in high concentrations of taurine suggests that taurine could bind to GABA receptors in the ENS as well as in the brain.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P8 GnRH産生細胞株における炎症性サイトカインの発現に対するTNF-αの影響 Effect of TNF-α on the expression of inflammatory cytokines in immortalized gonadotrophin-releasing hormone neurons ^○大谷佐知¹, Momo Enomoto², Yusuke Minato², Seishi Maeda², Hideshi Yagi² 1.兵庫医療大学　共通教育センター, 2.Department of Anatomy and Cell Biology, Hyogo College of Medicine ^○Sachi Kuwahara-Otani¹, Momo Enomoto², Yusuke Minato², Seishi Maeda², Hideshi Yagi² 1.General Education Center, Hyogo University of Health Sciences, 2.Department of Anatomy and Cell Biology, Hyogo College of Medicine Gonadotropin-releasing hormone (GnRH) neurons secrete an important hypothalamic neuropeptide that control of the reproduction and fertility. It is known that peripheral inflammation and stress can influence reproduction at the level of hypothalamus. We have previously reported that immortalized GnRH neurons, GT1-7 cells express some inflammatory cytokines and their receptors as shown by RT-PCR analysis. It is suggested that inflammatory cytokines exert direct effects upon GnRH neurons via specific receptors and GnRH neurons synthesize cytokines. Neuronal expression of cytokines is an area of active investigation in the contexts of development, disease, and normal neuronal function. In this study, we examined the effect of TNF-α in GT1-7 cells. GT1-7 cells were found to express TNF receptor 1 (TNFR1), analyzed by immunofluorescence and western blot. To investigate whether TNF-α activates NF-κB in GT1-7 cells, we applied recombinant mouse TNF-α (rmTNF-α) and studied the intracellular localization of NF-κB p65 using immunocytochemistry. In untreated cells NF-κB p65 was totally inactive and retained in the cytoplasm. Administration of rmTNF-α induced a nuclear NF-κB p65 translocation. We investigated whether TNF-α promotes the expression of inflammatory cytokines in GT1-7 cells. Stimulation of GT1-7 cells with rmTNF-α increased the expression of TNF-α mRNA, but not IL-1β and IL-6. Our observations indicate that TNF-α exert direct effects upon GnRH neurons via its specific receptor and there is a possibility that TNF-α acts on GnRH neurons through an autocrine pathway.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P9 高機能自閉スペクトラム症のマクロファージにおけるTNF-α発現異常 TNF-α expression aberration of M1/M2 macrophages in adult high functioning autism spectrum disorder ^○Yamauchi Takahira¹, Kazuki Okumura¹, Yoshinori Kayashima¹, Rio Ishida¹, Ryohei Takada¹, Michihiro Toritsuka¹, Naoko Kishimoto¹, Masato Takahashi¹, Takashi Komori¹, Yasunari Yamaguchi¹, Kazuhiko Yamamuro¹, Sohei Kimoto¹, Toshifumi Kishimoto¹, Manabu Makinodan¹ 1.Department of Psychiatry, Nara Medical University, 2.Akitsukonoike Hospital ^○Yamauchi Takahira¹, Kazuki Okumura¹, Yoshinori Kayashima¹, Rio Ishida¹, Ryohei Takada¹, Michihiro Toritsuka¹, Naoko Kishimoto¹, Masato Takahashi¹, Takashi Komori¹, Yasunari Yamaguchi¹, Kazuhiko Yamamuro¹, Sohei Kimoto¹, Toshifumi Kishimoto¹, Manabu Makinodan¹ 1.Department of Psychiatry, Nara Medical University, 2.Akitsukonoike Hospital The etiology of autism spectrum disorder (ASD) is complex, and its pathobiology is characterized by enhanced inflammatory activities; however, the precise pathobiology and underlying causes of ASD remain unclear. This study was performed to identify inflammatory indicators useful for diagnosing ASD. The mRNA expression of cytokines, including tumor necrosis factor-α (TNF-α), was measured in cultured M1 and M2 macrophages from patients with ASD (n = 29) and typically developed (TD) individuals (n = 30). Additionally, TNF-α expression in the monocytes of patients with ASD (n = 7), showing aberrations in TNF-α expression in M1/M2 macrophages and TD individuals (n = 6), was measured. TNF-α expression in M1 macrophages and the TNF-α expression ratio in M1/M2 macrophages were markedly higher in patients with ASD than in TD individuals; however, this increase was not observed in M2 macrophages (M1: sensitivity = 34.5%, specificity = 96.7%, area under the curve = 0.74, positive likelihood ratio = 10.34; ratio of M1/M2: sensitivity = 55.2%, specificity = 96.7%, area under the curve = 0.79, positive likelihood ratio = 16.55). Additionally, TNF-α expression did not significantly differ in monocytes from patients with ASD and TD individuals. In conclusion, further studies on TNF-α expression in cultured macrophages may improve the understanding of ASD pathobiology.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P10 口腔内炎症はパーキンソン病発症に関与するのか Role of oral inflammation in the pathogenesis of Parkinson disease ^○永井雅代¹, Masaaki Hirayama³, Makoto Naoi¹, Wakako Maruyama¹ 1.愛知学院大学心身科学部健康栄養学科, 2.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 3.blank spot ^○Masayo Shamoto-Nagai¹, Masaaki Hirayama³, Makoto Naoi¹, Wakako Maruyama¹ 1.Department of Health and Nutrition、Faculty of Psycological & Physical Science、Aichi Gakuin University, 2.Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, 3.blank spot Objective: Association of entero-microbiome and relating chronic enteric inflammation with the pathogenesis of Parkinson disease (PD).is gathering attention. Oral cavity is the most rostral part of the enteric canal, but limited number of studies have been done in concern to PD. Periodontal disease, a common chronic inflammation in the aged increases the risk of geriatric disorders, including neurodegenerative diseases. Myeloperoxidase (MPO), an enzyme released from activated neutrophils migrated to the saliva, is known to play a central role in the primary defense in the oral cavity, and is proposed a biomarker candidate for periodontal disorders. In this paper, the occurrence of oral inflammation was studied by analyzing salivary MPO activity in PD patients. Materials and Methods: Saliva samples were obtained from the aged patients in the elderly care facility (n = 13), PD patients (n = 117), patients of REM sleep behavior disorder (RBD), prodromal PD patients (n = 11), and control (n =68). MPO activity and protein level in the saliva was quantified spectrometrically and ELISA assay, respectively. The study protocol was approved by Nagoya University Ethics Committee. (Authorization number: 8277). Results: MPO level increased in patients with periodontitis even in the early stage. Salivary MPO activity was significantly increased in PD patients (means: 5.90 ± 7.48 Δ650 nm/min/ml, median: 3.16) compared to RBD patients (3.01 ± 2.14, 2.14) and control (4.39 ± 9.08, 2.75). Conclusion: Salivary MPO is applicable biomarker of periodontitis. Chronic oral inflammation in PD may increase oxidative stress at peripheral nerve ending distributing in the oral cavity and produce toxic oxidatively-modified protein relating the pathogenesis of PD. Conflict of interest: No

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P11 胎児期の有機リン系農薬曝露が小脳発達に与える影響 Effects of fetal exposure to organophosphorus pesticide, chlorpyriphos, on cerebellar development ^○羽谷航¹, Rie Matsufusa¹, Yasunari Kanda², Sachiko Yoshida¹ 1.豊橋技術科学大学応用化学・生命工学課程学部4年, 2.National Institute of Health Sciences ^○Wataru Haya¹, Rie Matsufusa¹, Yasunari Kanda², Sachiko Yoshida¹ 1.Toyohashi University of Technology, 2.National Institute of Health Sciences It has been reported that chemical exposure in utero increases the risk of developing Autism Spectrum Disorder (ASD) and some mental disorders. The cerebellum is an organ that controls motor functions, intentions, and emotions and has been shown to be a central site in the development of ASD. We have observed changes in the function and structure of the cerebellum and hippocampus as well as behavior in rats exposed to valproic acid (VPA) during the fetal period. In the present study, we focus on chemicals suspected of developmental neurotoxicity of organophosphorus pesticides. Chlorpyrifos (CPF), an organophosphate pesticide used as a termite exterminator, is suspected to be a cause of ASD development. Therefore, as a part of elucidating the mechanism of the induction of developmental neurotoxicity by pesticide exposure, we evaluate the effects of pesticide exposure by observing the cerebellum of pups born from mother rats exposed to CPF and discuss the mechanism quantitatively. Pregnant rats on gestation day 16 (G16) were acutely administrated with 5 mg/kg of CPF, or chronically administrated from G5 to G20 with a total 5 mg/kg of CPF. Male offspring were fixed on a postnatal day 7 (P7) to P14, and their cerebellum was observed by confocal microscopy. CPF exposure resulted in Purkinje cell (PC) irregularity and excess-folding of cerebellar lobule V and VI layers; however, the number of PC was not decreased. We will discuss the mechanism by which CPF causes neurological disorders from the perspective of inflammation and epigenetics. Understanding the mechanism of action of chemical-induced neurological disorders will offer the possibility of recovering from them.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P12 Th17細胞マスター転写因子レチノイン酸関連オーファン受容体γt過剰が中枢神経系および行動に及ぼす影響 Retinoic acid-related orphan receptor-γt overexpression affect the murine central nervous system and behaviors. ^○佐々木哲也¹, Aki Takahashi², Satoru Takahashi³, Yosuke Takei¹ 1.国立大学法人　筑波大学　医学医療系　生命医科学域　解剖学・神経科学研究室, 2.Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, 3.Faculty of Medicine, Department of Anatomy and Embryology, University of Tsukuba ^○Tetsuya Sasaki¹, Aki Takahashi², Satoru Takahashi³, Yosuke Takei¹ 1.Laboratory of Anatomy and Neuroscience, Faculty of Medicine, University of Tsukuba, 2.Laboratory of Behavioral Neuroendocrinology, University of Tsukuba, 3.Faculty of Medicine, Department of Anatomy and Embryology, University of Tsukuba T helper 17 (Th17) cells are a subset of CD4+ T cells that produce interleukin (IL)-17A. Recent studies showed that an increase in circulating IL-17A causes cognitive dysfunction, although it is unknown how increased systemic IL-17A affects brain function. Using transgenic mice overexpressing RORγt, a transcription factor essential for differentiation of Th17 cells (RORγt Tg mice), we examined changes in the brain caused by chronically increased IL-17A resulting from excessive activation of Th17 cells. RORγt Tg mice exhibited elevated Rorc and IL-17A mRNA expression in the colon, as well as a chronic increase in circulating IL-17A. We found that the immunoreactivity of Iba1 and density of microglia were lower in the dentate gyrus of RORγt Tg mice compared with wild-type mice. However, GFAP+ astrocytes were unchanged in the hippocampi of RORγt Tg mice. Levels of synaptic proteins were not significantly different between RORγt Tg and wild-type mouse brains. In addition, novel object location test results indicated no difference in preference between these mice. Our findings indicate that a continuous increase of IL-17A in response to RORγt overexpression resulted in decreased microglia activity in the dentate gyrus, but had only a subtle effect on murine hippocampal functions.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P13 LOTUSと自閉症スペクトラム症との関連性 Association of LOTUS, an endogenous NgR1 antagonist, with ASD-like behavior in mice ^○孫博雅, 西田遼平,竹居光太郎横浜市立大学大学院生命医科学研究科 ^○Sun Boya, 遼平西田 Yokohama City University 　Nogo signaling is involved in reduction of synaptic density via binding of Nogo to Nogo receptor-1 (NgR1). Recently we found that the lateral olfactory tract usher substance (LOTUS), an endogenous antagonist of NgR1, increases synaptic density by blockade of Nogo signaling. 　On the other hand, cerebral cortex of autistic patients showed higher 　synaptic density when compared to healthy person, thereby a close relationship between increased synaptic density and psychiatric disorder such as autism spectrum disorder (ASD)．　Therefore, we hypothesized that increase of synaptic density by LOTUS expression may give rise to autism spectrum disorder (ASD)-like behavior and increase of LOTUS expression may be involved in pathogenesis of ADS. To address this issue, we examined ADS-like behaviors in wild type (WT), LOTUS gene knockout (LOTUS-KO), and LOTUS overexpression transgenic (LOTUS-Tg) mice. 　First, we examined the effect of LOTUS on anxiety-like behavior in open field test, and no difference was found among each genotype. 　We next examined the sociability by using social interaction test and found that LOTUS-Tg mice tended to show lower social interactions and were sensitive to social stress, whereas LOTUS-KO mice showed higher sociability and were not sensitive to social stress. 　Cliff avoidance test for evaluating the ability of danger recognition showed that LOTUS-Tg mice had difficulty of danger recognition, while LOTUS-KO mice easily recognized danger. 　The data suggest that LOTUS-overexpressing mice may show, similar to autistic patients, a lack of sociability, strong social stress sensitivity, and lower dangerous cognitive ability. Thus, it can be considered that the increase of synaptic density by LOTUS overexpression is related to ADS-like pathogenesis.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P14 閉経後の高脂肪食に対する食物依存性の形成 Food addiction with high-fat diet after menopause ^○小宇田智子 Division of Nursing, Faculty of Nursing, Tokyo Healthcare University ^○Tomoko Koda Division of Nursing, Faculty of Nursing, Tokyo Healthcare University Estrogen plays an essential role in regulating appetite and eating behavior, and strongly influences body fat distribution. The Japanese diet has changed with the times, and many believe that the spread of western pattern diet is generally characterized by a high intake of high-fat dairy products. These foods can activate our brain reward neurocircuitry, which can create a highly rewarding experience that can make these foods difficult to stop eating when we full. In this study, we turned our attention to food addiction with a high-fat diet (HFD) after menopause. Ovariectomized (OVX: O) female mice and non-OVX (NO) were housed in a controlled environment and had free access to the control diet (10 kcal% fat: C) for 7 days and then divided randomly into 2 groups respectively. The OC and NOC were fed the C, and the OH and NOH were fed an HFD (40 kcal% fat) for 128 days. Bodyweight and food intake were measured during the experimental period. The white/black box test was used for food addiction with the HFD. The brain tissues were collected for histological assay to assess dopamine receptor d1 (D1) and dopamine transporter (DAT) expression in the brain. Food intakes of each group were almost the same during the experimental period. After 7 days from the onset of an HFD for OVX mice, the bodyweight was exponentially increased. The OH increased the time spent white area of the two-compartment white/black test box, and rearing behavior and move were considerably increased in the white area, however, there were no significant differences. D1 and DAT expressions are presently being analyzed. In conclusion, our data show that menopausal women may develop food addiction with an HFD. However, further study is required to revealed changes in nerve functions after menopause.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P15 運動量とセロトニンを介した前頭前野神経活動の関連 Analysis of relationship between locomotor activity and serotonin-mediated prefrontal activity ^○畦地裕統¹, Susumu Takahashi² 1.同志社大学脳科学研究科認知行動神経機構部門, 2.Laboratory of Cognitive and Behavioral Neuroscience, Graduate School of Brain Science, Doshisha University ^○Hirotsugu Azechi¹, Susumu Takahashi² 1.Organization for Research Initiatives and Development, Doshisha University, 2.Laboratory of Cognitive and Behavioral Neuroscience, Graduate School of Brain Science, Doshisha University Hyperactivity is a behavioral abnormality often observed in rodent models of psychiatric or developmental disorders, but its mechanisms remain unclear. One of the systems involved in the control of locomotor activity is the serotonin system which is also well known to regulate various brain functions such as mood, sleep, and memory. Optogenetic studies have reported changes in locomotor activity due to the activation of serotonin neurons in the dorsal raphe nucleus. However, the dorsal raphe nucleus projects serotonin neurons to some brain regions, and it is unclear how serotonin neurons projecting to which brain regions from the dorsal raphe nucleus affect locomotor activity. This study focused on the relationship between locomotor activity and neural activity of the prefrontal cortex, one of the serotonergic projection regions from the dorsal raphe nucleus. We are analyzing the relationship between locomotor activity and prefrontal electrophysiological properties in FH/HamSlc rats, a substrain of the Fawn-hooded rat that exhibits both brain serotonin dysfunction and hyperactivity, and Long-Evans rats as control by combining multi-neuron recordings under free-moving in the open field with optogenetic manipulation of prefrontal serotonin neurons. We report preliminary results on the relationship between locomotor activity and prefrontal neural activity.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P16 QPRTの欠損はキノリン酸によるドパミン作動性神経系における酸化ストレスを増強し、運動・認知機能障害を惹起する QPRT Deficit Leads Motor and Cognitive Dysfunction through Increase of Oxidative stress in the Dopaminergic Neurvous System by Quinolinic Acid ^○毛利彰宏¹, Moe Niijima¹, Kazuo Kunisawa¹, Takano Kazuki², Masayuki Yamada², Tomoaki Teshigawara³, Hisayoshi Kubota¹, Mami Hirakawa¹, Yuko Mori³, Masato Hoshi³, Hidetsugu Fujigaki³, Yasuko Yamamoto¹, Masaya Hasegawa¹, Hitomi Kurahashi¹, Kuniaki Saito³, Toshitaka Nabeshima⁴ 1.Department of Regulatory Science for Evaluation and Development of Pharmaceuticals and Devices, Fujita Health University Graduate School of Health Sciences., 2.Department of Molecular Imaging, Fujita Health University Graduate School of Health Science, 3.Department of Disease Control and Prevention, Fujita Health University Gradu ate School of Health Science, 4.Advanced Diagnostic System Research Laboratory, Fujita Health University Gra duate School of Health Science, 5.Japanese Drug Organization of Appropriate Use and Research ^○Akihiro Mouri¹, Moe Niijima¹, Kazuo Kunisawa¹, Takano Kazuki², Masayuki Yamada², Tomoaki Teshigawara³, Hisayoshi Kubota¹, Mami Hirakawa¹, Yuko Mori³, Masato Hoshi³, Hidetsugu Fujigaki³, Yasuko Yamamoto¹, Masaya Hasegawa¹, Hitomi Kurahashi¹, Kuniaki Saito³, Toshitaka Nabeshima⁴ 1.Department of Regulatory Science for Evaluation and Development of Pharmaceuticals and Devices, Fujita Health University Graduate School of Health Sciences., 2.Department of Molecular Imaging, Fujita Health University Graduate School of Health Science, 3.Department of Disease Control and Prevention, Fujita Health University Gradu ate School of Health Science, 4.Advanced Diagnostic System Research Laboratory, Fujita Health University Gra duate School of Health Science, 5.Japanese Drug Organization of Appropriate Use and Research Quinolinic acid phosphoribosyltransferase (QPRT) metabolizes quinolinic acid (QA) to nicotinamide adenine nucleotide (NAD+) via kynurenine pathway. QA is a excitotoxic substance that activate N-methyl-D-aspertate (NMDA) receptors and NAD+ is essential for cell survival. In this study, we evaluated QPRT knock out (KO) mice to explore the physiological role of QPRT in central nervous system. QPRT KO mice demonstrated motor deficits (decrease of locomotor activity, decrease of duration time to maintain balance on the rotarod, wide stance in footprint pattern test) and cognitive deficits (decrease of spontaneous alternation behavior in Y-maze test, and prolongation of latency to enter the target hole in the Barnes-maze test). But emotional change was not observed except for decrease in number of buried marbles in marble burying test. Dopaminergic dysfunction was observed in prefrontal cortex, nucleus accumbens and striatum of QPRT KO mice. Dopamine D1 receptor agonist (SKF81297)-induced hyperactivity is not observed in QPRT KO mice. Dopamine D2 receptor antagonist (raclopride)-induced catalepsy is more sensitive in QPRT KO mice. The activation of dopaminergic function by methylphenidate attenuated the impairment of short-term memory and hypoactivity of QPRT KO mice. QPRT KO mice showed increased level of QA in serum but normal level of NAD+ in brain. QA-mediated NMDA receptor signaling (phosphorylation of CaMK2 and activation of calpain) and oxidative stress were enhanced in prefrontal cortex, nucleus accumbens and striatum of QPRT KO mice. These results suggested that deficiency of QPRT lead motor and cognitive deficits associated with dopaminergic dysfunction via QA-induced calpain activation and oxidative stress.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P17 BTBR マウスにおけるルールシフト学習の障害と内側前頭前野における興奮性/抑制性バランスの不均衡 Dysfunction of rule-shift learning and Excitatory/Inhibitory balance in the medial prefrontal cortex in the BTBR T+ Itpr3tf/J mouse model of autism spectrum disorder ^○池原実伸¹, Kazuhiko Yamamuro¹, Kazuya Okamura¹, Yuki Noriyama¹, Yasunari Yamaguchi¹, Nozomi Endo¹, Taketoshi Sugimura¹, Mayumi nishi¹, Yasuhiko Saito¹, Manabu Makinodan¹ 1.Department of Psychiatry, Nara Medical University, 2.Department of Anatomy and Cell Biology, Nara Medical University, 3.Department of Neurophysiology, Nara Medical University ^○Minobu Ikehara¹, Kazuhiko Yamamuro¹, Kazuya Okamura¹, Yuki Noriyama¹, Yasunari Yamaguchi¹, Nozomi Endo¹, Taketoshi Sugimura¹, Mayumi nishi¹, Yasuhiko Saito¹, Manabu Makinodan¹ 1.Department of Psychiatry, Nara Medical University, 2.Department of Anatomy and Cell Biology, Nara Medical University, 3.Department of Neurophysiology, Nara Medical University Previous several studies reported that disrupting cross-hemisphericγsynchrony between prefrontal parvalbumin interneuron impairs rule shift learning and enhances persistent behavior. In human, patients with autism spectrum disorder exhibit obsessional behavior. Although BTBR mice, a mouse model of autism, also showed obsessional behavior, the neural circuit has been unknown. In this study, we performed Water T maze and found that persistent behavior was significantly enhanced in BTBR mice compared to C57BL/6 mice at 60-70 days. To elucidate its neural circuit, we recorded excitatory and inhibitory synaptic transmission from pyramidal cells in layer Ⅴ/Ⅵ of the medial prefrontal cortex(mPFC) using the whole-cell patch-clamp technique. We found that both spontaneous and miniature excitatory post-synaptic currents frequencies significantly increased in BTBR mice compared to C57BL/6 mice. In contrast, both spontaneous and miniature inhibitory post-synaptic currents frequencies significantly reduced in BTBR mice compared to C57BL/6 mice. These results indicate that disrupted excitatory/inhibitory balance in synaptic transmission in pyramidal cells of layer Ⅴ/Ⅵ of the mPFC in BTBR, and may be involved in the enhancement of persistent behavior in BTBR mice.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P18 先天的な防御行動を切替えるための神経回路の解析 Neural circuits for switching innate defensive behaviors ^○森川勝太¹, Kazuki Katori¹, Ai Nakashima¹, Haruki Takeuchi^1,4, Yuji Ikegaya^1,2,3 1.東京大学　薬学研究科, 2.Institute for AI and Beyond, The University of Tokyo, 3.Center for Information and Neural Networks, National Institute of Information and Communications Technology, 4.Social Cooperation Program of Evolutional Chemical Safety Assessment System, LECSAS, Graduate School of Pharmaceutical Sciences, University of Tokyo ^○Shota Morikawa¹, Kazuki Katori¹, Ai Nakashima¹, Haruki Takeuchi^1,4, Yuji Ikegaya^1,2,3 1.Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2.Institute for AI and Beyond, The University of Tokyo, 3.Center for Information and Neural Networks, National Institute of Information and Communications Technology, 4.Social Cooperation Program of Evolutional Chemical Safety Assessment System, LECSAS, Graduate School of Pharmaceutical Sciences, University of Tokyo Innate behaviors are triggered by the activation of specific neural circuits due to sensory input, independently of the animal′s learning and experience. The detailed neural circuits underlying innate behavior have not been identified. In this study, we used rabies virus tracing and in vivo calcium imaging techniques to identify the neural circuits responsible for innate behaviors. We found that glutamatergic neurons in the amygdala piriform transition area (APir) were activated upon presentation of predator odor, which induces automatic defensive behaviors and that the increased neural activity was associated with the onset of flight behaviors. These phenomena were not replicated in the entorhinal cortex or the ventral hippocampus, which are peripheral regions of APir, but rather the activity of these brain regions was suppressed. Furthermore, suppression of neural activity of APir cells projecting to the central nucleus of the amygdala significantly reduced the latency and frequency of switching from defensive behavior to flight behavior. Furthermore, retrograde virus tracing revealed that APir is innervated by the olfactory bulb mitral cells, which are innervated by glueneberg ganglion, a putative sensor region for alarm pheromones. This study promotes our understanding of the circuit mechanism by which predator odors are transformed into behavioral expression.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P19 健常対象者と統合失調症患者の死後脳における視空間作業記憶ネットワーク回路におけるアクチン関連およびOXPHOS関連遺伝子の発現解析 Expression of actin signaling and OXPHOS-related transcripts across the cortical visuospatial working memory network in unaffected comparison and schizophrenia subjects ^○紀本創兵¹, 橋本隆紀^2,3, 坪本真², 山口泰成¹, 岸本年史¹, David A. Lewis³ 1.Department of Psychiatry, Nara Medical University, 2.Department of Psychiatry and Behavioral Science, Kanazawa University Graduate School of Medical Sciences, 3.Department of Psychiatry, University of Pittsburgh ^○SOHEI KIMOTO¹, 橋本隆紀^2,3, 坪本真², 山口泰成¹, 岸本年史¹, David A. Lewis³ 1.Department of Psychiatry, Nara Medical University, 2.Department of Psychiatry and Behavioral Science, Kanazawa University Graduate School of Medical Sciences, 3.Department of Psychiatry, University of Pittsburgh Background:Visuospatial working memory (WM) is mediated by a distributed cortical network that is functionally impaired in schizophrenia. Within one node of this network, the dorsolateral prefrontal cortex (DLPFC), altered regulation of the actin assembly in dendritic spines and lower energy production via mitochondrial oxidative phosphorylation (OXPHOS) system have been suggested in subjects with schizophrenia. However, little is known about the extent to which similar alterations are present in other regions of the visuospatial WM network. Methods:Transcript levels of a subset of actin signaling (CDC42, BAIAP2, ARPC3 and ARPC4) and OXPHOS-related (ATP5H, COX4I1, COX7B, and NDUFB3) molecules were determined by quantitative PCR in total gray matter from DLPFC and three other cortical regions in the visuospatial WM network of 20 matched pairs of unaffected comparison (UC) and schizophrenia subjects. Results:In UC subjects, the cross-regional expression patterns of actin signaling transcripts were consistent with a greater actin assembly in the DLPFC than in the other regions of the WM network. In contrast, all OXPHOS-related transcripts showed expression levels that were lowest in DLPFC and progressively decreased from posterior to anterior regions. In schizophrenia subjects, CDC42 and ARPC4 levels were lower in all regions, BAIAP2 levels higher only in V1, and ARPC3 levels unaltered across regions. In contrast, levels of all OXPHOS-related transcripts were lower in schizophrenia with the magnitude of the disease effect decreasing from posterior to anterior regions. Conclusions: Differential alterations in actin assembly and energy production across cortical regions may contribute to visuospatial WM deficits in schizophrenia.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P20 オキシトシンはバルプロ酸暴露マウス仔の母親分離による仔の超音波発声のcomplexタイプを変調する。 Oxytocin administration modulates the complex type of ultrasonic vocalisation of mice pups prenatally exposed to valproic acid ^○辻隆宏¹, Chiharu Tsuji² 1.Department of ophthalmology, University of Fukui, 2.Research Center for Child Mental Development, Kanazawa University. ^○Takahiro Tsuji¹, Chiharu Tsuji² 1.Department of ophthalmology, University of Fukui, 2.Research Center for Child Mental Development, Kanazawa University. Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterised by　communication disability with no curative treatment. Maternal separation-induced　ultrasonic vocalisation (USV) was widely used to assess communication disability between pups and dams. Particularly, USV calls in many genetically modified ASD model mice were altered. Previously, we demonstrated that mice pups exposed to valproic acid in utero (VPA pups) showed decreased number of USV calls on postnatal day 11 and were rescued by subcutaneous injection of oxytocin. However, the qualitative change of USV calls by oxytocin has not been evaluated in VPA pups. In the present study, we examined the duration of oxytocin effect and analysed the altered pattern of USV calls using VPA pups. The oxytocin administration increased the total number of USV calls and the effect persisted up to 120 min in VPA pups. The pattern analysis revealed that the increase in the number of complex calls also persisted up to 120 min. These results suggested that oxytocin had a prolonged effect on USV calls, mainly on complex calls, in VPA pup, showing that oxytocin could recover their social modality to respond to maternal separation.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P21 ラットの卵巣摘出による自発運動量の低下に対するラクトフェリンの予防効果 Preventive effect of lactoferrin on decreased spontaneous activity by ovariectomy in rats ^○Megumi Furukawa¹, Nobuo Izumo¹, Yuna Ishiyama¹, Haruna Kurono¹, Takeaki Nitto¹, Tatsuo Hoshino¹, Takayuki Manabe¹ 1.Center for pharmaceutical education, Yokohama University of Pharmacy, 2.Laboratory of Pharmacotherapy, Yokohama University of Pharmacy, 3.Laboratory of Functional Materials, Yokohama University of Pharmacy, 4.Department of Nursing, Faculty of Nursing, Chukyogakuin University, 5.NRL Pharma, Inc. ^○Megumi Furukawa¹, Nobuo Izumo¹, Yuna Ishiyama¹, Haruna Kurono¹, Takeaki Nitto¹, Tatsuo Hoshino¹, Takayuki Manabe¹ 1.Center for pharmaceutical education, Yokohama University of Pharmacy, 2.Laboratory of Pharmacotherapy, Yokohama University of Pharmacy, 3.Laboratory of Functional Materials, Yokohama University of Pharmacy, 4.Department of Nursing, Faculty of Nursing, Chukyogakuin University, 5.NRL Pharma, Inc. The female hormone estradiol affects the central nervous system disorders, such as depression and dementia. Hormone replacement therapy (HRT) is used to treat symptoms associated with female menopause. However, the administration of estradiol is reported to increase the risk of breast and endometrial cancers, it is important to find a novel therapy of disorders related the female hormone. We have already reported that ovariectomized (OVX) rats reduced the spontaneous activity during the dark period due to the decease of serotonin release in the amygdala (Behav Brain Res. 2012, 227, 1-6). In this study, we examined the potential of lactoferrin (LF) on the recovery of less spontaneous activity seen in rats with OVX-induced despair-like behaviors. 8-week-old female OVX rats were administered LF (300mg/kg) five days a week for 6 weeks from the day after surgery. After administration was completed, spontaneous locomotor activity in the dark period, immobility time in a forced swim test, and release amount of dopamine and serotonin in the brain were measured. LF was found to improve OVX-induced decreases in locomotor activity and increases in immobility time in the forced swim test. Furthermore, the administration of LF elicited significant recovery of decreased dopamine and serotonin release in the brains of OVX group rats. These results strongly suggest that LF improved OVX-induced decreases in momentum during the dark period and, moreover, that release of dopamine and serotonin in the brain was involved in this effect.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P22 ストレス誘発型突発性難聴モデルマウスの確立 Establishment of model mice in stress-induced sudden hearing loss ^○小山佳久¹, Takuya Sumi^1,3, Shoichi Shimada^1,2 1.Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, 2.Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, 3.Department of Cell Biology, Osaka University Graduate School of Medicine ^○Yoshihisa Koyama¹, Takuya Sumi^1,3, Shoichi Shimada^1,2 1.Department of Neuroscience and Cell Biology, Osaka University Graduate School of Medicine, 2.Addiction Research Unit, Osaka Psychiatric Research Center, Osaka Psychiatric Medical Center, 3.Department of Cell Biology, Osaka University Graduate School of Medicine Sudden hearing loss is cryptogenic acute sensorineural deafness. Due to the significant increase in the number of patient and low treatment efficiency, it is urgent to develop the radical therapeutic agent. Although it is essential to establish a model animal for hearing loss, models such as mainstream drug administration and noise exposure cannot completely explain the mechanism of the disease. We focused on “mental stress” as a cause of hearing loss and investigated whether repetitive cold-treated mice could be stress-induced deafness model animals. At this presentation, we would like to discuss the achievements to date and future prospects.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P23 慢性社会的敗北ストレスにより誘発されるうつ様行動において小脳血液-脳関門の破綻が関与する Involvement of cerebellum blood-brain barrier disruption in the depression-like behaviors induced by chronic social defeat stress. ^○小菅愛加¹, Kazuo Kunisawa¹, Hiroyuki Tezuka², Masato Hoshi³, Nanaka Morita⁴, Tomoki Kawai¹, Yasuko Yamamoto⁴, Kuniaki Saito^4,5,6, Toshitaka Nabeshima^5,6, Akihiro Mouri^1,6 1.藤田医科大学大学院保健学研究科, 2.Department of Cellular Function Analysis, Research Promotion and Support Headquarters, Fujita Health University, 3.Department of Biochemical and Analytical Science, Fujita Health University Graduate School of Health Sciences, 4.Department of Disease Control and Prevention, Fujita Health University Graduate School of Health Sciences, 5.Advanced Diagnostic System Research Laboratory, Fujita Health University Graduate School of Health Science, 6.Japanese Drug Organization of Appropriate Use and Research (J-DO) ^○Aika Kosuge¹, Kazuo Kunisawa¹, Hiroyuki Tezuka², Masato Hoshi³, Nanaka Morita⁴, Tomoki Kawai¹, Yasuko Yamamoto⁴, Kuniaki Saito^4,5,6, Toshitaka Nabeshima^5,6, Akihiro Mouri^1,6 1.Department of Regulatory Science for Evaluation & Development of Pharmaceuticals & Devices, Fujita Health University Graduate School of Health Sciences, 2.Department of Cellular Function Analysis, Research Promotion and Support Headquarters, Fujita Health University, 3.Department of Biochemical and Analytical Science, Fujita Health University Graduate School of Health Sciences, 4.Department of Disease Control and Prevention, Fujita Health University Graduate School of Health Sciences, 5.Advanced Diagnostic System Research Laboratory, Fujita Health University Graduate School of Health Science, 6.Japanese Drug Organization of Appropriate Use and Research (J-DO) Neuroinflammation contributes to the pathogenesis of major depressive disorder (MDD). However, the pathogenic mechanisms of neuroinflammation remain poorly understood. The blood-brain barrier (BBB) is an important physiological barrier that separates the central nervous system (CNS) from the peripheral circulation. Previous studies have shown elevated levels of pro-inflammatory cytokines in the serum of patients with MDD. Peripheral inflammation can disrupt the BBB, resulting in the enhancement of neuroinflammation response in the brain. In the present study, we investigated the involvement of BBB disruption in the chronic social defeat stress (CSDS), by using a mouse model of MDD. The adult C57BL/6J mouse was exposed to aggressor ICR mouse for 10 consecutive days. One day after the last stress exposure, CSDS reduced the SI ratios in the social interaction test, while increased immobility time in the forced swimming test and significantly increased infiltration of Evans Blue dye, the marker of microvascular leakage, in the cerebellum of mice suggesting that the disruption of cerebellar BBB in CSDS mice. Further, CSDS significantly increased IL-1β expression following microglial activation in the cerebellum. The spleen is crucial for the peripheral immune system. Importantly, the spleen weights in the CSDS mice were significantly larger than those of control mice. To investigate the role of spleen in the neuroinflammation accompanying BBB disruption in the cerebellum, we will investigate the microglial profiles in the cerebellum of splenectomized CSDS mice. Our results suggest that CSDS may increase the microglial activation in the cerebellum through BBB disruption induced by peripheral inflammation, which may be responsible for the pathogenesis of MDD.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P24 発達段階の大脳新皮質において、11β-hydroxysteroid dehydrogenase type1 をコードするHsd11b1の発現領域は一時的に拡大する The transient expansion of Hsd11b1-expression region in the developing mouse neocortex ^○土井美幸¹, Yuichiro Oka^1,2, Makoto Sato^1,2,3 1.Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2.Department of Child Development, United Graduate School of Child Development, Osaka University, 3.Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University ^○Miyuki Doi¹, Yuichiro Oka^1,2, Makoto Sato^1,2,3 1.Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, 2.Department of Child Development, United Graduate School of Child Development, Osaka University, 3.Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University Corticosterone (CORT) is known as a stress-related hormone, important for the maintaining the homeostasis. The systemic CORT level is regulated by Hypothalamus-Pituitary-Adrenal gland (HPA) -axis. On the other hand, the local CORT level is regulated by hydroxysteroid dehydrogenase type1 (11β-HSD1) encoded by Hsd11b1. It is reported that 11β-HSD1 affect the brain function. However, the Hsd11b1 expression region in the neonatal mouse brain has not been fully investigated. Here we examined the Hsd11b1 expression region in the developing neocortex, and found that the distribution pattern of the Hsd11b1 positive cells changed dynamically during postnatal development. In detail, at postnatal day 0 (P0), Hsd11b1 positive cells were observed only in the deep layer of the somatosensory cortex. The expression area gradually expanded from P3 to P8, and then it was gradually narrowed. Finally, we observed the Hsd11b1 expression region only in primary somatosensory cortex layer5 at P26 and thereafter. Furthermore, to examine the effect of systemic CORT level on the Hsd11b1 expression, we administered CORT to feeding dams for 10 consecutive days. It increased the systemic CORT level in their pups, and the number of the Hsd11b1 positive cells in the neocortex were decreased compared with that of the control group. Recently, it has been reported that the early life stresses affect development of neural circuit and brain function. From our observation, it is likely that early life stresses affect the expression level of Hsd11b1 in the neocortex, which may prevent normal brain development.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P25 ヒト脳オルガノイドへ低酸素・低グルコース/再酸素化を行うことによる虚血マーカーと脂質代謝の遺伝子変動 Human cerebral organoids under oxygen-glucose deprivation and reoxygenation affect gene expression differences of ischemic markers and lipid metabolism ^○岩佐直毅¹, Tatsuhide Tanaka², Akio Wanaka², Eiichiro Mori³, Kazuma Sugie¹ 1.Department of Neurology, Nara Medical University, 2.Department of Anatomy and Neuroscience, Nara Medical University, 3.Department of Future Basic Medicine, Nara Medical University ^○Naoki Iwasa¹, Tatsuhide Tanaka², Akio Wanaka², Eiichiro Mori³, Kazuma Sugie¹ 1.Department of Neurology, Nara Medical University, 2.Department of Anatomy and Neuroscience, Nara Medical University, 3.Department of Future Basic Medicine, Nara Medical University Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional mono-culture systems.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P26 社会敗北ストレス負荷されたマウスにおける甘味嗜好性の低下は有郭乳頭での甘味受容体の発現変化を伴う Alteration of sweet taste receptor expression in circumvallate papillae of mice with decreased sweet taste preference induce by social defeat stress ^○長澤一樹, Yuka Takahata, Waka Yoshimoto, Eri Kuwagaki, Yukie Yamada, Katsuhito Morito, Kentaro Takayama 京都薬科大学衛生化学分野 ^○Kazuki Nagasawa, Yuka Takahata, Waka Yoshimoto, Eri Kuwagaki, Yukie Yamada, Katsuhito Morito, Kentaro Takayama Department of Environmental Biochemistry, Kyoto Pharmaceutical University Depressive disorder induces sweet taste preference as anhedonia, but its details are not clarified yet. In this study, we investigated whether decrease of sweet taste preference was accompanied with alteration of sweet taste receptor expression in circumvallate papillae (CP) of social defeat stress (SDS)-subjected mice. Male BALB/c mice subjected to SDS for 10 days exhibited decrease of sociability and increase of immobility times in social interaction and forced swim tests, respectively. In these SDS-susceptible mice, a two-bottle choice test with sucrose solution revealed reduction of sweet taste preference, while expression of sweet taste receptors, T1R2 and T1R3, in their CP was greater than in the case of control with no apparent alteration of morphology of taste buds and numbers of type II-taste cells. In contrast, there was no alteration of sweet taste receptor expression in CP of SDS-susceptible male C57BL/6J mice without decrease of sweet taste preference. Together with the finding that the body weight gain of SDS-susceptible BALB/c, but not C57BL/6J, mice were apparently less than that of control ones, it is suggested that expression of sweet taste receptors in CP of mice with decreased sweet taste preference is upregulated to compensate for stress-induced increase of energy expenditure.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P27 ApoE分泌におけるプレセニリンの不可欠な作用 Presenilin is essential for ApoE secretion ^○鄒鶤¹, Sadequl Islam¹, Yang Sun¹, Yuan Gao¹, Tomohisa Nakamura¹, Noriyuki Kimura¹, Etsuro Matsubara¹, Kensaku Kasuga¹, Takeshi Ikeuchi¹, Taisuke Tomita¹, Makoto Michikawa¹ 1.名古屋市立大学医学研究科, 2.Department of Neurology, Faculty of Medicine, Oita University, 3.Department of Molecular Genetics, Brain Research Institute, Niigata University, 4.Laboratory of Neuropathology and Neuroscience, Faculty of Pharmaceutical Sciences, University of Tokyo ^○Kun Zou¹, Sadequl Islam¹, Yang Sun¹, Yuan Gao¹, Tomohisa Nakamura¹, Noriyuki Kimura¹, Etsuro Matsubara¹, Kensaku Kasuga¹, Takeshi Ikeuchi¹, Taisuke Tomita¹, Makoto Michikawa¹ 1.Department of Biochemistry, Graduate School of Medical Sciences, 2.Department of Neurology, Faculty of Medicine, Oita University, 3.Department of Molecular Genetics, Brain Research Institute, Niigata University, 4.Laboratory of Neuropathology and Neuroscience, Faculty of Pharmaceutical Sciences, University of Tokyo Mutations in presenilin (PS) lead to abnormal generation of amyloid -β-protein (Aβ), which is the major cause of familial Alzheimer’s disease (FAD); however, whether PS is involved in the pathogenesis of sporadic AD (SAD) is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear-transport receptor. Moreover, expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal controls. Our findings suggest that PS contributes to the pathogenesis of SAD by regulating ApoE secretion.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P28 ラット臍帯血幹細胞のコンドロイチン硫酸/デルマタン硫酸糖鎖の特徴 Structural features of chondroitin sulfate/dermatan sulfate in stem cells derived from rat umbilical cord blood ^○中西圭子¹, Kyohei Higashi¹, 3¹, Toshihiko Toida⁴, Masato Asai¹ 1.Department of Disease Model, Institute for Developmental Research, Aichi Developmental Disability Center, 2.Department of Pediatrics, Central Hospital, Aichi Developmental Disability Center, 3.Faculty of Pharmaceutical Science, Tokyo University of Science, 4.Center for Preventive Medical Sciences, Chiba University ^○Keiko Nakanishi¹, Kyohei Higashi¹, 3¹, Toshihiko Toida⁴, Masato Asai¹ 1.Department of Disease Model, Institute for Developmental Research, Aichi Developmental Disability Center, 2.Department of Pediatrics, Central Hospital, Aichi Developmental Disability Center, 3.Faculty of Pharmaceutical Science, Tokyo University of Science, 4.Center for Preventive Medical Sciences, Chiba University Chondroitin sulfate (CS) and its isomeric variant, dermatan sulfate (DS) are complex glycosaminoglycans (GAGs) with repeating disaccharide units and the major constituent of the extracellular matrix in the central nervous system (CNS) as well as other tissues. There are several CS/DS-disaccharide units with a different number and position of sulfation. Of these, the highly sulfated disaccharides have been shown to bind to several growth factors and to be involved in neurite outgrowth, neural stem cell proliferation, and neural protection. We have previously shown that neural stem/progenitor cells (NSPCs) plus chondroitinase (Chase) ABC, a CS degrading enzyme, improved neonatal hypoxic-ischemic (HI) brain injury more effectively than did NSPCs. The result indicates the possible beneficial effects of CS and/or DS. In addition, intraperitoneal administration of stem-cell-enriched umbilical cord blood cells (SCE-UCBCs) expanded from rat UCBCs attenuated HI brain injury in neonatal rats. However, the CS and/or DS of hematopoietic stem cells (HSCs) have not been fully understood. To know the involvement of CS on stem cell physiological function, we analyzed GAGs on mononuclear cells of rat umbilical cord blood cells (UCB-MNCs) and SCE-UCBCs. CS was detected in vasculatures of rat umbilical cord at E19 by immunohistochemistry. Disaccharide composition analysis revealed that CS/DS were abundant in both UCB-MNCs and SCE-UCBCs, while the amount of heparin sulfate (HS) was less. The major component of CS in UCBCs was the A-unit.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P29 発達遅滞児から同定されたNFIA^K125Eミスセンス突然変異がもたらす機能変化の解析 Analysis of functional changes caused by a novel NFIA^K125E missense mutation identified from children with developmental delay ^○佐貫理佳子¹, Tomoko Uehara², Yurie Ogura³, Atsushi Yokoyama⁴, Takeshi Yoshida⁴, Hiroshi Futagawa⁵, Hiroshi Yoshihashi⁵, Mamiko Yamada², Hisato Suzuki², Toshiki Takenouchi⁶, Kohei Matsubara¹, Hiromi Hirata³, Kenjiro Kosaki², Toshiyuki Takano-Shimizu¹ 1.京都工芸繊維大学, 2.Center for Medical Genetics, Keio University School of Medicine, 3.Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 4.Department of Pediatrics, Kyoto University Graduate School of Medicine, 5.Division of Medical Genetics, Tokyo Metropolitan Children′s Medical Center, 6.Department of Pediatrics, Keio University Hospital ^○Rikako Sanuki¹, Tomoko Uehara², Yurie Ogura³, Atsushi Yokoyama⁴, Takeshi Yoshida⁴, Hiroshi Futagawa⁵, Hiroshi Yoshihashi⁵, Mamiko Yamada², Hisato Suzuki², Toshiki Takenouchi⁶, Kohei Matsubara¹, Hiromi Hirata³, Kenjiro Kosaki², Toshiyuki Takano-Shimizu¹ 1.Department of Applied Biology, Kyoto Institute of Technology, 2.Center for Medical Genetics, Keio University School of Medicine, 3.Department of Chemistry and Biological Science, College of Science and Engineering, Aoyama Gakuin University, 4.Department of Pediatrics, Kyoto University Graduate School of Medicine, 5.Division of Medical Genetics, Tokyo Metropolitan Children′s Medical Center, 6.Department of Pediatrics, Keio University Hospital NFIA (Nuclear Factor 1 A-type) is a transcription factor that belongs to the NFI family. Nonsense mutations or gene deletions in NFIA are known to cause neurodevelopmental disorders referred to as NFIA -related disorder. However, patients with heterozygous missense mutations have not been reported. In the present study, we found two unrelated patients with the typical phenotype of NFIA-related disorder had a missense mutation, p.Lys125Glu (K125E), in the NFIA gene. We analyzed the K125E mutation in NFIA using the model organisms Drosophila and zebrafish. In Drosophila, ectopic expression of wild-type human NFIA in the eye-antenna disc caused the antenna-to-leg transformation. On the other hand, NFIA^K125E did not show any abnormality at all. In zebrafish, nfia-deficient embryos were injected with wild-type and K125E human NFIA mRNA, respectively. As a result, the wild-type NFIA rescued the loss of the commissural axon, while NFIA^K125E did not. We further analyzed the promoters of genes whose expression is regulated by NFIA. The wild-type NFIA repressed the HES1 promoter activity in an NFIA dose-dependent manner, whereas the NFIA^K125E showed no such repressive function. These results led us to conclude that the alteration of NFIA^K125E is a mutation that causes a loss of function in NFIA. We think that our analysis using multiple model systems provides advantages in elucidating the pathogenesis of rare and undiagnosed diseases based on genomic information.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P30 ネオニコチノイド、アセタミプリドの神経毒性と腸内細菌叢の変化 Neurotoxicity of neonicotinoid, Acetamiprid and changes in the gut microbiota. ^○岡田紗依¹, Christine Lee Li Mei¹, Thomas Tiong¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.応用化学・生命工学系生命機能科学研究室, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences ^○Sae Okada¹, Christine Lee Li Mei¹, Thomas Tiong¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.Department of Applied Chemistry and Life Science, Toyohashi University of Technology, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences Neonicotinoids, a new class of insecticides, mimic the chemical structure of nicotine, which binds to the nicotinic acetylcholine receptor (nAChR) to provide highly systemic insecticidal effects. Due to differences in nAChR susceptibility between insects and mammals, neonicotinoids have been seen as safer insecticides than organophosphate compounds; however, in recent years, there have been reports suggesting developmental neurotoxicity of neonicotinoids. In a previous study, acute prenatal exposure to acetamiprid (ACE), one of the neonicotinoids, with different doses (20 mg/kg, 40 mg/kg, or 60 mg/kg) showed that a misalignment and excessive folding of Purkinje cells (PC) between lobule V and VI in the cerebellar vermis. These neuronal alterations are sometimes deeply related to microbiome change due to the gut-brain axis, which is an immune network consisting of the gut microbiota, peripheral and central nervous system, and immune system. ACE could perturb the peripheral nervous system and the microbiome environment. In this study, we investigate the changes in the gut microbiota of rats, dams, and pups, acutely or chronically exposed to ACE. ACE 2.5 mg/kg-day (total of 40 mg/kg) were chronically administered to Wistar rats on the 4th to 19th day of gestation (G4-G19). Stool samples were collected at gestation day 6, 13, 15, 17, and 20 (G6, G13, G15, G17, G20) for dams and day 28 after birth (P28) for pups. Changes in the gut microbiota of dams and pups were then analyzed. Our data suggest some inflammatory reactions in the gut with ACE administration would change the gut microbiota of offspring.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P31 外傷性脳損傷における2ccPAによる血液漏出の抑制の分子機構 The molecular mechanisms underlying the rededucion of blood leakage by 2ccPA in traumatic brain injury ^○遠藤美沙紀¹, Mari Nakashima^1,2, Mari Gotoh^2,3, Kimiko Murakami-Murofushi³, Hiroko Ikeshima-Kataoka⁴, Yasunori Miyamoto^1,2 1.お茶の水女子大学大学院人間文化創成科学研究科ライフサイエンス専攻, 2.Institute for Human Life Innovation, Ochanomizu University, 3.Research division of human welfare science, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University ^○Misaki Endo¹, Mari Nakashima^1,2, Mari Gotoh^2,3, Kimiko Murakami-Murofushi³, Hiroko Ikeshima-Kataoka⁴, Yasunori Miyamoto^1,2 1.Graduate School of Humanities and Sciences, Ochanomizu University, 2.Institute for Human Life Innovation, Ochanomizu University, 3.Research division of human welfare science, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University Traumatic brain injury (TBI) results from an impact to the head and it induces haemorrhage. We found that stable derivative of cyclic phosphatidic acid (cPA) : 2-carbacyclic phosphatidic acid (2ccPA) suppresses serum immunoglobulin extravasation after a stab wound injury as a murine TBI model. cPA is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol. In this study, we aimed to examine the molecular mechanisms underlying the reduction of haemorrhage by 2ccPA after TBI. First, to investigate the effect of 2ccPA on blood leakage, we injected Evans blue (EB) into the tail vein of TBI model mice and quantified the amount of leakage of EB around the injury. We found that 2ccPA suppressed the extravascular leakage of EB caused by stab wounds. Next, to explore this mechanism, RNA-sequencing was performed with RNA extracted from the region around the injury at 1 and 3 days after TBI. Administration of 2ccPA promoted an increase in the mRNA expression of Neuropeptide Y Receptor Y5 (NPY5r) at 1 day after TBI. By using the real-time RT-PCR, we also confirmed the up-regulation of NPY5r by 2ccPA, suggesting that NPY5r may promote the vasoconstriction in the injured region. Furthermore, We found that administration of 2ccPA promoted the expression of tenascin-C (TN-C), an extracellular matrix protein, from astrocytes. Recntly, it has been reported that TN-C causes fibrin accumulation by down-regulation of tissue plasminogen activator. This report support the notion that the up-regulation of TN-C by 2ccPA may promote fibrin accumulation, and decrease the hemorrhage after TBI. These results suggest that 2ccPA suppresses blood leakage via promoting vasoconstriction and regulating fibrin accumulation after TBI.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P32 hiPSC3D培養モデルを用いた内在性タウタンパク質凝集誘導系の開発 Developing a 3D hiPSC-derived culture model system for inducing endogenous tau aggregation ^○平峯勇人¹, Sumihiro Maeda², Mitsuru Ishikawa², Ikuko Koya³, Ryosuke Nagashima¹, Seiji Shiozawa^3,4, Mari Nakamura⁵, Manabu Itoh¹, Hideyuki Okano² 1.JSR Corporation, 2.Department of Physiology, Keio University School of Medicine, 3.Center for Integrated Medical Research, Keio University School of Medicine, 4.Institute of Animal Experimentation, Kurume University School of Medicine, 5.Department of Pharmacology, University of California San Diego School of Medicine ^○Hayato Hiramine¹, Sumihiro Maeda², Mitsuru Ishikawa², Ikuko Koya³, Ryosuke Nagashima¹, Seiji Shiozawa^3,4, Mari Nakamura⁵, Manabu Itoh¹, Hideyuki Okano² 1.JSR Corporation, 2.Department of Physiology, Keio University School of Medicine, 3.Center for Integrated Medical Research, Keio University School of Medicine, 4.Institute of Animal Experimentation, Kurume University School of Medicine, 5.Department of Pharmacology, University of California San Diego School of Medicine Tauopathies are neurodegenerative diseases characterized by the intracellular accumulation of tau protein aggregates in the brains. Multiple failures of clinical trials based on the results of mouse models indicated the requirement of more appropriate disease models of human cells. In this study, we aimed to establish 3D human iPSC-derived culture system harboring multiple cell types with close contact and adult tau isoform (3R and 4R) expression which are resembling the in vivo situation because in AD brains 3R and 4R tau are co-aggregated. At first, we introduced MAPT N279K/P301S/IVS10+16 mutations to healthy donor-derived hiPSC by genome-editing technology. Then, cerebral organoids were formed from the hiPSC to induce various neural cell types. Next, the organoids were once dissociated to single cells and plated onto low attachment plates to induce cell clustering. Finally, we obtained multiple cell clusters, i.e. 3D culture model, containing various neural cells with close contact. The scRNA-seq analysis revealed that the 3D culture consisted of excitatory/inhibitory neurons, astrocytes, oligodendrocyte precursor cells, and so on. After the addition of recombinant tau seeds to initiate tau aggregation, we could induce endogenous tau aggregation mainly in the 3D model of mutant hiPSC. In addition, we found that the triple mutation increased the expression of 4R tau, implied the co-expression of 3R and 4R tau with the mutations enhanced tau pathogenesis. Our findings indicated that the close contact of neural cells in the 3D model enabled the propagation of tau aggregates among human neurons and triggered endogenous tau aggregation. This 3D culture model using mutant hiPSC will serve as a platform for both basic research and drug discovery targeting tau aggregation.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P33 牛車腎気丸（ゴシャジンキガン)はp38-TNFシグナル伝達を介して中枢神経での神経疾患の炎症を緩和する Go-sha-jinki-Gan (GJG) Mitigates Inflammation via p38-TNF Signaling in the CNS ^○蒋詩桜¹, Kousuke Baba^1,2, Tatsusada Okuno¹, Mitsuru Kinoshita¹, Chi-Jing Choong¹, Hideki Hayakawa¹, Hiroshi Sakiyama¹, Kensuke Ikenaka¹, Seiichi Nagano^1,3, Tsutomu Sasaki^1,3, Munehisa Shimamura^1,4, Yoshitaka Nagai⁵, Keisuke Hagihara⁶, Hideki Mochizuki¹ 1.Department of Neurology, Graduate School of Medicine, Osaka University, 2.Department of Neurology, Faculty of Medicine, University of Toyama, 3.Department of Neurotheraputics, Graduate School of Medicine, Osaka University, 4.Department of Health Development and Medicine, Graduate School of Medicine, Osaka University, 5.Department of Neurology, School of Medicine, Kinki University, 6.Department of Advanced Hybrid medicine, Graduate School of Medicine, Osaka University ^○Shiying Jiang¹, Kousuke Baba^1,2, Tatsusada Okuno¹, Mitsuru Kinoshita¹, Chi-Jing Choong¹, Hideki Hayakawa¹, Hiroshi Sakiyama¹, Kensuke Ikenaka¹, Seiichi Nagano^1,3, Tsutomu Sasaki^1,3, Munehisa Shimamura^1,4, Yoshitaka Nagai⁵, Keisuke Hagihara⁶, Hideki Mochizuki¹ 1.Department of Neurology, Graduate School of Medicine, Osaka University, 2.Department of Neurology, Faculty of Medicine, University of Toyama, 3.Department of Neurotheraputics, Graduate School of Medicine, Osaka University, 4.Department of Health Development and Medicine, Graduate School of Medicine, Osaka University, 5.Department of Neurology, School of Medicine, Kinki University, 6.Department of Advanced Hybrid medicine, Graduate School of Medicine, Osaka University [Objective]Go-shajinki-Gan (GJG) is a traditional Japanese Kampo formula which has been widely used in clinical settings. Previously, we have reported that GJG is able to ameliorate inflammation by suppressing production of TNF-a and phosphorylated p38 (p-p38) in the peripheral nervous system (PNS). This time we used experimental autoimmune encephalomyelitis (EAE) mouse model and MPTP induced Parkinson’s disease mouse model to investigate anti-inflammatory effect of GJG in the central nervous system (CNS) by focusing on p38-TNF signaling pathway, especially in the pathological and biochemical analyses. [Methods] C57BL/6J female mice were divided into CT, EAE and EAE+GJG groups randomly. EAE+GJG groups were pre-treated with GJG 2 weeks before immunization. After 28 days of immunization, mice were collected. In MPTP mice model, C57BL/6J male mice were divided into CT, MPTP and MPTP+GJG groups randomly. MPTP (15mg/kg) injection (4times/2h interval) was performed and samples were collected a week later. Pathological and biochemical analyses of p38 and TNF-a were performed. [Results] GJG suppressed inflammatory cell infiltration, number of Iba1 positive cells, production of TNF-a and p-p38 both in EAE mouse model and MPTP mouse model in pathological and biochemical analyses. In addition, GJG improved motor deficit of MPTP mice in behavioral test. [Conclusions]Our data indicated that GJG not only ameliorates inflammation in the PNS, but also mitigates inflammatory neurological disorders by suppressing p-p38 and production of TNF-a in the CNS.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P34 ヒトPHYHIPL遺伝子の Ser19Stop SNPは、マウス小脳機能の異常をもたらす The Ser19Stop single nucleotide polymorphism (SNP) of human PHYHIPL affects the cerebellum in mice ^○定方哲史¹, Takuro Horii², Jun-Na Hirota³, Yoshitake Sano³, Yo Shinoda⁴, Ayumu Konno⁵, Hirokazu Hirai⁵, Yasuki Ishizaki⁶, Hajime Hirase⁷, Izuho Hatada², Teiichi Furuichi³, Hisako Sugimoto¹ 1.群馬大学大学院医学系研究科大学院教育研究支援センター, 2.Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan., 3.Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan., 4.Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan., 5.Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan., 6.Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan., 7.Center for Translational Neuromedicine, Faculty of Medical and Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. ^○Tetsushi Sadakata¹, Takuro Horii², Jun-Na Hirota³, Yoshitake Sano³, Yo Shinoda⁴, Ayumu Konno⁵, Hirokazu Hirai⁵, Yasuki Ishizaki⁶, Hajime Hirase⁷, Izuho Hatada², Teiichi Furuichi³, Hisako Sugimoto¹ 1.Education and Research Support Center, Gunma University Graduate School of Medicine, 2.Laboratory of Genome Science, Biosignal Genome Resource Center, Institute for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan., 3.Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan., 4.Department of Environmental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan., 5.Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan., 6.Department of Molecular and Cellular Neurobiology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan., 7.Center for Translational Neuromedicine, Faculty of Medical and Health Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. The HapMap Project is a major international research effort to construct a resource to facilitate the discovery of relationships between human genetic variations and health and disease. The Ser19Stop single nucleotide polymorphism (SNP) of human phytanoyl-CoA hydroxylase-interacting protein-like (PHYHIPL) gene was detected in HapMap project and registered in the dbSNP. PHYHIPL gene expression is altered in global ischemia and glioblastoma multiforme. However, the function of PHYHIPL is unknown. We generated PHYHIPL Ser19Stop knock-in mice and found that PHYHIPL impacts the morphology of cerebellar Purkinje cells (PCs), the innervation of climbing fibers to PCs, the inhibitory inputs to PCs from molecular layer interneurons, and motor learning ability. Thus, the Ser19Stop SNP of the PHYHIPL gene may be associated with cerebellum-related diseases.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P35 慢性予測不能軽度ストレスによる行動変化におけるミクログリアに関連したキヌレニン代謝の関与 Involvement of microglia-associated kynurenine metabolism in chronic unpredictable mild stress-induced behavioral change ^○長谷川眞也¹, Akihiro Mouri^1,4, Kazuo Kunisawa¹, Hisayoshi Kubota¹, Hitomi Kurahashi¹, Yasuko Yamamoto², Kuniaki Saito^2,3,4, Toshitaka Nabeshima^3,4 1.藤田医科大学保健学研究科保健学専攻レギュラトリーサイエンス分野, 2.Department of Diseases Control and Prevention, Fujita Health University Graduate School of Health Sciences, Aichi, Japan., 3.Advanced Diagnostic System Research Laboratory, Fujita Health University Graduate School of Health Sciences, Aichi, Japan., 4.Japanese Drug Organization of Appropriate Use and Research, Aichi, Japan. ^○Masaya Hasegawa¹, Akihiro Mouri^1,4, Kazuo Kunisawa¹, Hisayoshi Kubota¹, Hitomi Kurahashi¹, Yasuko Yamamoto², Kuniaki Saito^2,3,4, Toshitaka Nabeshima^3,4 1.Department of Regulatory Science for Evaluation ＆ Development of Pharmaceuticals ＆ Devices, Fujita Health University Graduate School of Health Sciences, Aichi, Japan., 2.Department of Diseases Control and Prevention, Fujita Health University Graduate School of Health Sciences, Aichi, Japan., 3.Advanced Diagnostic System Research Laboratory, Fujita Health University Graduate School of Health Sciences, Aichi, Japan., 4.Japanese Drug Organization of Appropriate Use and Research, Aichi, Japan. Chronic stress contributes to the pathogenesis of major depressive disorder (MDD). L-tryptophan is metabolized via the kynurenine pathway (KP), highly regulated by cytokines. In the KP, kynurenine (KYN) is metabolized to 3-hydroxykynurenine (3-HK) by kynurenine 3-monooxygenase (KMO) and to kynurenic acid (KA) by kynurenine aminotransferase (KAT). Several studies have demonstrated the alternation of KP is associated with the pathogenesis of MDD. We investigated whether KP is changed by chronic unpredictable mild stress (CUMS). In terms of CUMS, mice were exposed to 9 kinds of stressors for 4 weeks. Mice showed decrease in social interaction time by CUMS. We investigated changes in the mRNA expression of KP enzymes and cytokines in the prefrontal cortex (PFC) by quantitative real-time PCR. Immediately after CUMS, KMO and interleukin-1β (IL-1β) mRNA were decreased in the PFC. KMO is mainly localized in microglia which is known to release IL-1β. The mRNA of Iba-1, a marker for microglia was decreased immediately after CUMS in the PFC. These data suggested decrease of KMO and IL-1b were associated with decline of microglia by CUMS. One week after CUMS, KATⅡ mRNA was increased in the PFC. Thus, the alternation of KP would eventually shift from 3-HK to KA. Because KA has antagonistic activity to α7-nicotinic acetylcholine receptor (α7nAChR), effects of nicotinic agonists on impairment of social interaction were investigated. Nicotine (Nic), galantamine (Gal:α7nAChR allosteric modulator) or varenicline (Var:α4β2nAChR partial agonist) was administrated before exposure to each stressor of CUMS. Nic and Gal, but not Var attenuated decrease in social interaction time. In conclusion, CUMS-induced social impairment may be induced by increased KA after change in KP.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P36 脳オルガノイドを用いたアルツハイマー病モデルの作製 Modeling Alzheimer’s disease in forebrain organoids ^○嶋田弘子¹, Yuta Sato^1,4, Aki Shimozawa², Tomoko Shindo³, Shinsuke Shibata^3,5, Takahiro Kondo¹, Hirofumi Aoyagi², Jyunro Kuromitsu², Hideyuki Okano¹ 1.Department of Physiology, Keio University School of Medicine, 2.Eisai-Keio innovation lab for dementia, Keio University School of Medicine, 3.Electron Microscope Laboratory, Keio University School of Medicine, 4.Graduate School of Science and Technology, Keio University, 5.Division of Microscopic Anatomy, Graduate School of Medical and Dental Sciences, Niigata University ^○Hiroko Shimada¹, Yuta Sato^1,4, Aki Shimozawa², Tomoko Shindo³, Shinsuke Shibata^3,5, Takahiro Kondo¹, Hirofumi Aoyagi², Jyunro Kuromitsu², Hideyuki Okano¹ 1.Department of Physiology, Keio University School of Medicine, 2.Eisai-Keio innovation lab for dementia, Keio University School of Medicine, 3.Electron Microscope Laboratory, Keio University School of Medicine, 4.Graduate School of Science and Technology, Keio University, 5.Division of Microscopic Anatomy, Graduate School of Medical and Dental Sciences, Niigata University Alzheimer’s disease (AD) is the most common cause of age-related neurodegenerative dementia associated with progressive memory impairment and cognitive damage. While access to patient brain tissues is limited, human induced pluripotent stem cell (iPSC) technology is a promising approach for the generation of in vitro human AD models. To study disease mechanisms, develop drug-screening system and evaluate candidate molecules, we generated 3D forebrain organoids from AD-iPSCs and analyzed Ab and Tau pathology. The obtained AD-organoids recapitulated Ab pathology but were missing tau aggregation. Neurodegenerative diseases characterized by the pathological accumulation of tau are collectively termed tauopathy. P301L is the most common mutation in Tau linked to FTDP-17 and has higher aggregation ability than Tau-WT. In order to generate tauopathy models using brain organoids, we overexpressed full length tau carrying the P301L mutation into forebrain organoids. Immunofluorescence showed that phospho-tau and aggregated tau levels were dramatically increased in Tau-P301L overexpressed organoids compared to GFP transduced controls. Moreover, tau filaments were detected in soma and neurites by Immunoelectron microscopy. We have successfully recapitulated amyloid b pathology in AD-patient derived brain organoid, and tau pathology in Tau-P301L overexpressed brain organoids. These models can be used as a platform for studying the molecular processes of Alzheimer’s disease, developing drug screening approaches and evaluating candidate molecules.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P37 グリホサート投与ラットおよび酪酸回復モデルにおける腸内細菌叢の変化 Examining the association between a model of butyrate recovery in glyphosate-treated rats and changes in flora in a single dose of butyrate. ^○稲川貴也¹, Thomas Tiong Kwong Soon¹, Ken Futagami¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.豊橋技術科学大学　応用化学・生命工学専攻　生命機能科学研究室, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences ^○Takaya Inakawa¹, Thomas Tiong Kwong Soon¹, Ken Futagami¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.Department of Applied Chemistry and Life Science, Toyohashi University of Technology, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences Glyphosate (GLY) is a major component of herbicides; however, we observed high-dose prenatal exposure to GLY induced neurological and neurobehavioral abnormalities, and butyric acid (BA) treatment after birth alleviated the neurotoxicity caused by GLY exposure. In this study, we examined the alteration of the gut microbiome in GLY-treated or GLY-BA-treated Wistar rats. Rat dams were treated with GLY (250 mg/kg-bw) on embryonic day 16 (E16), and some pups were treated additionally with BA (400 mg/kg-bw) from postnatal day 3 (P3) to P9. We also examined the effect of chronic GLY exposure (15.625 mg/kg-day, E5~E20) and low chronic GLY exposure (1 mg/kg-day, from E2 to E21). Fecal samples were collected from E7 to E21 for dams and P28 for pups. 16S rRNA sequencing was performed on DNA extracted from the fecal samples to profile the changes in the gut flora. The result showed that the Ruminococcaceae family of butyrate-producing bacteria tended to decrease in the GLY-treated group, and the chronic treatment group tended to decrease even more. We suggested that GLY exposure altered the gastrointestinal homeostasis and intestinal permeability.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P38 hiPS細胞を用いた、アルツハイマー病様培養モデルによる表現型解析及び17β-エストラジオールよる表現型変化の解析 Evaluation of Alzheimer’s disease model phenotypes and the effects of 17β-estradiol on neurons derived from human-iPS cells ^○Sopak Supakul, Sumihiro Maeda, Hideyuki Okano Department of Physiology, Keio University ^○Sopak Supakul, Sumihiro Maeda, Hideyuki Okano Department of Physiology, Keio University 　Alzheimer’s disease (AD) is a neurodegenerative disease that is associated with cognitive decline and has a high prevalence of more than 30 million patients worldwide. While several clinical trials on AD treatments have failed to achieve the pleasant outcomes, it is believed that the heterogenous nature of disease mechanism of AD determines the different drug responses and disease mechanisms among the AD patients. Therefore, the generation of the robust disease model of AD as a platform for understanding AD heterogeneity has been strongly suggested. Here, we generated the iPSCs-derived cortical neurons including the excitatory and inhibitory neurons. The donors are comprised of familial and sporadic subtypes (sAD lines were generously provided by the Goldstein Laboratory, UCSD) through the newly established feeder-free induction method. These iPSC-derived neurons demonstrated the disease phenotypes of AD such as increased amyloid-beta (Aβ) peptides evaluated by ELISA of secreted Aβ42 and Aβ40, neuronal hyperactivation evaluated by calcium imaging using Fluo-8 indicator, and morphological changes of the neurites. Besides, we evaluated the non-cell autonomous effect of sex dimorphism coming from sex hormone by treating the with 17β-estradiol, we showed that 17β-estradiol treatment for 15 minutes increased neuronal activity. These findings indicated that (i) the disease modeling of AD from hiPSC can be achieved by this newly established feeder-free induction method, (ii) hiPSC-derived AD neurons respond to sex steroid hormones, (iii) the current AD models can be used to investigate both autonomous and non-autonomous effects coming from sex dimorphism.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P39 バルプロ酸誘因うつ・不安様行動におけるCdk5活性の役割 The role of the Cdk5 activity in valproic acid-induced anxiety- and depression-like behaviors ^○高橋美由紀¹, Ran Wei^1,2, Kanae Ando², Toshio Ohshima¹, Shin-ichi Hisanaga² 1.Department of Life Science and Medical Bio-Science, Waseda University, 2.Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University ^○Miyuki Takahashi¹, Ran Wei^1,2, Kanae Ando², Toshio Ohshima¹, Shin-ichi Hisanaga² 1.Department of Life Science and Medical Bio-Science, Waseda University, 2.Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizure, migraine and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated by p35 or p39 in neurons, and plays a role in a variety of neuronal functions including psychiatric behaviors. We previously reported that VPA suppressed the Cdk5 activity by reducing expression of p35, leaving p39 without the changes. To ask for the role of Cdk5 to VPA-induced psychiatric behaviors, in this study we used p39 knockout (KO) mouse and investigated the VPA-induced anxiety- and depression-like behaviors. When VPA was chronically administrated for 2 weeks, p35 was decreased in p39 KO mice as well as WT mice. p39 KO mice treated chronically with VPA exhibited less anxiety- and depression-like behaviors, compared with WT mice. These results suggested that the pharmacological action of VPA on anxiety- and depression-like behaviors is mediated, at least a part, by the Cdk5 activity.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P40 神経伝達物質放出に対する生理活性ペプチド因子ヒューマニンの影響 The effect of Humanin, a bioactive peptide, on neurotransmitter release ^○新倉貴子, Nozomi Morita, Ayari Kozuma, Natsumi Ikegawa, Nobuyuki Sasakawa 上智大学　理工学部 ^○Takako Niikura, Nozomi Morita, Ayari Kozuma, Natsumi Ikegawa, Nobuyuki Sasakawa Department of Information and Communication Sciences, Faculty of Science and Technology, Sophia University Humanin (HN) is a 24-residue polypeptide and initially identified as a neuroprotective factor against Alzheimer’s disease (AD) -related insults. HN suppresses neuronal death caused by amyloid beta, an AD-associated cytotoxic insult, in vitro and ameliorates memory deficit of AD mouse models. HN is a secretive peptide and the HN level in circulation decreases age-dependently. It is thus assumed that the change in HN level is implicated in aging-associated cognitive decline. S14G-HN, a highly potent HN derivative, suppresses memory deficit caused by muscarinic receptor antagonist in normal mice, suggesting that HN may affect neurotransmission. To understand the effect of HN in neurotransmission, we first examined the acetylcholine (ACh) level in the hippocampus of mice by microdialysis. Intraperitoneal injection of S14G-HN increased ACh level in hippocampus, while no change was observed in physical activity. We next examined the effect of HN on neurotransmitter release in vitro using bovine adrenal chromaffin cells. S14G-HN increased the amount of total catecholamine released from chromaffin cells by the stimulation of ACh, whereas no effect was observed by the treatment of C8A-HN, a neuroprotection-defective HN derivative. Further, we performed amperometric analysis to examine the exocytotic kinetics in chromaffin cells. S14G-HN increased the frequency of secretory events and changed some other parameters in response to the ACh stimulation. These results suggest that HN functions as a modulator of neurotransmitter release under the normal physiological condition.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P41 自閉症モデルマウスにおける自律神経系の異常とGABAA受容体遺伝子との関係 Abnormality of autonomic nervous system and relationship with GABAA receptor genes in autism model mice. ^○延廣香澄¹, Jin Nakatani¹, Futoshi Toyoda², Yasuhiro Go³, Shin-ichi Horike⁴, Natsu Koyama⁵, Seiji Hitoshi⁵, Toru Takumi⁷, Ikuo Tooyama⁶, Shigehiro Morikawa⁶, Toshiro Inubushi⁶, Toshinori Sawano¹, Hidekazu Tanaka¹ 1.Department Pharmacology, Ritsumeikan University, 2.Department Cell Physiology, Shiga University of Medical Science, 3.Department Brain Sciences, National Institute of Natural Sciences, 4.Advanced Science Research Center, Kanazawa University, 5.Department Integrative Physiology, Shiga University of Medical Science, 6.Molecular Neuroscience Research Center, Shiga University of Medical Science, 7.Department of physiology and cell biology, Kobe University ^○Kasumi Nobuhiro¹, Jin Nakatani¹, Futoshi Toyoda², Yasuhiro Go³, Shin-ichi Horike⁴, Natsu Koyama⁵, Seiji Hitoshi⁵, Toru Takumi⁷, Ikuo Tooyama⁶, Shigehiro Morikawa⁶, Toshiro Inubushi⁶, Toshinori Sawano¹, Hidekazu Tanaka¹ 1.Department Pharmacology, Ritsumeikan University, 2.Department Cell Physiology, Shiga University of Medical Science, 3.Department Brain Sciences, National Institute of Natural Sciences, 4.Advanced Science Research Center, Kanazawa University, 5.Department Integrative Physiology, Shiga University of Medical Science, 6.Molecular Neuroscience Research Center, Shiga University of Medical Science, 7.Department of physiology and cell biology, Kobe University Autism spectrum disorder (ASD) is a heritable, multistage prenatal disorder. Also, it is known for neurodevelopment at disorder. We established copy number variation (CNV) model of ASD mice that contain mouse chromosome 7 duplication, corresponding to human chromosome 15q11-13. We found that our CNV model mice have abnormal autonomic nervous system. We suspect that excessive expression of GABAA receptor (GABAAR) cause this abnormality. In order to test this hypothesis, we performed in situ hybridization（ISH）to evaluate the expression level of GABAAR in CNV model mice. First, we prepared GABAAR cRNA probe and stained in adult hippocampal region. We confirmed that our cRNA probe was specifically hybridized with GABAAR mRNA. Next, we performed ISH in rostral ventrolateral medulla (RVLM). RVLM is known to a part of the medulla oblongata. This region suppresses peripheral sympathetic nerve activity via GABA. From this experiment, we found that expression level of GABAAR increased in RVLM. Also, it corresponded with its copy number. From these results, we concluded that CNV model mice have the abnormal autonomic nervous regulation, may be due to the excess expression of GABAAR in RVLM.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P42 NSFを欠損するマウスでは自閉症様行動障害および海馬のシナプス異常を示す NSF deficient mice leads to autism like behavior with hippocampal synaptic dysfunction ^○謝敏かく¹, Iwata Keiko^1,2,3, Ishikawa Yasuyuki⁴, Fukazawa Yugo^1,5, Matsuzaki Hideo^1,2,3 1.Division of development of mental functions, Research Center for Child Mental Development, 2.Life Science lnnovation Center, University of Fukui, Fukui 910-1193, Japan, 3.United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Osaka 565-0871, Japan, 4.Department of Systems Life Engineering, Maebashi Institute of Technology, Gunma, 371-0816, Japan, 5.Division of Brain Structures and function, Department of Morphological and Physiological Sciences, Graduate School of Medical Sciences, University of Fukui, Fukui 910-1193, Japan ^○Xie Min-Jue¹, Iwata Keiko^1,2,3, Ishikawa Yasuyuki⁴, Fukazawa Yugo^1,5, Matsuzaki Hideo^1,2,3 1.Division of development of mental functions, Research Center for Child Mental Development, 2.Life Science lnnovation Center, University of Fukui, Fukui 910-1193, Japan, 3.United Graduate School of Child Development, Osaka University, Kanazawa University, Hamamatsu University School of Medicine, Chiba University and University of Fukui, Osaka 565-0871, Japan, 4.Department of Systems Life Engineering, Maebashi Institute of Technology, Gunma, 371-0816, Japan, 5.Division of Brain Structures and function, Department of Morphological and Physiological Sciences, Graduate School of Medical Sciences, University of Fukui, Fukui 910-1193, Japan Autism, characterized by profound impairment in social interactions and communicative skills, is the most common neurodevelopmental disorder. Abnormalities synaptic plasticity have been revealed in numerous genetically modified mice that have molecular construct validity to human autism spectrum disorders, but its underlying molecular mechanisms remain controversial. Our previous reported that NSF mRNA expression tended to be reduced in post-mortem brains and was significantly reduced and correlated with the severity of the clinical symptom in lymphocytes of ASD individuals, (Iwata et al 2014). In this study, we firstly generated the NSF+/- mice and investigated their behavioral, neurotransmitter and neurophysiological phenotypes. We assessed the autistic like behaviors using the three-chambered task and ultrasonic vocalizations analysis. We found that the social interaction and communication reduced in the NSF+/- mice, compared with the wild mice. Additionally, we examined AMPA receptors location in the synapse of the NSF+/- mice by using freeze-fractured replica-immunolabeling study, and revealed the significant decrease in postsynaptic expression of AMPA receptors in CA1 of the hippocampus. Interesting, we also found that postsynaptic density (PSD) ultrastructure was impaired in the CA1 of the NSF+/- mice. Furthermore, to examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. The long-term depression (LTD) impaired in the CA1 of the NSF+/- mice, but was normal long-term potentiation (LTP). It is suggested that NSF is regulators of synaptic plasticity and synaptic protein trafficking, might be related to the pathophysiology of autistic behavior.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P43 合成カンナビノイドCP55940がシナプス形成にもたらす発達神経毒性：ドレブリン局在変化の解析 Effects of a synthetic cannabinoid, CP55940, on synaptogenesis of cultured hippocampal neurons: imaging analysis of drebrin immunocytochemistry. ^○間瀬省吾¹, Toshinari Mitsuoka^2,3, Noriko Koganezawa², Hiroyuki Yamazaki², Yuuichi Kato¹, Izuo Tsutsui¹, Hiroshi Kawabe², Yuko Sekino¹ 1.群馬大学医学系研究科薬理学分野, 2.Department of Pharmacology, Gunma University Graduate School of Medicine, 3.Department of Pharmacy Administration, Faculty of Pharmaceutical Science, Hokkaido University of Science ^○Shogo Mase¹, Toshinari Mitsuoka^2,3, Noriko Koganezawa², Hiroyuki Yamazaki², Yuuichi Kato¹, Izuo Tsutsui¹, Hiroshi Kawabe², Yuko Sekino¹ 1.Endowed Laboratory of Human Cell-Based Drug Discovery, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 2.Department of Pharmacology, Gunma University Graduate School of Medicine, 3.Department of Pharmacy Administration, Faculty of Pharmaceutical Science, Hokkaido University of Science In this study, we investigate hazard risks of early-life exposure to cannabinoids using cultured rat hippocampal neurons and drebrin immunocytochemistry. Drebrin plays a key role in dendritic spine formation of neurons and is responsible for the morphological plasticity of dendritic spines. In this study, effects of CP55940 on the distribution of drebrin in developing neurons. Hippocampal neurons prepared from frozon embryonic rats hippocampal neurons (SKY neuron, AlzMed, Inc., Tokyo) were incubated in 96-well microplates. After 7, 14 or 21days, neurons were treated with CP55940 (from 100 nM to 10µM). After the process of immunocytochemistry to visualize drebrin, MAP2 and cell nucleus, neuron number, dendrite length, and drebrin clusters were automatically quantified using our algorithms. CP55940 significantly changed the ratio of high intensity drebrin clusters as well as increased the numbers of drebrin clusters. On the other hand, the treatment of 10µM CP55940 for 7, 14 days significantly reduced neuron numbers. It has been discussed how cannabinoids affect the developing brain, and lead to poorer cognitive and emotional outcomes in adulthood. In Japan, a study group of the Ministry of Health, Labor and Welfare has been studying the risks because the abuse of cannabis by young people becomes more serious. Identification of drebrin clusters and analysis of their numbers and distribution are promising to detect hazard risk of cannabis. This work was supported by the Japan Chemical Industry Association (JCIA) Long-range Research Initiative (LRI) to YS , and the Regulatory Science Research Grant from MLHW (2020-2022) to YS		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P44 GluA2/3 AMPA受容体複合体に対する光不活性化技術 Optical inactivation technology for GluA2/3 AMPA receptor complex. ^○實木亨, Kiwamu Takemoto Department of Biochemistry, Graduate School of Medicine, Mie University ^○Susumu Jitsuki, Kiwamu Takemoto Department of Biochemistry, Graduate School of Medicine, Mie University Excitatory and inhibitory neurotransmitter receptors usually form protein complex by various combinations with subunits and express their spatio-temporal function. For example, AMPA type glutamate receptors (AMPA-Rs), which are well known to be important glutamate receptors for learning, are composed of variable combinations of four subunits, GluA1-4. Among them, GluA1 homomer, GluA1/2 and GluA2/3 were known to be expressed in adult brain. AMPA-Rs with GluA1 subunits require plasticity-inducing protocols and NMDA-Rs activation to be driven into synapses and serve to enhance neurotransmission. In contrast, GluA2/3 complex continuously replace synaptic receptors in a manner that maintains transmission (Shi, S-H. et al. Cell 2001, Takahashi, T. et al. Science 2003　etc.).　In addition, subunit combinations affect the functions of ion channel. Complexes lacking GluA2 subunit shows calcium permeability and high single channel conductance (Dingledine R et al. Pharmacol Rev 1999, Coombs ID et al. J. Neurosci. 2012). These observations support the idea that AMPA-Rs complexes should have different physiological functions in vivo. To elucidate their complex-specific functions in vivo, we have developed an optical technology for acute inactivation of synaptic GluA1 homomer in vivo by chromophore assisted light inactivation (Takemoto et al. Nat. Biotechnol. 2017). This technology enabled us to elucidate the function of GluA1 homomer in the acquisition of contextual fear memory in hippocampus. Based on this study, we are currently developing a CALI method for GluA2/3, an AMPA receptor that does not contain GluA1.　In this presentation, we will discuss our new CALI technology for GluA2/3 receptors.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P45 背側海馬と腹側海馬歯状回におけるCadherin-13の局在と発現量の比較 Comparison of Cadherin-13 expression in dorsal and ventral dentate gyrus in hippocampus ^○山本愛実, Mika Hojo, Toshinori Sawano, Yumika Miyagi, Jin Nakatani, Hidekazu Tanaka 立命館大学 ^○Manami Yamamoto, Mika Hojo, Toshinori Sawano, Yumika Miyagi, Jin Nakatani, Hidekazu Tanaka Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University Dorsal and ventral hippocampus are supposed to owe distinct functions. Consistently, we have found that the dendritic arborizations of the granule cells in the dorsal dentate gyrus (dDG) is more complex than those in the ventral (v) DG. The dendritic spine density is higher in vDG than in dDG. Cadherin-13 (Cdh13) mRNA is expressed in vDG about twice as much as dDG using microarray analysis. CDH13 belongs to the cadherin superfamily and is anchored to the cellular membrane through glycosylphosphatidylinositol. It is also reported that the knockdown of Cdh13 reduces synaptic density of cultured hippocampal neurons. In this study, we described the detailed localizations of Cdh13 mRNA and protein in the hippocampus and confirmed the difference in the expression levels between dDG and vDG. Cdh13 mRNA was uniformly and weakly expressed throughout the granule cell layer of dDG and vDG, but there was no significant difference in signal intensity between dDG and vDG. CDH13 protein was expressed higher in the granule cell and molecular layers of vDG than in dDG. In particular, there were strong signals in the distal two-thirds of the molecular layer of vDG. It is possible that CDH13 in vDG may be involved in the function of dendrites that receive inputs from the entorhinal cortex. In addition, since it has been reported that CDH13 is upregulated in the amygdala of depressed humans and mice, we would like to discuss whether social defeat stress is related to Cdh13 expression in the hippocampus.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P46 神経回路形成因子LOTUSによるアミロイドβタンパク質受容体PirBの制御 Regulation of PirB, an amyloid β protein receptor by a neural circuit formation factor LOTUS ^○川口祐生¹, Yuji Kurihara¹, Junpei Matusbayashishi¹, Yutaka kawakami¹, Kohtaro Takei¹ 1.生命医科学研究科　生体機能医科学研究室　竹居研, 2.Department of molecular biology graduate school of pharmaceutical sciences Nagoya City Unversity ^○Yuki Kawaguchi¹, Yuji Kurihara¹, Junpei Matusbayashishi¹, Yutaka kawakami¹, Kohtaro Takei¹ 1.Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science, 2.Department of molecular biology graduate school of pharmaceutical sciences Nagoya City Unversity It has been recently reported that paired immunoglobulin like receptor B（PirB） acts as a receptor of amyloid beta (Aβ) protein, known as a main risk factor of Alzheimer´s disease (AD). We identified lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of PirB. Previously, We reported that LOTUS inhibits the binding of Aβ to PirB in Cos7 cells. In this study, we examined whether LOTUS suppresses Aβ-induced decrease of spine density and cofilin dephosphorylation in cultured hippocampal neurons. We first found that LOTUS inhibits Aβ-induced decrease of spine density. Though the spine density is reduced by Aβ treatment, the reduction was suppressed by overexpression of LOTUS by gene transfection in cultured hippocampal neurons derived from the wild type mouse. It is known that the decrease of spine density is caused by Aβ-induced cofilin dephsphorylation via PirB. We then examined effect of LOTUS expression level on cofilin dephosphorylation induced by Aβ treatment. We found that LOTUS overexpression inhibits cofilin dephosphorylation induced by Aβ treatment. The data suggest that LOTUS suppresses Aβ-induced decrease of synaptic density through blockade of cofilin dephosphorylation, thereby possibly alleviating Aβ-induced pathogenesis. This inhibitory effect by LOTUS leads to the idea that LOTUS may be a possible therapeutic agent for the progression of AD pathology.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P47 高次脳機能に関わる成熟脳におけるミクログリアによるシナプス貪食 Modulation of mature brain functions by microglia through synaptic elimination by phagocytosis ^○田中潤也愛媛大学医学系研究科　分子細胞生理学 ^○Junya Tanaka Department of Molecular and Cellular Physiology, Ehime University We found that microglia suppress appearance of Parkinson's disease (PD) symptoms through synaptic phagocytosis in a rat 6-OHDA-induced hemi-PD model. Although activated microglia in the substantia nigra parc compacta (SNc) are well known to exert proinflammatory toxic effects on dopaminergic neurons, microglial activation was more pronounced in SN pars reticulata (SNr) than in the SNc. Microglia in the SNr were more activated in terms of phagocytosis than those in the SNc, suggesting that the former microglia suppress PD-induced motor deficits by eliminating glutamatergic synapses from the subthalamic nuclei, which are hyperactivated in PD (DOI: 10.1002/glia.23199). Furthermore, we noticed that microglia in the normal mature rat cerebral cortex have larger cell bodies at ZT0 than those at ZT12. During sleep onset or ZT0. Synaptosomes prepared at ZT0 were more densely decorated with complement C3 or MFG-E8, which were assumed to be phagocytosed by microglia (DOI: 10.1002/glia.23698). This diurnal changes in microglia may be related to the diurnal changes in noradrenaline contents and glucocorticoid levels in the circulation. Thus, microglia may be deeply involved in the normal functioning of neural circuits through synaptic phagocytosis. Furthermore, we found that cFos expression in neurons in the medial prefrontal cortex of the Lister hooded rat (LHR), a new model animal of attention deficit/hyperactivity disorder (ADHD), was increased (DOI: 10.1016/j.neuint.2020.104857). Microglia density in the same region is low, and individual cell size and CD11b expression were also reduced. We are now investigating the relationship between the ADHD-like behaviors and microglial synaptic phagocytosis.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P48 大脳皮質発達過程におけるオートファジー関連分子Wdr45の役割 Role of Wdr45, the autophagy-related gene, in the development of mouse cerebral cortex ^○野田万理子, Hidenori Ito, Kou-ichi Nagata 愛知県医療療育総合センター　発達障害研究所　分子病態研究部 ^○Mariko Noda, Hidenori Ito, Kou-ichi Nagata Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center WDR45 is highly conserved during evolution and essential in autophagy. WDR45 gene de novo mutations are reported to cause β-propeller protein-associated neurodegeneration (BPAN), which is a subtype of neurodegeneration with brain iron accumulation (NBIA). The clinical feature of BPAN is global developmental delay in early childhood, with adult-onset neurodegeneration (including dystonia, dementia and parkinsonism). These mutations lead lower expression of WDR45 protein. However, molecular pathophysiological mechanism of BPAN in developmental stage remains largely unknown. We here examined the role of Wdr45 in mouse brain development. We first performed immunohistochemistry and western blotting to determine the Wdr45 protein expression profile in the developing mouse brain. Wdr45 was expressed throughout the developmental process and it changed intracellular expression pattern in a developmental stage-dependent manner. Acute silencing of Wdr45 by in utero electroporation was then carried out to determine the knock down effects on corticogenesis. While it had little effects on excitatory neuron positioning, dendritic arbor formation and synaptogenesis were severely impaired. Based on these results, we propose that Wdr45 is critical for the maturation of cortical excitatory neurons and is likely to be related to pathophysiology of BPAN.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P49 定常状態および脳梗塞モデル成体マウスにおけるArcadlin mRNAの発現解析 Expression analysis of Arcadlin mRNA in intact and ischemic brain of adult mice ^○井上翔太¹, Yosuke Inoue¹, Toshinori Sawano¹, Natsumi Yamaguchi¹, Akinori Takayama¹, Shinobu Inagaki^2,3, Jin Nakatani¹, Hidekazu Tanaka¹ 1.立命館大学大学院　薬理学研究室, 2.United Graduate School of Child Development, Osaka University, 3.Department of Physical Therapy, Osaka Yukioka College of Health Science ^○Shota Inoue¹, Yosuke Inoue¹, Toshinori Sawano¹, Natsumi Yamaguchi¹, Akinori Takayama¹, Shinobu Inagaki^2,3, Jin Nakatani¹, Hidekazu Tanaka¹ 1.Pharmacology Laboratory, Department of Biomedical Sciences, College of Life Sciences, Ritsumeikan University, 2.United Graduate School of Child Development, Osaka University, 3.Department of Physical Therapy, Osaka Yukioka College of Health Science The morphological plasticity of neuron serves as the adaptive capabilities of central nervous system to various stimuli. Ischemic stroke alters the neuronal morphology, and these changes are involved in the pathogenesis and recovery process. Arcadlin/Paraxial protocadherin/Protocadherin-8 is a regulator of dendritic spine density. However, the involvement of Arcadlin in the ischemic stroke remains to be investigated. Currently, we revealed the expression pattern of Arcadlin mRNA after cerebral ischemia using in situ hybridization histochemistry. Cerebral ischemia upregulated Arcadlin mRNA in the piriform cortex, dentate gyrus, CA3, entorhinal cortex, olfactory area, prefrontal cortex, insular cortex, amygdala, and septohippocampal nucleus. Arcadlin long variant and short variant have been reported. Arcadlin short variant reduces the spine density, but long variant does not. In this study, we demonstrated that ischemia induced Arcadlin upregulation was significant in Arcadlin short variant rather than long variant. These results suggest that Arcadlin is involved in the dendritic spine density in the ischemic brain.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P50 Arcadlinが海馬CA3錐体細胞の樹状突起スパインに及ぼす影響 Preventive effect of lactoferrin on decreased spontaneous activity by ovariectomy in rats ^○河前なつみ Pharmacology Laboratory, Graduate School of Life Sciences, Ritsumeikan University ^○Natsumi Kawamae Pharmacology Laboratory, Graduate School of Life Sciences, Ritsumeikan University The female hormone estradiol affects the central nervous system disorders, such as depression and dementia. Hormone replacement therapy (HRT) is used to treat symptoms associated with female menopause. However, the administration of estradiol is reported to increase the risk of breast and endometrial cancers, it is important to find a novel therapy of disorders related the female hormone. We have already reported that ovariectomized (OVX) rats reduced the spontaneous activity during the dark period due to the decease of serotonin release in the amygdala (Behav Brain Res. 2012, 227, 1-6). In this study, we examined the potential of lactoferrin (LF) on the recovery of less spontaneous activity seen in rats with OVX-induced despair-like behaviors. 8-week-old female OVX rats were administered LF (300mg/kg) five days a week for 6 weeks from the day after surgery. After administration was completed, spontaneous locomotor activity in the dark period, immobility time in a forced swim test, and release amount of dopamine and serotonin in the brain were measured. LF was found to improve OVX-induced decreases in locomotor activity and increases in immobility time in the forced swim test. Furthermore, the administration of LF elicited significant recovery of decreased dopamine and serotonin release in the brains of OVX group rats. These results strongly suggest that LF improved OVX-induced decreases in momentum during the dark period and, moreover, that release of dopamine and serotonin in the brain was involved in this effect.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P51 神経細胞の脂質代謝におけるORP6の役割 The involvement of Oxysterol-binding protein related protein (ORP) 6 in the transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons ^○望月信弥, Harukata Miki, Ruyun Zhou, Yasuko Noda Department of Anatomy, Jichi Medical University ^○Shinya Mochizuki, Harukata Miki, Ruyun Zhou, Yasuko Noda Department of Anatomy, Jichi Medical University Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of Group III ORP family, localizes in membrane contact sites between ER and other organelles and is relevant to the non-vesicular exchange of lipid including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the counter-transported lipid in exchange of PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of phosphatidylserine (PS) marker but not filipin staining free cholesterol in primary cultured cerebellar neurons. Treatment of PI4KⅢα inhibitor in Neuro 2A cells specifically inhibited the synthesis of PI4P and caused the loss of PI4P marker at plasma membrane (PM), which also suppressed the localization of ORP6 and the PS marker at PM. These data collectively suggest the involvement of ORP6 in the transport of PI4P and PS.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P52 膀胱尿路上皮は大腸菌由来LPSをATPを介して神経シグナルに変換し排尿反射亢進をもたらす Bladder urothelium converts bacterial lipopolysaccharide information into neural signaling via an ATP-mediated pathway to enhance the micturition reflex for rapid defense ^○近藤誠¹, Norichika Ueda^1,2, Kentaro Takezawa^1,2, Hiroshi Kiuchi², Noriyoshi Usui¹, Yoshihisa Koyama¹, Yukiko Nakamura¹, Norio Nonomura², Shoichi Shimada¹ 1.Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, 2.Department of Urology, Graduate School of Medicine, Osaka University ^○Makoto Kondo¹, Norichika Ueda^1,2, Kentaro Takezawa^1,2, Hiroshi Kiuchi², Noriyoshi Usui¹, Yoshihisa Koyama¹, Yukiko Nakamura¹, Norio Nonomura², Shoichi Shimada¹ 1.Department of Neuroscience and Cell Biology, Graduate School of Medicine, Osaka University, 2.Department of Urology, Graduate School of Medicine, Osaka University When bacteria enter the bladder lumen, a first-stage active defensive mechanism flushes them out. Although urinary frequency induced by bacterial cystitis is a well-known defensive response against bacteria, the underlying mechanism remains unclear. In this study, using a mouse model of acute bacterial cystitis, we demonstrate that the bladder urothelium senses luminal extracellular bacterial lipopolysaccharide (LPS) through Toll-like receptor 4 and releases the transmitter ATP. Moreover, analysis of purinergic P2X2 and P2X3 receptor-deficient mice indicated that ATP signaling plays a pivotal role in the LPS-induced activation of L6–S1 spinal neurons through the bladder afferent pathway, resulting in rapid onset of the enhanced micturition reflex. Thus, we revealed a novel defensive mechanism against bacterial infection via an epithelial-neural interaction that induces urinary frequency prior to bacterial clearance by neutrophils of the innate immune system. Our results indicate an important defense role for the bladder urothelium as a chemical-neural transducer, converting bacterial LPS information into neural signaling via an ATP-mediated pathway, with bladder urothelial cells acting as sensory receptor cells.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P53 ラクトフェリンは、ERK経路を介したPC12細胞の神経突起伸長を促進する Lactoferrin promotes PC12 cells neurite outgrowth via ERK signaling pathways ^○Nobuo Izumo¹, Megumi Furukawa¹, Daichi Nagashima¹, Masahiro Toho¹, Haruna Kurono¹, Noa Mizukami¹, Tatsuo Hoshino¹, Takayuki Manabe¹ 1.Laboratory of Pharmacotherapy, Yokohama University of Pharmacy, 2.Center for pharmaceutical education, Yokohama University of Pharmacy, 3.Department of Nursing, Faculty of Nursing, Chukyogakuin University, 4.Laboratory of Functional Materials, Yokohama University of Pharmacy, 5.NRL Pharma, Inc. ^○Nobuo Izumo¹, Megumi Furukawa¹, Daichi Nagashima¹, Masahiro Toho¹, Haruna Kurono¹, Noa Mizukami¹, Tatsuo Hoshino¹, Takayuki Manabe¹ 1.Laboratory of Pharmacotherapy, Yokohama University of Pharmacy, 2.Center for pharmaceutical education, Yokohama University of Pharmacy, 3.Department of Nursing, Faculty of Nursing, Chukyogakuin University, 4.Laboratory of Functional Materials, Yokohama University of Pharmacy, 5.NRL Pharma, Inc. Lactoferrin (LF) is a protein that is rich in breast milk. Its action has attracted great attention as a potential functional factor to not only enhances immune function but also acts on the central nervous system. However, the molecular mechanism underlining neurite outgrowth of LF is unclear. In this study, effect of LF on rat adrenal pheochromocytoma PC12 cells neurite outgrowth was determined using morphometry, while MAPK signaling pathways related to neurite outgrowth of PC12 cells were investigated based on the addition of inhibitors. In addition, mRNA expression level of neuro factor was measured by Real-time RT-PCR. PC12 cells seeded onto 24-well plate (1×10⁴cells/well) were cultured in 10% FBS DMEM.　After 24h,the cells were incubated for 3 days in serum free DMEM containing LF (100nM) with/without PD98059.　On day1 and 3, morphometric analysis of the neurites and length was performed by Neurocyte Image Analyzer software (KURABO). Our findings indicated that LF enhanced PC12 cells neurite outgrowth in a dose-dependent manner. Furthermore, we found that LF may cause ERK activity because the addition of PD98059 suppressed neurite outgrowth. The mRNA expression of NF-L was significantly enhanced after treatment of LF. These results indicate that LF induces ERK activation, increases NF-L gene expression level , and promotes neurite outgrowth on PC12 cells.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P54 タウタンパク質からアプローチする神経軸索内輸送の分子メカニズム A study of molecular mechanism for the axonal transport employing the Tau protein as a model cargo ^○中田里奈穂, 御園生裕明同志社大学　脳科学研究科 ^○里奈穂中田, 裕明御園生同志社大学脳科学研究科病態脳科学分野チャネル病態生理部門 The abnormal localization and deposition of tau protein is a hallmark of Alzheimer disease. Tau is known to be transported to the axon by the slow axonal transport and localized to the axons of neurons (Kubo et al., 2019), but in Alzheimer disease, it accumulates abnormally in cell bodies and dendrites. In this study, we aim to understand the mechanism of the abnormal localization of tau protein that occurs in neurodegeneration through understanding of the molecular mechanism of tau transport and how it may be disrupted. We used an inducible expression system we developed and human tau tagged with a photoconvertible fluorescent protein, Dendra2, to visualize the molecular behaviors and transport of tau in primary cultured rat hippocampal neurons. To visualize the transport of tau to the axon, we photoconverted Dendra2-tau, which is expressed at a low level in dendrites, and analyzed whether and how the dendritic tau migrates into the axon. The current results show that the tau moves from a dendrite to the axon at approximately 0.05 um/s (4 mm per day), which is roughly the same speed reported for the slow axonal transport. This transport was inhibited by depleting ATP in the cell, indicating that the transport process is energy-dependent. Based on these results, in order to clarify the mechanism of tau axonal transport, we would like to devise fluorescent probes to better visualize axonal transport more clearly. To substantiate that the disruption of transport underlies the pathological mislocalization of tau, we will also investigate how pathological phosphorylation and mutations affect the transport and localization of tau. Understanding the transport mechanism may add new insights into how changes in localization affect pathology in Alzheimer disease.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P55 Rosa26ノックイン型タウオパチー認知症モデルマウスの作出 Development of Rosa26-knockin P301L human tau expressing mice ^○矢内凜, Takeharu Minamihisamatsu, Masafumi Shimojo, Naruhiko Sahara 国立研究開発法人量子科学技術研究開発機構量子医学・医療部門放射線医学総合研究所脳機能イメージング研究部 ^○Rin Yanai, Takeharu Minamihisamatsu, Masafumi Shimojo, Naruhiko Sahara Department of Functional Brain Imaging, National Institutes for Quantum and Radiological Science and Technology Tauopathy is characterized by the fibrillar tau accumulation in central nervous system. Neurofibrillary lesions strongly correlated with cognitive deficits in neurodegenerative diseases. Animal models of tauopathy are essential for preclinical studies of Alzheimer’s disease and related dementias. Overexpression of familial mutant tau has been utilized to drive tauopathy and disease-relevant phenotypes in a number of different animal models that have established a role of this protein in neurodegeneration. The rTg4510mouse line is one of the popular tauopathy models (Santacruz et al. 2005). This model uniquely employed the CaMKIIa promoter-driven tetracycline transactivator (tTA) transgene to drive 0N4R P301L tau expression in the forebrain and to allow conditional repression of the transgenic tau expression through the use of doxycycline, tetracycline analog. Recent studies revealed that transgenic insertion/deletion mutations may affect pathogenesis of tauopathy in the rTg4510 mice (Gamache et al. 2019). To overcome this issue, we generate a novel tauopathy mouse model, in which 1N4R P301L human tau was integrated in the Rosa26 safe harbor gene and expressed by cross-breeding with tTA mice under C57BL/6J background. Successfully, this Rosa26-KI tau mouse expresses P301L human tau in the forebrain with ~4-fold of mouse tau. A homozygous mouse for P301L human tau gene developed pretangle pathology in hippocampus at 13 months of age. Although this model delayed tau pathology to compare with rTg4510 mice, either leaky expression of P301L human tau or dentate granule neuron degeneration was absent in this model. Further characterizations are ongoing for the usage of preclinical examinations.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P56 新規外傷性脳損傷モデルCHIMERAを用いたタウオパチー病理の研究 Tauopathy pathology in a novel traumatic mouse brain injury model ^○木村妙子¹, Masami Masuda-Suzukake¹, Hashimoto Masashi¹, Shin-ichi Hisanaga², Masato Hasegawa¹ 1.Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science, 2.Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University ^○Taeko Kimura¹, Masami Masuda-Suzukake¹, Hashimoto Masashi¹, Shin-ichi Hisanaga², Masato Hasegawa¹ 1.Department of Brain and Neurosciences, Tokyo Metropolitan Institute of Medical Science, 2.Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University Tauopathy is a group of neurodegenerative diseases, including Alzheimer's disease (AD), characterized by aggregates of tau in neurons and glial cells. The pathological mechanism of tauopathy is not known yet. Recently, chronic traumatic brain injury (CTE) caused by traumatic brain injury (TBI) is shown to be also a tauopathy. Different from other tauopathies, we can follow the disease progression of CTE because the onset and cause are clear. However, most previous CTE models are invasive and did not represent sports injuries and traffic accident injuries. Here, we used a novel TBI model called CHIMERA (closed-head impact model of engineered rotational acceleration). Firstly, we established TBI conditions that could cause tau pathology using wild-type mice. With mild repeated TBI, mice displayed axonal damages such as varicosities and bulbs in optic tract at 7 days after TBI. Iba1 and GFAP-positive inflammation was also observed at the axon injury sites, but hyperphosphorylated tau (AT8) positive pathology was not. Then, we used tau Tg (Tau Tg) mice, which express tau with pathological Ser mutation at Pro301 and accumulate tangles of hyperphosphorylated tau at 6 months. Tau Tg mice also showed axonal inflammation at 1~2 months after TBI, whereas tau pathology was not observed. In contrast, Tau Tg mice increased tau hyperphosphorylation at the IV and V layers in the motor and sensory cortex, where human TBI has tau pathology. The IV, V layer neurons extends long axons to spinal cord. We are now studying whether their long axons are damaged by TBI.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P57 異なったタウオパチー神経変性疾患ではタウのリン酸化も異なっている Distinct phosphorylation profiles of tau in brains of patients with different tauopathies ^○久永眞市¹, Nastaran Samimi², Taeko Kimura³, Tomoyasu Matsubara⁴, Yuko Saito⁴, Shigeo Murayama,⁴, Masato Hasegawa³, Kanae Ando,¹ 1.東京都立大学理学研究科生命科学専攻神経分子機能研究室, 2.Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, 3.Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, 4.Department of Neuropathology (the Brain Bank for Aging Research), Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, ^○Shin-ichi Hisanaga¹, Nastaran Samimi², Taeko Kimura³, Tomoyasu Matsubara⁴, Yuko Saito⁴, Shigeo Murayama,⁴, Masato Hasegawa³, Kanae Ando,¹ 1.Laboratory of Molecular Neuroscience, Department of Biological Sciences, Tokyo Metropolitan University, 2.Noncommunicable Diseases Research Center, Fasa University of Medical Sciences, 3.Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, 4.Department of Neuropathology (the Brain Bank for Aging Research), Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tauopathies are neurodegenerative diseases that are characterized by pathological accumulation of tau protein. Tau is hyperphosphorylated in the brain of tauopathy patients, and this phosphorylation is proposed to play a role in disease development. However, it has been unclear whether phosphorylation is different among different tauopathies. Here, we investigated the phosphorylation states of tau in several tauopathies, including corticobasal degeneration (CBD), Pick’s disease (PiD), progressive supranuclear palsy (PSP), argyrophilic grain dementia (AGD) and Alzheimer’s disease (AD). Analysis of tau phosphorylation profiles using Phos-tag SDS-PAGE revealed distinct phosphorylation of tau in different tauopathies, whereas similar phosphorylation patterns were found within the same tauopathy. For PSP, we found two distinct phosphorylation patterns suggesting that PSP may consist of two different related diseases. Immunoblotting with anti-phospho-specific antibodies showed different site-specific phosphorylation in the temporal lobes of patients with different tauopathies. AD brains showed increased phosphorylation at Ser202, Thr231 and Ser235, PiD brains showed increased phosphor-Ser202, and AGD brains showed increased phosphor-Ser396. The cis conformation of the peptide bond between phospho-Thr231 and Pro232 (cis ptau) was increased in AD and AGD. These results indicate that while tau is differently phosphorylated in tauopathies, a similar pathological mechanism may occur in AGD and AD patients. The present data provide useful information regarding tau pathology and diagnosis of tauopathies.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P58 新免疫染色法を用いた発生期マウスにおけるリン酸化タウの組織学的解析 Histological analysis analyses of phosphorylated tau in developmental mouse by unique immunostaining method ^○東優人¹, 宮坂知宏^1,2 1.Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan, 2.Center for Research in Neurodegenerative Diseases, Doshisha University, Kyoto, Japan ^○Yuto Higashi¹, 知宏宮坂^1,2 1.Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, Kyoto, Japan, 2.Center for Research in Neurodegenerative Diseases, Doshisha University, Kyoto, Japan Tau is one of the microtubule-associated proteins that localize in axons. Its physiological function is believed to bind on microtubules and stabilize them. There are numerous evidence indicating that the function of tau on microtubule were regulated by phosphorylation. For example, physiologically, tau is known to be highly phosphorylated in developmental brains. Recent studies showed that the optimal phosphorylation state and timing of dephoshporylation are essential for accurate microtubule binding and axonal localization of tau, although its details in vivo remain unclear because of the technical limitation. Immunohistochemically, phosphorylated tau deposited in pathological inclusions were easily labeled. In contrast, preaggregated phosphorylated tau is hard to detect by conventional immunohistochemical procedures. Previously, we had reported that the novel protocol that are consisted with fresh frozen section, fumigation fixation by formalin, and detergent pretreatment, enable to label phosphorylated tau in tissue by specific anti-phospho-tau antibodies. Here we analyzed the tissue distribution of phosphorylated/dephosphorylated tau in developing mice. Using our method, AT8 positive phosphorylated tau was diffusively localized throughout the neuronal cell bodies at postnatal day 7 (P7) and then rapidly dephosphorylated at P9. Immunolabeling using RTM38, an anti-total tau antibody, showed that the tau axonal distribution was established by P14. Thus, dephosphorylation of tau at Ser202 and Thr205 were precede from completion of its axonal distribution. This study provides an insight of the significance of phosphorylation in the mechanisms of tau axonal localization.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P59 神経細胞におけるタウの微小管結合を低下させるリン酸化サイトの同定 Identification of tau phosphorylation sites which cause its abnormal microtubule binding in neurons ^○姚芷薇¹, Ayaka Tatsumoto¹, Tomohiro Torii², Hiroaki Misonou², Tomohiro Miyasaka¹ 1.同志社大学　生命医科学研究科　医生命システム専攻, 2.Laboratory of Ion Channel Pathophysiology, Graduate School of Brain Science, Doshisha University ^○Zhiwei Yao¹, Ayaka Tatsumoto¹, Tomohiro Torii², Hiroaki Misonou², Tomohiro Miyasaka¹ 1.Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University, 2.Laboratory of Ion Channel Pathophysiology, Graduate School of Brain Science, Doshisha University Tau is a microtubule-associated protein (MAP) that is localized to the axon, and is also known as a component of neurofibrillary tangles in Alzheimer's disease (AD). Because the pathologically deposited tau is hyper-phosphorylated and lost its functions on microtubules (MT), dysregulation of MT-binding of tau is considered to be a key step to cause neurodegeneration in AD brains. However, critical phosphorylation site(s) of tau that affects MT-binding of tau in neuronal cells had remain obscure. Here we addressed whether the combinations of phospho-mimetic mutation of eight Ser/Thr sites in the proline-rich region 2 disrupts both of MT-binding and axonal localization of tau. We subclassed these phosphorylation sites into three groups, Ser198-Thr205 (4E-tau), Thr212/Ser214 (2E1-tau), Thr231/Ser235 (2E2-tau), and created pseudo-phosphorylation mutants. Wild-type and the mutant tau proteins tagged with GFP were transiently expressed in rat primary cultured neurons, and their molecular dynamics were analyzed using a fluorescence recovery after photobleaching (FRAP) assay. We found that the 2E1-tau exhibited higher mobility than other mutants in FRAP analyses, indicating reduced binding of 2E1-tau onto MTs. Because the double phosphorylation in Thr212/Ser214 sites has been specifically identified in pathologically deposited abnormal tau, they may be essential for the abnormal behaviors of tau in the pathogenesis of AD.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P60 ダイレクトリプログラミングによるヒト血管内皮細胞から神経細胞への誘導 Human blood endothelial cells require extra molecules, in addition to NeuroD1 known as the pioneer factor, to be converted into neurons ^○松田(伊藤) 花菜江, Taito Matsuda, Kinichi Nakashima 九州大学医学研究院　基盤幹細胞学分野 ^○Kanae Matsuda-Ito, Taito Matsuda, Kinichi Nakashima Department of Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu University Direct reprogramming is the technology to convert somatic cells from the original lineage to another by bypassing pluri/multi-potent stem cell state. To achieve this, targeted cell lineage-specific transcription factors that can rewrite epigenetic signatures, such as DNA methylation and histone modifications, are often forcibly expressed in the original cell types. We have previously reported that mouse microglia can be converted into neurons by the expression of a single transcription factor, NeuroD1, through its pioneering activity. However, it is necessary to convert human somatic cells into neurons when considering the clinical application to treat patients suffering neurological disorders. Recently, it has become apparent that human cells require more factors than those for model animal cells to be converted into other lineages. Consistently, we also found that NeuroD1 alone cannot convert human blood endothelial cells (HBECs) into neurons in contrast to mouse microglia. HBECs can be obtained from human in a minimally invasive manner and are even commercially available. Therefore, it is considered that HBECs are suitable as a human cell source to investigate reprogramming factors for direct conversion of human cells into neurons. We, therefore, screened genes that allow us to induce neurons from HBECs and found that HBECs can be converted into neurons by expressing several transcription factors in addition to NeuroD1. These converted neuronal cells expressed neuronal markers, e.g., DCX, TUBB3. In conclusion, our findings reinforce the notion that human cells require additional factors to induce direct reprogramming and we should take this into account for the effective conversion of human cells to develop therapeutic strategies for neurological diseases.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P61 哺乳類大脳皮質発生において、サブプレートニューロンが皮質板下に整然と配置されるメカニズム How are the subplate neurons properly positioned below the cortical plate in the developing mammalian cortex? ^○吉永怜史¹, Kazuhiro Ishii¹, Mayumi Okamoto³, Takaki Miyata³, Kazunori Nakajima¹, Ken-ichiro Kubo^1,2 1.東京慈恵会医科大学解剖学講座, 2.Department of Anatomy, The Jikei University School of Medicine, 3.Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University ^○Satoshi Yoshinaga¹, Kazuhiro Ishii¹, Mayumi Okamoto³, Takaki Miyata³, Kazunori Nakajima¹, Ken-ichiro Kubo^1,2 1.Department of Anatomy, Keio University School of Medicine, 2.Department of Anatomy, The Jikei University School of Medicine, 3.Department of Anatomy and Cell Biology, Graduate School of Medicine, Nagoya University In the developing mammalian cerebral cortex, the earliest-generated subplate (SP) neurons first reside in the preplate (PP), which is split into a superficial layer called the marginal zone (MZ), and a deep layer, the SP, upon the formation of the cortical plate (CP). SP neurons contribute to white matter neurons, which are increased in some patients with psychiatric disorders. The subplate specific genes during development also show an enrichment for association with these disorders. Some reports suggested that SP neurons are positioned below the CP passively since “layer VI neurons” split the PP. However, it has not been experimentally verified whether the neurons that split the PP are the layer VI neurons. How the SP layer is properly formed remains to be elucidated. Here, we specifically labeled future SP neurons with FlashTag technology, which labels cells that undergo mitosis on the ventricular surface with high temporal resolution, to describe migratory and positional profiles. Labeled cells were first observed in the PP and then in the CP and MZ upon the formation of the CP; finally, they moved down below the CP 1-2 days later, suggesting downward movement of some SP neurons through the CP. This observation was independently validated by ontogenetic observations using in utero electroporation, immunohistochemistry, and in situ hybridization. Furthermore, SP neuron-specific expression of a dominant negative form of PI3-kinase resulted in abnormal positioning above and below the SP. These observations suggest that future SP neurons actively migrate to form a distinct layer below the CP. It would be intriguing to explore the possibility that mispositioned SP neurons play an important role in the pathogenesis of psychiatric disorders.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P62 自閉スペクトラム症責任遺伝子DEAF1がマウス大脳皮質発達に果たす役割 Role of DEAF1, a possible responsible gene for autism spectrum disorders, in mouse brain development ^○西條琢真, 浜田奈々子, 永田浩一愛知県医療療育総合センター発達障害研究所分子病態研究部 ^○琢真西條, 奈々子浜田, 浩一永田愛知県医療療育総合センター発達障害研究所分子病態研究部 The DEAF1 (Deformed Epidermal Autoregulatory Factor 1) gene, encoding a transcription factor, has been shown to be a possible responsible gene for autism spectrum disorders, intellectual disability and other neurodevelopmental disorders. We here analyzed the role of DEAF1 in the development of mouse cerebral cortex. Acute knockdown of mouse DEAF1 with the in utero electroporation technique caused suppression of dendritic arborization in cortical layer II/III pyramidal neurons during brain development. This phenotype was rescued by an RNAi-resistant version of DEAF1. Electrophysiological analyses using the whole-cell patch-clamp technique showed that DEAF1 knockdown reduced both firing rate and excitatory synaptic transmission in cortical layer II/III pyramidal neurons. These results strongly suggest that 1) DEAF1 plays an essential role in corticogenesis and 2) its functional defects cause structural and functional impairment of cortical neurons, which is related to the pathophysiology of neurodevelopmental disorders.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P63 発生期神経幹細胞の細胞周期及び分化、増殖へのKlf5遺伝子への影響 Effect of Klf5 gene on cell cycle, differentiation and proliferation of embryonic neural stem cells ^○黒田杏理¹, Hiroaki Kitagawa¹, Asmaa Abdullah¹, Takahiro Fuchigami¹, Yoshitaka Hayashi¹, Masatsugu Ema², Seiji Hitoshi¹ 1.生理学講座統合生理学部門, 2.Human Disease Models Research Center for Animal Life Science, Shiga University of Medical Science ^○Anri Kuroda¹, Hiroaki Kitagawa¹, Asmaa Abdullah¹, Takahiro Fuchigami¹, Yoshitaka Hayashi¹, Masatsugu Ema², Seiji Hitoshi¹ 1.Department of Integrative Physiology, Shiga University of Medical Science, 2.Human Disease Models Research Center for Animal Life Science, Shiga University of Medical Science Klf5 is a member of Krüppel-like factor (Klf) family of transcription factors and plays a significant role in differentiation, maintaining an undifferentiated state and control of cell cycle in stem cells. We have been analyzing the function of Klf5 by culturing cells from the ganglionic eminence of mouse embryonic brains, where neural stem cells (NSCs) reside. The number of NSCs and their self-renewal ability were evaluated by a colony-forming neurosphere assay. We examined Klf5 overexpression (OE) mouse embryos and found that the number of neurospheres derived from single NSCs was decreased, indicating that Klf5 gene suppressed the self-renewal of NSCs. Hence, we next performed RNA-seq analysis using E15.5 mouse brain to identify Klf5 related genes. GO analysis of RNA-seq results revealed the altered expression of genes related to the cell cycle and Notch signaling. To further analyze effects of Klf5 on the cell cycle in detail, we overexpressed Klf5 gene in Neuro2a cells, in which BrdU and EdU was incorporated, and calculated the cell cycle length from the difference between BrdU and EdU uptake time. The cell cycle length of the control Neuro2a cells was 33 hours, whereas that of Klf5-overexpressing cells was decreased to 25 hours (p=0.013), suggesting that Klf5 is a critical factor to regulate the cell cycle. We are now performing qRT-PCR and in situ hybridization of Hes1 and Hes5, using E15.5 Klf5 OE mouse brains, to analyze the relationship between Klf5 expression and Notch signaling activation. Furthermore, we are examining the cell cycle in vivo by injecting BrdU at E15.5 to ask whether or not Klf5 overexpression changes the proliferation and differentiation of Pax6+ NSCs and Tbr2+ progenitor cells.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P64 発達期ラット小脳に対するHDAC阻害剤投与が及ぼすエピジェネティックな変化とグルタミン酸恒常性 Epigenetic alteration and glutamate homeostasis due to prenatal administration of various HDAC inhibitors to developmental rat cerebellum ^○松井紗羅沙¹, Kengo Tanaka¹, Akari Adachi¹, Misaki Iwanaga¹, Yasunari Kanda², Sachiko Yoshida¹ 1.応用化学生命工学専攻　生命機能科学研究室, 2.National Institute of Health Sciences ^○Sarasa Matsui¹, Kengo Tanaka¹, Akari Adachi¹, Misaki Iwanaga¹, Yasunari Kanda², Sachiko Yoshida¹ 1.Department of Applied Chemistry and Life Science, Toyohashi University of Technology, 2.National Institute of Health Sciences Prenatal chemical exposure is one of the causes of developmental neurotoxicities, such as autism spectrum disorder (ASD) or mental disorders. Valproate (VPA), a widely used anti-epileptic drug and a well-known ASD inducer, has an HDAC inhibition effect, and we have observed VPA-administrated rat pups showed the alteration of Purkinje cell development and the excess folding in the cerebellar lobules. In this study, we administrated various HDAC inhibitors to prenatal rat at embryonic day 16 (E16); Vorinostat (SAHA) 25, or 50 mg/kg i.p., Entinostat (MS-275) 2.0 mg/kg p.o., butylate (BA) 200 mg/kg p.o., and VPA 600 mg/kg p.o., and observed the cerebellum of pups at postnatal day 7 (P7) or P14. Both SAHA-, BA-, and VPA-administrated pups showed the alteration of Purkinje cell development and the excess folding of the cerebellar lobule, whereas MS-275-administrated pups showed a slight developmental alteration. In VPA- and BA-administrated animals, H3K9me expression was decreased in the molecular layer at P7. Additionally, both GFAP and GLAST expressed higher in these animals than in control animals. H3K9me or tri-me are known as efficient histone methylation deeply related to astrocyte development. These data suggest that the administration of HDAC inhibitors would alter astrocyte and Bergmann glia maturation epigenetically and change glutamate homeostasis in the molecular layer.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P65 生後中枢神経系及び脊髄損傷後におけるFLRT2の発現 Expression analysis of FLRT2 in postnatal central nervous system development and after spinal cord injury ^○Li Juntan¹, Yo Shinoda², Shuhei Ogawa³, Shunsuke Ikegaya¹, Shuo Li^1,4, Yukihiro Matsuyama⁴, Kohji Sato¹, Satoru Yamagishi¹ 1.Department of Organ & Tissue Anatomy, Hamamatsu University School of Medicine, 2.Department of Enviromental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 3.Division of Experimental Animal Immunology, Research Institute for Biomedical Science, Tokyo University of Science, 4.Department of Orthopedic Surgery, Hamamatsu University School of Medicine ^○Juntan Li¹, Yo Shinoda², Shuhei Ogawa³, Shunsuke Ikegaya¹, Shuo Li^1,4, Yukihiro Matsuyama⁴, Kohji Sato¹, Satoru Yamagishi¹ 1.Department of Organ & Tissue Anatomy, Hamamatsu University School of Medicine, 2.Department of Enviromental Health, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 3.Division of Experimental Animal Immunology, Research Institute for Biomedical Science, Tokyo University of Science, 4.Department of Orthopedic Surgery, Hamamatsu University School of Medicine FLRT2, a member of the fibronectin and leucine-rich transmembrane protein (FLRT) family, is necessary for different developmental processes through acting as homophilic cell adhesion molecules, heterophilic repulsive ligands of Unc5/Netrin receptors, and synaptogenic function by binding to latrophilins. Although FLRT2 function in regulating cortical migration at the late gestation stage was analyzed, not much is yet known about FLRT2 expression pattern during central nervous system (CNS) development. Here we utilized Flrt2-LacZ knock-in mice to provide a detailed analysis of FLRT2 expression during postnatal CNS development. At an early postnatal stage, FLRT2 expression is mainly restricted in several regions of the striatum and deep layer of the cerebral cortex. In adulthood, FLRT2 expression was more prominent in the cerebral cortex, hippocampus, pyramidal layer of the piriform area (PIR), the nucleus of the lateral olfactory tract (NLOT) and the ventral medial nucleus of the thalamus (VM), whereas it was decreased in the striatum. Notably, in the hippocampus, FLRT2 expression was highly confined in the CA1 region and partly overlayed with PSD95, but not in the CA3 and DG. However, electrophysiological assays in hippocampus of FLRT2 conditional knock-out mice using Emx1-Cre showed no significant changes in hippocampal synaptic plasticity. Lastly, we observed temporally FLRT2 expression around the lesion site at 7 days after thoracic spinal cord injury. These dynamic FLRT2 expression may enable to function the multiple FLRT2 activities, such as cell adhesion, repulsion, and synapse formation in different regions during CNS development and after spinal cord injury.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P66 神経再生を促進するiPS細胞を用いた新規人工神経の開発 Developing a novel medical material with using human iPS-derived nerve fibers for enhancing the regeneration of the nerves ^○芝田晋介¹, Takayuki Nishijima^3,4, Hiroo Kimura⁴, Kentaro Okuyama^1,2,3, Tomoko Shindo^2,3, Manabu Hayatsu¹ 1.Division of Microscopic Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, 2.Electron Microscope Laboratory, Keio University School of Medicine, 3.Department of Physiology, Keio University School of Medicine, 4.Department of Orthopaedical surgery, Keio University School of Medicine ^○Shinsuke Shibata¹, Takayuki Nishijima^3,4, Hiroo Kimura⁴, Kentaro Okuyama^1,2,3, Tomoko Shindo^2,3, Manabu Hayatsu¹ 1.Division of Microscopic Anatomy, Graduate School of Medical and Dental Sciences, Niigata University, 2.Electron Microscope Laboratory, Keio University School of Medicine, 3.Department of Physiology, Keio University School of Medicine, 4.Department of Orthopaedical surgery, Keio University School of Medicine When the nerve is damaged due to cancer surgery or traumtic injury, it is necessary to perform surgical operation to cure. At present, the most common treatment is to carry out the self-nerve transplantation with a part of patient's peripheral sensory nerve. Self-nerve removal leads to the various defects, such as loss of the sensory perception carried by the removed doner sensory nerve. Our nerve regenerative project team has been conducting collaborative research for many years, trying the several challenging tasks by taking advantage of each other's advanced technologies as surgeon and imaging specialists. In recent years, we have been working on developing a new treatment for peripheral nerve deficits that promotes functional recovery by transplantation. The novel nerve organoid culture method with the excellent patented technology was utilized in this project, and we tried to develop a novel artificial nerve for nerve transplantation. Human iPS cells were induced to differentiate into neurons and cultured using a special culture device. The novel artificial nerve was transplanted into the sciatic nerve defect of rats with optimized transplantation surgery, the motor function was evaluated over time, and the condition of peripheral nerve regeneration by transplantation was evaluated at several weeks after transplantation. The histological evaluation is performed in detail using immunostaining and an electron microscope to evaluate the effect of this novel transplantation therapy. In the future, we hope new artificial nerve prepared in advance will be transplanted if a major peripheral nerve is cut due to traumatic injury or surgical operation of cancer, without any loss of patient's own peripheral nerve.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P67 トリデオキシシチジンによる神経幹細胞の増殖促進とプロテオミクス解析による変動因子の探索 Proteomics analysis of trideoxycytidine-induced proliferation of neural stem cells ^○石本尚大¹, Naoto Matsumura¹, Yusuke Masuo¹, Keisuke Kiriyama², Keita Sutoh², Yukio Kato¹ 1.金沢大学薬学系, 2.Fordays Co., Ltd. ^○Takahiro Ishimoto¹, Naoto Matsumura¹, Yusuke Masuo¹, Keisuke Kiriyama², Keita Sutoh², Yukio Kato¹ 1.Faculty of Pharmacy, Kanazawa University, 2.Fordays Co., Ltd. Oral intake of nucleic acid fraction (NAF) of hydrolyzed salmon milt extract has been reported to enhance learning and memory in mice with concomitant increase in expression of nestin, a marker for neural stem cells (NSCs), in hippocampus. In the present study, we tried to elucidate NAF-induced proliferation in mouse primary cultured NSCs. We focused on trideoxyribonucleotides because they are highly contained in NAF compared with other nucleotides. The NSCs were exposed to each trideoxyribonucleotide for 3 days, and proliferative activity was evaluated. Among all 64 trideoxyribonucleotides examined, only trideoxycytidine (CCC) significantly increased proliferative activity of NSCs. Quantification of CCC and its metabolites in the culture medium by LC-MS/MS showed that CCC was gradually metabolized to deoxycytidine monophosphate and deoxycytidine. Since exposure to these metabolites did not increase proliferation of NSCs, CCC itself may promote the proliferation. To evaluate the proteins involved in such proliferative activity, proteomics analysis was performed in NSCs incubated with CCC for 3 days. CCC significantly upregulated 81 proteins and downregulated 248 proteins, which were subjected to enrichment analysis in the database KEGG PATHWAY. PI3K-Akt signaling pathway, oxidative phosphorylation, and cell cycle were found to be significant enriched pathways. We performed knockdown of candidates related to these pathways by siRNA transfection. Among the candidates, transfection of siRNA for Eef1a2 significantly suppressed the CCC-induced proliferation of NSCs. Because Eef1a2 has been reported to activate PI3K-Akt signaling pathway, Eef1a2 and PI3K-Akt signaling would be one of the possible proteins and/or signaling pathway involved in CCC-induced proliferation in NSCs.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P68 デスモソーム関連タンパク質Plakophilin-2のマウス海馬神経細胞における局在と機能 Localization and function of Plakophilin-2, desmosome-related protein in mouse hippocampal neurons ^○雑賀智菜実, Akinori Takayama, Toshinori Sawano, Kenshiro Uemura, Nana Koyama, Jin Nakatani, Hidekazu Tanaka 立命館大学 ^○Chinami Saika, Akinori Takayama, Toshinori Sawano, Kenshiro Uemura, Nana Koyama, Jin Nakatani, Hidekazu Tanaka Department of Biomedical Sciences, Collage of Life Sciences, Ritsumeikan University Desmosomes are cell-cell adhesive structures with a well-known role in forming strong intercellular adhesion in tissues subject to mechanical stress, such as the heart and skin. Plakophilin-2, a member of the armadillo protein family, is a desmosomal component protein that functions to anchor other desmosomal components and intermediate filament to the desmosomal structure. There are a few reports that Plakophilin-2 is also expressed in the central nervous system, especially in the hippocampus, but desmosomes are not found in hippocampal neurons. This suggests that Plakophilin-2 in the brain may have different functions from those in the heart and skin. We started this study by confirming the detailed localization of Plakophilin-2 mRNA in the mouse hippocampus using in situ hybridization and aimed to explore the new function of Plakophilin-2 in the brain. Strong signals were detected in neuronal cell bodies in the pyramidal cell layer of the hippocampal CA1, CA2, and CA3, and weak signals were detected in neuronal cell bodies in the granular cell layer of the hippocampal dentate gyrus. Western blotting showed that Plakophilin-2 protein was also expressed in the mouse hippocampus. In addition, the detailed subcellular localization of Plakophilin-2 protein in cultured hippocampal neurons from embryonic rats will be investigated. We would like to discuss a plausible function of Plakophilin-2 in the mouse hippocampus based on the detailed localization data.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P69 自閉症原因遺伝子MED13Lの神経発達における役割と変異がもたらす病態形成メカニズムの解明 Essential role of MED13L, a responsible gene for autism spectrum disorders, in brain development ^○浜田奈々子, Koh-ichi Nagata 愛知県医療療育総合センター発達障害研究所分子病態研究部 ^○Nanako Hamada, Koh-ichi Nagata Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Developmental Disability Center The Mediator complex is a large complex of proteins that is evolutionarily conserved and ubiquitously expressed, and functions as a conserved interface between RNA polymerase II and gene-specific transcription factors. MED13L, encoded by the Mediator complex subunit 13-like gene (MED13L), is a subunit of the Mediator complex. As for human diseases, MED13L gene mutations-related intellectual disability (ID) is a new syndrome that is characterized by ID, motor developmental delay, speech impairment, hypotonia and facial dysmorphism. We here analyzed the physiological role of MED13L in the developing mouse cerebral cortex and pathophysiological significance of the gene mutations with developing mouse cerebral cortices. Acute knockdown of MED13L with in utero electroporation caused poor dendritic arbor formation of pyramidal neurons during the developmental stage. This phenotype was rescued by RNAi-resistant wild type MED13L but not by p.T2162M mutant, derived from an ID patient. Both the dendritic spine density and the ratio of mature spine were reduced in the when MED13L was silenced. Taken together, MED13L was found to play a pivotal role during corticogenesis through the regulation of dendritic development and subsequent synaptic network formation. The results obtained indicate that deficits in the MED13L function may contribute to the etiology of ID and other neurodevelopmental disorders with MED13L gene abnormalities.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P70 神経回路形成因子LOTUSはBDNFにより発現増加する Expression of LOTUS, a neural circuit formation factor, is increased by BDNF ^○松林潤平, Yuki Kawaguchi, Kohtaro Takei 横浜市立大学大学院生命医科学研究科生体機能医科学研究室竹居グループ ^○Junpei Matsubayashi, Yuki Kawaguchi, Kohtaro Takei Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science 　Neurons in the central nervous systems (CNS) undergo limited axonal regeneration after trauma and neurological disorder. Lateral olfactory tract usher substance (LOTUS) contributes to axonal tract formation in developing brain and axonal regeneration in the adult brain as an endogenous Nogo receptor-1 (NgR1) antagonist. Therefore, LOTUS is expected to be useful for future therapy of the CNS damage such as spinal cord injury and brain ischemia. However, the expression level of LOTUS is down-regulated and decreased by the CNS damage. How LOTUS expression is regulated remains to be clarified. In this study, we examined molecular mechanism of regulation in LOTUS expression and found that brain-derived neurotrophic factor (BDNF) increased LOTUS expression in cultured hippocampal neurons. First, LOTUS expression were assessed by real-time PCR and immunoblotting in cultured mouse hippocampal neurons treated with BDNF and found that exogenous application of BDNF increased LOTUS at mRNA and protein levels in a dose-dependent manner. We also found that pharmacological inhibitor of TrkB, a receptor of BDNF suppressed BDNF-induced increase of LOTUS expression. Furthermore, BDNF-induced increased LOTUS expression enhanced antagonistic action to NgR1 function in in vitro axonal regeneration assay in cultured hippocampal neurons. These findings suggest that BDNF may act as a positive regulator of LOTUS expression through TrkB and that BDNF treatment may inhibit NgR1 function by increase of LOTUS expression. Thus, BDNF may have synergistic effect on functional recovery from CNS damage through positive regulation of LOTUS expression.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P71 直接誘導法を用いた、ヒト細胞由来in vitro疾患モデルの作製とTau遺伝子変異の比較・検討 Elucidation of Tauopathy Pathogenesis With Next Generation in vitro Disease Model ^○伊津野舞佳¹, Sumihiro Maeda¹, Taro Tsujimura¹, Karch M. Celeste², Hideyuki Okano¹ 1.慶應義塾大学医学部, 2.Department of Psychiatry, Washington University in St. Louis ^○Maika Itsuno¹, Sumihiro Maeda¹, Taro Tsujimura¹, Karch M. Celeste², Hideyuki Okano¹ 1.Department of Physiology, Keio University School of Medicine, 2.Department of Psychiatry, Washington University in St. Louis Neurodegenerative disorders characterized by Tau protein accumulation in brain are called Tauopathies. This study focuses on how Tau plays its role in Tauopathy pathogenesis, for it is reported that Tau accumulation highly correlates with cognitive impairment. Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17 (FTDP-17) is a Tauopathy caused by tau gene mutations. Among these mutations, only few, including the R406W tau (MAPT)mutation, develop Alzheimer’s Disease (AD)-like features without Aβaccumulation. Innovative in vitro model of human neurons, iN (induced Neuron), is used in this study. The most prevalent way to generate neurons from human somatic cells, is to establish neurons via induced Pluripotent Stem Cells (iPSCs). However, cells are rejuvenated through the reprogramming process generating iPS cells. In contrast, iNs are directly induced neurons from somatic cells, such as fibroblasts or blood cells. This technology bypasses the reprogramming process, so the aging signatures in original cells are thought to be preserved in the generated iNs. For the effective use of directly converted neurons from human cells, and to understand how the aging process impacts on the disease pathogenesis, iNs present a unique opportunity to add a new perspective to research. iNs were generated by miRNA-based conversion, from fibroblasts of R406W mutation carriers, and non-carriers in the pedigree carrying the R406W mutation. These iNs expressed neuronal markers within two weeks. Moreover, age-dependent abnormalities in the neuronal nuclei were observed. These findings implied that the iNs induced by our method can be a model to investigate the interaction of aging and disease-causing mutations.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P72 細胞外マトリックスを含有する人工足場を用いた傷害脳組織における新生ニューロンの移動促進 Promotion of neuronal migration in injured brain using extracellular matrix-containing artificial scaffolds ^○長瀬次郎¹, Chikako Nakajima¹, Sayuri Nakamura¹, Masato Sawada^1,2, Kazunobu Sawamoto^1,2 1.名古屋市立大学神経発達・再生医学分野, 2.Division of Neural Development and Regeneration, National Institute for Physiological Sciences, National Institutes of Natural Sciences ^○Jiro Nagase¹, Chikako Nakajima¹, Sayuri Nakamura¹, Masato Sawada^1,2, Kazunobu Sawamoto^1,2 1.Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Sciences, 2.Division of Neural Development and Regeneration, National Institute for Physiological Sciences, National Institutes of Natural Sciences Newly generated neurons (neuroblasts) in the postnatal ventricular-subventricular zone (V-SVZ) are highly motile and migrate into the olfactory bulb under the physiological states. After brain insults, like cerebral infarction and ischemic stroke, neuroblasts migrate also toward the injury sites and differentiate into mature neurons. Promoting neuroblast migration toward sites of injury by optimizing intrinsic and extrinsic mechanisms leads to functional recovery in gait behavior. Brain injury is associated with gliogenesis and alteration of the local extracellular matrix components, which impedes neuroblast migration. It is thus essential to develop means to enhance neuroblast migration for better recovery from brain injuries. ECM family member proteins act as regulators of neuronal functions, like axonal guidance, axon extension, synaptic development, and maturation. However, their functions in neuroblast migration are not yet well known. In this research, we evaluated the effects of ECMs on neuroblast migration. We found that recombinant ECMs in hydrogel scaffolds promote migration of neuroblasts derived from V-SVZ explant in vitro. Further, we validated that neuroblasts increase their migration speed when in contact with the ECM-containing scaffold material. Finally, we investigated the effects of in vivo application of injectable scaffold material. As a result, the density of regenerated neuroblasts in the injured cortex was significantly higher in the treated brain than that of the control. These results suggest that ECM-containing scaffolds will be useful as a novel material to promote postnatal neuroblast migration in brain injury, which may lead to enhance functional recovery.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P73 エピジェネティック因子Trim28による胎生期マウス神経幹細胞の分化制御機構 Neural stem cell fate regulation by an epigenetic factor Trim28 during mouse brain develpment ^○中川拓海 Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu Univeristy ^○Takumi Nakagawa Stem Cell Biology and Medicine, Graduate School of Medical Sciences, Kyushu Univeristy During development of mammalian cerebral cortex, neural stem cells (NSCs) have a higher potency to self-renew in the early gestation, but gradually change their property; they differentiate into neurons in mid- and into glial cells (astrocytes and oligodendrocytes) in late-gestation. This temporal change of NSC property is regulated by epigenetic modifications, such as DNA methylation and histone modifications. We have previously shown that DNA demethylation near glial genes is prerequisite for acquisition of potential to differentiate into glial cells by NSCs. Besides DNA demethylation, we also identified developmental progression-dependent gain of DNA methylation regions (GDMRs) near neuron-related genes, however, the effects of this DNA remethylation and factors which implicated in this epigenetic change are remain unknown. In this study, using our whole genome bisulfite sequence data of directly isolated NSCs from Sox2-EGFP mice, together with public ChIP-seq database, we searched transcription factors that have potential to bind GDMRs to repress neuronal potential of NSCs. Consequently, we found that transcriptional repressor Tripartite motif-containing protein 28 (Trim28) frequently binds to the GDMRs. We confirmed that knock down of Trim28 increased pro-neuronal gene Neurog1 expression in NSCs, enhancing their neuronal differentiation. Despite our expectation, Trim28 know down didn’t change DNA methylation status in Neurog1 locus, but it increased H3K27 acetylation, a gene activation-associated histone modification. Since Trim28 is known to associate with histone deacetylase, we are currently exploring the function of Trim28 in NSCs’ fate regulation through the histone acetylation-related mechanism.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P74 成体脳海馬神経新生における ASIC1aの役割 Involvements of acid-sensing ion channel-1a in hippocampal adult neurogenesis ^○熊本奈都子, Yasuhiro Shibata, Takashi Ueda, Shinya Ugawa 名古屋市立大学医学系研究科機能組織学分野 ^○Natsuko Kumamoto, Yasuhiro Shibata, Takashi Ueda, Shinya Ugawa Department of Neurobiology and Anatomy, Nagoya City University ASIC1a(acid-sensing ion channel-1a), a proton-gated cation channel widely expressed in central neurons, postsynaptically receives extracellular protons released from synaptic vesicles, thereby modulating neurotransmission. On the other hand, it is well known that adult hippocampal neurogenesis is enhanced after ischemic brain injury accompanied by local tissue acidosis. We hypothesized that proton might regulate the neurogenesis through the activation of ASIC1a, and subsequently identified ASIC1a transcripts in mouse hippocampal newborn neurons from early stages of the neurogenesis. Interestingly, BrdU (bromodeoxyuridine) assay revealed higher proliferation rate of neural stem/precursor cells (NPCs) in ASIC1a knockout (KO) mice, whereas the number of surviving cells 28 days post-injection (dpi) of BrdU was decreased in the mice. These data suggest that ASIC1a exerts a negative role toward cell proliferation, or impaired survival of newborn neurons in the KO mice conversely enhances proliferation of NPCs to compensates for the shortfall. To clarify when the number of BrdU-positive (BrdU+) cells starts to decline, we quantified the numbers of BrdU+ cells at 14 dpi and 21 dpi in the KO mice. However, the differences in the numbers between the KO and WT mice at both dpis were not statistically significant. Next, we evaluated the number of apoptotic cells in the KO hippocampus by counting the number of cells expressing cleaved caspase-3 and found that the KO mice exhibited marginal increase in cell death. Our results indicate the possibility that ASIC1a would be necessary for the long-term survival of newly generated neurons in the adult hippocampus.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P75 ヘパラン硫酸プロテオグリカンシンデカン-3はマウス小脳顆粒前駆細胞の細胞周期脱出を制御する Heparan sulfate proteoglycan syndecan-3 regulates the cell cycle exit of mouse cerebellar granule progenitor cells ^○渡邉雛¹, Natsumi Ikeda¹, Ayaka Fukuda², Kei Hashimoto³, Yasunori Miyamoto^1,2,4 1.お茶の水女子大学大学院　人間文化創成科学研究科　ライフサイエンス専攻, 2.Department of Biology, Ochanomizu University, 3.Academic Production, Ochanomizu University, 4.Institute for Human Life Innovation, Ochanomizu University ^○Hiina Watanabe¹, Natsumi Ikeda¹, Ayaka Fukuda², Kei Hashimoto³, Yasunori Miyamoto^1,2,4 1.Graduate School of Humanities and Sciences, Ochanomizu University, 2.Department of Biology, Ochanomizu University, 3.Academic Production, Ochanomizu University, 4.Institute for Human Life Innovation, Ochanomizu University In the developing mouse cerebellum, the cell cycling of cerebellar granule precursor cells (CGCPs) is regulated tempo-spatially. CGCPs proliferate in the outer granular layer (EGL) on the surface and exit the cell cycle in the inner EGL. After that, the GCGPs complete the differentiation in the inner granule layer (IGL). Our previous study indicated that syndecan-3 (Syn3), a heparan sulfate proteoglycan, is expressed in the developing CGCPs in the inner EG and is involved in the cell cycling of CGCPs. However, how Syn3 regulates the cell cycling of CGCP remains largely unclear. In this study, we aimed to clarify the effect of Syn3 on the cell cycling of CGCPs by the knockdown (KD) and overexpression (over) of Syn3. First, in the 3, 4, and 5-day primary cultured CGCPs, the effects of KD and over of Syn3 on the expression of a regulator of cell cycle exit, p27Kip1 were analyzed. The Syn over significantly increased the ratio of p27Kip1 positive cells, and the Syn KD significantly reduced that in 3-day culture. Next, we examined the effect of Syn on the cell cycle exit using immunostaining for a 24 h-labelled BrdU and Ki67. As a result, the Syn KD significantly increased the efficiency of cell cycle exit of CGCP, and the Syn over significantly suppressed that in 4-day and 5-day culture. These results indicated that Syn3 regulates the cell cycle exit of CGCP. In addition, we examined the ligand candidate of Syn3, and focused on pleiotrophin (PTN), which has been shown to bind to the heparan sulfate chain of Syn3 and activate it. When PTN was added to the primary cultures of CGCPs, Ki67 and BrdU staining showed an increase of the percentage of Ki67-negative and BrdU-positive cells. This result suggests that PTN may be served as a ligand for Syns in CGCPs during development.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P76 新規中枢神経再生因子の探索 Identification of the novel factor that regulate central nervous system regeneration ^○樋口京香¹, Shogo Tanabe¹, Minoru Narita^2,4, Rieko Muramatsu¹ 1.国立精神神経医療研究センター　神経研究所　神経薬理研究部, 2.Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 3.Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 4.Division of Cancer Pathophysiology, National Cancer Center Research Institute ^○Kyoka Higuchi¹, Shogo Tanabe¹, Minoru Narita^2,4, Rieko Muramatsu¹ 1.Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 2.Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, 3.Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 4.Division of Cancer Pathophysiology, National Cancer Center Research Institute Spinal cord injury (SCI) induces devastating and persistent neurological deficits due to the disconnection of neural networks. Although, in general, the regeneration of the neural networks is limited in the central nervous system (CNS), recent studies revealed that slight but spontaneous repair of neural networks have been observed in the adult CNS. As this compensatory neural network contributes to the attenuation of neurological deficits after SCI, promotion of the formation of compensatory neural networks could be an effective therapeutic strategy. However, the molecular mechanism mediating the spontaneous formation of compensatory neural networks has not been fully understood. In this study, we explored the novel factor which mediates the formation of compensatory neural networks using databases and in vitro screening system. To identify the factor promoting the formation of compensatory neural networks, we extracted 20 candidate genes, which are associated with neurological dysfunctions and are highly expressed in neurons of CNS. We performed neurite outgrowth assay using siRNA library, and found that synaptotagmin 4 (Syt4) is involved in neurite outgrowth of mouse cortical neuron in vitro. We next examined whether Syt4 mediates the formation of compensatory neural networks in vivo. We suppressed Syt4 expression in the motor area by injecting adeno-associated virus coding shRNA against Syt4, and assessed whether Syt4 is involved in repair of injured neural networks and attenuation of neurological deficits. In this presentation, we will show an update about the result of the role of Syt4 on the neural networks regeneration after SCI.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P77 ケタミン代謝産物による持続的な抗うつ作用の分子神経メカニズム Molecular and neural mechanisms underlying the sustained antidepressant actions of ketamine metabolites ^○九野(川竹) 絢子, Erina Ishimori, Haiyan Li, Yusuke Sakai, Yuki Funayama, Hiromichi Inaba, Naoya Oishi, Toshiya Murai, Shusaku Uchida 京都大学 ^○Ayako Kawatake-, Erina Ishimori, Haiyan Li, Yusuke Sakai, Yuki Funayama, Hiromichi Inaba, Naoya Oishi, Toshiya Murai, Shusaku Uchida Kyoto University There is a growing body of evidence indicating that ketamine shows rapid and long-lasting antidepressant effects. Neurobiological insights into ketamine efficacy shed new light on the mechanisms underlying antidepressant efficacy. However, mechanisms underlying the sustained antidepressant-like effects remain unclear. Elucidating this mechanism is the key to finding new therapeutic targets and developing therapeutic patterns. Of note, the metabolism of (R, S)-ketamine to (2R, 6R)- or (2S, 6S)-hydroxynorketamine (R-HNK or S-HNK) has been demonstrated to be essential for the antidepressant effects. However, the antidepressant effects of HNK enantiomers and their underlying mechanism remain to be elusive. Here we characterized behavioral effects of S-HNK in mice and investigated neural and molecular mechanisms related to the long-lasting actions by S-HNK. We found that S-HNK has rapid and sustained antidepressant effects in C57BL/6 mice, as assessed by tail-suspension test. The antidepressant-like behaviors by S-HNK treatment were also observed in BALB/c mice subjected to social defeat stress. Importantly, S-HNK did not exert hyperactivity and motor impairment in mice, suggesting low side effects. We also found that S-HNK treatment upregulates cFos expression in the medial prefrontal cortex, paraventricular thalamus, amygdala, and nucleus accumbens. At the molecular level, S-HNK treatment altered DNA methylation levels at the particular genes involved in synaptic plasticity. Together, our results suggest that the final metabolite of ketamine, S-HNK has sustained antidepressant effects at least in part by the epigenetic modifications of plasticity-related genes.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P78 hnRNPA3とともにGGGGCCリピートRNA分解促進に関与する分子の網羅的同定 Identification of molecules interacting with hnRNPA3, a factor promoting C9orf72 repeat RNA degradation in FTLD/ALS. ^○魚住亮太, Kohji Mori, Tomoko Yamashita, Shiho Gotoh, Tesshin Miyamoto, Manabu Ikeda Department of Psychiatry, Osaka University Graduate School of Medicine ^○Ryota Uozumi, Kohji Mori, Tomoko Yamashita, Shiho Gotoh, Tesshin Miyamoto, Manabu Ikeda Department of Psychiatry, Osaka University Graduate School of Medicine Intronic GGGGCC repeat expansion in C9orf72 is a most frequent genetic cause of familial frontotemporal lobar degeneration (FTLD)/amyotrophic lateral sclerosis (ALS). Neurodegeneration in C9orf72 FTLD/ALS is assumed to be caused by the toxicities of transcribed repeat RNA itself and its repeat-associated non-AUG (RAN) translation products, dipeptide repeat protein (DPR). hnRNPA3 is a heterogeneous nuclear ribonucleoprotein (hnRNP) identified as a GGGGCC repeat RNA binding factor. Although hnRNPA3 itself does not have apparent RNA degrading ability, increased hnRNPA3 expression led to a reduction of repeat RNA and DPR production, while reduction of hnRNPA3 induced accumulation of repeat RNA. Thus, hnRNPA3 promotes the degradation of GGGGCC repeat RNA. However, the exact mechanism by which hnRNPA3 promotes repeat RNA degradation is unclear. Liquid-liquid phase separation (LLPS) is mediated through weak and dynamic electrostatic and/or hydrophobic interactions between molecules. Since most hnRNPs including hnRNPA3 are known to undergo LLPS, we hypothesized that loose binding of hnRNPA3 with factors potentially involved in RNA degradation may facilitate hnRNPA3-mediated RNA degradation. To mark and purify loose binding factors of hnRNPA3, we introduced a state-of-art APEX2 technology which allows selective biotin labeling of proximate molecules. Biotinylated proteins in hnRNPA3-APEX2 expressing cells were purified with streptavidin pull-down for subsequent LC-MS/MS analysis. As a result, several candidate proteins were identified. This study is an attempt to elucidate the pathogenic repeat RNA degradation pathway in C9orf72 FTLD/ALS, and our results could be applied for the development of future FTLD/ALS treatment.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P79 LPS投与による発達神経毒性はエピジェネティック変異から遅発性の神経死に至る Developmental neurotoxicity due to prenatal LPS administration progresses from epigenetic alteration to delayed neuronal cell death ^○シャルマディヴェール¹, Haruko Ohtsuka¹, Kazunobu Tsunemoto², Thomas Tiong Kwong Soon¹, Yasunari Kanda², Sachiko Yoshida¹ 1.豊橋技術科学大学　吉田祥子　研究室, 2.National Institute of Health Sciences, Kawasaki, Japan ^○SHARUMADHI VELOO¹, Haruko Ohtsuka¹, Kazunobu Tsunemoto², Thomas Tiong Kwong Soon¹, Yasunari Kanda², Sachiko Yoshida¹ 1.Toyohashi University of Technology, Toyohashi, Japan, 2.National Institute of Health Sciences, Kawasaki, Japan Lipopolysaccharide (LPS), which is present in the outer membrane of Gram-negative bacteria, is a well-known pro-inflammatory factor via binding to TLR 4 and may cause some mental disorders. We have observed the developmental cerebellar degeneration and behavior of LPS-administrated rat (LPS-rat) offspring and discussed its pathology. Neonatal cerebellum of LPS- rat offspring showed high-level expression of inflammatory cytokines, TNFalpha, and IL-1 beta. Initially, the cytokine expression would be expected to induce neuronal cell death in offspring; however, the decrease of Purkinje cells appeared at postnatal day 14 (P14), which was not observed at P7. Moreover, the LPS-rat cerebellum showed excessive folding of cerebellar V and VI lobes, as same as the alteration in HDAC inhibitor-administrated animals (HDACi-rat). In this study, we observed the epigenetic changes in LPS-rat offspring. H3K9me expression was decreased in the molecular layer at P7, and GFAP expressed higher in LPS-rat than in control animals. H3K9me or tri-me are known as efficient histone methylation deeply related to astrocyte development. Because H3K9me decrease was also observed in HDACi-administrated rat cerebellum, and both cerebella showed excessive folding of cerebellar V and VI lobes at P7, early astrocytes or Bergmann glia development would induce developmental alteration of lobule folding both in LPS-rat and HDACi-rat. On the other hand, the number of Purkinje cells in LPS-rat showed a decrease at P14, as opposed to them in HDACi-rat. Additionally, microglia was increased rapidly from P7 to P14 in the LPS-rat cerebellum. These data suggest that developmental neurotoxicity due to LPS administration would have two phases; epigenetic alteration and delayed neuronal cell death.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P80 オキシトーシス/フェロトーシスを阻害し後期エンドソームおよびリソソームを標的とする新規神経保護化合物N,N-ジメチルアニリン誘導体の同定 Identification of novel neuroprotective N,N-dimethylaniline derivatives targeting to late endosomes/lysosomes that prevent oxytosis/ferroptosis ^○髙橋麻由¹, Yoshiyuki Tsunekawa¹, Kentaro Oh-hashi^1,2, Kyoka Kawaguchi¹, Masumi Hayazaki¹, Miyu Watanabe¹, Hiroshi Takemori^1,2, Kyoji Furuta^1,2, Yoko Hirata^1,2 1.Graduate School of Natural Science and Technology, Gifu University, 2.United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University ^○Mayu Takahashi¹, Yoshiyuki Tsunekawa¹, Kentaro Oh-hashi^1,2, Kyoka Kawaguchi¹, Masumi Hayazaki¹, Miyu Watanabe¹, Hiroshi Takemori^1,2, Kyoji Furuta^1,2, Yoko Hirata^1,2 1.Graduate School of Natural Science and Technology, Gifu University, 2.United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University Oxidative stress has been implicated in the aging process and the progression of many neurodegenerative disorders. Currently, there are no treatments to cure neurodegenerative diseases. We previously reported that a novel oxindole compound, GIF-0726-r, effectively prevents endogenous oxidative stress, such as oxytosis/ferroptosis. Oxytosis/ferroptosis is a form of oxidative stress-induced programed cell death which is iron-dependent and different from apoptosis, necrosis, autophagy. In this study using two hundred compounds that were developed based on the structure-activity relationship of GIF-0726-r, we screened for the most potent compounds that prevent oxytosis/ferroptosis. Using submicromolar concentrations, we identified nine neuroprotective compounds that have N,N-dimethylaniline as a common structure but no longer contain an oxindole ring. The most potent derivatives, GIF-2114 and GIF-2197-r, did not affect glutathione levels, had no antioxidant activity in vitro, or ability to activate the Nrf2 pathway, but prevented oxytosis/ferroptosis via reducing reactive oxygen production and reducing ferrous ions. Furthermore, we developed fluorescent probes of GIF-2114 and GIF-2197-r to image their distribution in live cells and found that they preferentially accumulated in late endosomes/lysosomes, which play a central role in iron metabolism. These unique characteristics of N,N-dimethylaniline derivatives allow them to be candidates for further development as therapeutic interventions for oxidative stress-related diseases. Furthermore, the fluorescent probes GIF-2264 and GIF-2250-r can be useful tools to image late endosomes/lysosomes, as well as extracellular vesicles, such as exosomes, because the fluorescence of the compound itself is quenched in the culture medium.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P81 オキシインドール-クルクミンハイブリッド化合物のマウス海馬由来HT22細胞における神経保護作用 Neuroprotective effects of the oxindole-curcumin hybrid compound in mouse hippocampal HT22 cells ^○佐藤美夏¹, Takanori Ikawa², Kentaro Oh-hashi^1,2, Kyoji Furuta^1,2, Yoko Hirata^1,2 1.Graduate School of Natural Science and Technology, Gifu University, 2.United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University ^○Mina Sato¹, Takanori Ikawa², Kentaro Oh-hashi^1,2, Kyoji Furuta^1,2, Yoko Hirata^1,2 1.Graduate School of Natural Science and Technology, Gifu University, 2.United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University The oxindole compound GIF-2165X-G1 is a hybrid molecule composed of the oxindole skeleton of the neuroprotective compound GIF-0726-r and the polyphenolic skeleton of the antioxidant curcumin. In the previous study, we investigated the effect of GIF-2165X-G1 on endogenous oxidative stress in mouse hippocampal HT22 cells in comparison with GIF-0726-r and curcumin. GIF-2165X-G1 exhibited more potent neuroprotective activity than GIF-0726-r or curcumin and had less cytotoxicity than that observed with curcumin. Both GIF-0726-r and GIF-2165X-G1 were found to activate the Nrf2 pathway, the major cellular pathway that regulates the antioxidant defense system, and have ferrous ion chelating activity similar to that exhibited by curcumin. These results demonstrate that the introduction of the polyphenol skeleton of curcumin to the oxindole GIF-0726-r improves neuroprotective features. In this study, we further characterized the effect of GIF-2165X-G1 on γ-glutamylcysteine ligase (GCL) that belongs to phase II enzymes. GCL comprises catalytic (GCLC) and regulatory subunits and catalyzes the rate-limiting step in de novo GSH synthesis. GIF-2165X-G1 increased GCLC promoter activity, mRNA, and protein levels in HT22 cells. To analyze the mechanism of GCLC transcriptional activation, we cloned and characterized a 5 kb fragment of the 5´-franking promoter region of the mouse GCLC gene. Although GIF-2165X-G1 potently induced antioxidant response element (ARE)-driven transcription, the compound increased GCLC transcriptional activity through Sp1 pathway in a Keap1–Nrf2–ARE-independent manner. These results suggest that GIF-2165X-G1 itself and further modification of the compound are useful interventions for promoting neuronal survival by augmenting resistance to oxidative stress.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P82 外傷性脳損傷修復におけるビトロネクチンの神経保護作用 Neuroprotective effects of vitronectin in recovering from traumatic brain injury. ^○山下みのり¹, Kei Hashimoto³, Mari Nakashima^1,2, Hiroko Ikeshima-Kataoka⁴, Yasunori Miyamoto^1,2 1.お茶の水女子大学人間文化創成科学研究科ライフサイエンス専攻生命科学コース博士前期課程2年, 2.Graduate School of Humanities and Sciences, Ochanomizu University, 3.Academic Production, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University ^○Minori Yamashita¹, Kei Hashimoto³, Mari Nakashima^1,2, Hiroko Ikeshima-Kataoka⁴, Yasunori Miyamoto^1,2 1.Institute for Human Life Innovation, Ochanomizu University, 2.Graduate School of Humanities and Sciences, Ochanomizu University, 3.Academic Production, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University Traumatic brain injury (TBI), which is caused by physical trauma, induces　neuronal cell death and inflammation. Vitronectin (VN), one of cell adhesion proteins, plays roles in blood coagulation, fibrinolysis, and neuronal death in nervous and repairing systems. In the previous our study, it is reported that VN suppresses hemorrhage in TBI. However, the effect of VN on neuronal cell death has not clarified. We examined the effects of VN on neuroprotection from neuronal cell death and its underlying mechanism. Stab-wounded cerebral cortexes of 6-week old VN-KO mice were co-immunostained for activated caspase3 and NeuN. As a result, the percentage of apoptotic cells in the wounded regions of VN-KO mice was higher than that of wild-type mice, suggesting that VN suppresses the neuronal apoptosis in wounded region. Next, to investigate the underlying mechanism, we analyzed the effects of VN on the activation of astrocytes, which contribute to neuroprotection. The immunostaining level of GFAP, a marker of astrocyte activity, around the lesion was increased in VN-KO mice. Moreover, we analyzed the effect of VN and lipopolysaccharide (LPS) on the mRNA expression levels of proinflammatory cytokines in a primary culture of astrocytes. VN reduced the mRNA expression level of IL-1β in LPS-treated astrocytes. Next, the astrocyte conditioned　medium (ACM) was added to the primary cultured neurons and the state of cell death was confirmed by immunoreactivity for activated caspase3. The ACM from VN and LPS-treated astrocytes significantly reduced the neuronal cell death, compared with that of LPS-treated cells These results suggest that VN　suppresses the expression level of IL-1β, which is involved in neurotoxicity cytokines via astrocytes and has a neuroprotective effect.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P83 ショウジョウバエにおける老化依存的なカスパーゼ活性を制御するカスパーゼ近傍タンパク質の解析 Analysis of proximal proteins of caspases that are responsible for age-dependent activation in Drosophila ^○村本雅哉, Nomomi Hanawa, Natsuki Shinoda, Masayuki Miura Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo ^○Masaya Muramoto, Nomomi Hanawa, Natsuki Shinoda, Masayuki Miura Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo Caspases are cysteine proteases that play a central role in cell death. In the olfactory receptor neuron of Drosophila, we have previously showed that nerve cell death is caused during aging associated with caspase activation. On the other hand, caspases are known to have non-apoptotic functions in nervous system. Caspases are involved in the regulation of synaptic vesicles volume at presynaptic terminals and long-term depression at postsynaptic terminals. As caspase activation regulatory mechanisms, not only well-known caspase inhibitor, Diap1, but also Tango7 known as eIF3 subunit controls the degree of caspase activation. This suggests that the microenvironment surrounding the caspases is involved in its activation, and proximal proteins of caspases are putative activation regulators. We decided to use TurboID, which biotinylates proximal proteins, as a labeling tool for proteins in the vicinity of caspases. We fused TurboID with Drice, a Drosophila caspase, tagged by V5 and we used its knock-in line to comprehensively analyze neighboring proteins of Drice. We first observed the endogenous expression pattern of Drice in the adult brain with V5 antibody and Streptavidin. In the adult brain, Drice was highly expressed in several specific types of neuron including olfactory receptor neuron. We labeled proximal proteins by TurboID-mediated biotinylation and identified proteins by NeutrAvidin purification followed by mass spectrometry. It was found that many proximal proteins exist near the plasma membrane by GO analysis. Moreover, genetic manipulation of some of the proximal proteins alters age-dependent caspase activation patterns. Our results suggest that changes in the proximal proteins regulate age-dependent caspase activation in adult brain.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P84 オルガネラストレスはAβペプチド合成の変化およびCaspase3依存性アポトーシスを引き起こすーケミカルシャペロンの神経細胞保護効果の検討ー Organelle stress alters Aβ peptide production and induces Caspase3-dependent apoptosis -Possible protective effect of chemical chaperone in neuronal cells- ^○須賀圭¹, Sachiko Yamamoto-Hijikata¹, Yasuo Terao¹, Makoto Ushimaru¹ 1.杏林大学医学部化学教室, 2.Department of Medical Physiology, Faculty of Medicine, Kyorin University ^○Kei Suga¹, Sachiko Yamamoto-Hijikata¹, Yasuo Terao¹, Makoto Ushimaru¹ 1.Department of Chemistry, Faculty of Medicine, Kyorin University, 2.Department of Medical Physiology, Faculty of Medicine, Kyorin University Involvement of the dysfunction of neuronal cells caused by Golgi stress in neurodegenerative diseases such as Alzheimer’s disease (AD) is poorly understood. We have been focusing on the function of ER-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) in βAPP processing under cellular stress. We previously showed that ER and Golgi stress upregulate de novo synthesis of ER-Golgi SNAREs Syntaxin5 (Syx5), and the reduction of β-amyloid peptide (Aβ peptide) in neuronal cells. Conversely, down regulation of Syx5 protein was observed upon apoptosis which was due to the degradation by activated caspase-3. In addition, we reported that chemical chaperone 4-phenylbutyrate (4PBA) showed alleviation of caspase3-dependent apoptosis induced by ER stress. Last year, we reported that various Golgi stress inducers also cause upregulation of ER-Golgi SNAREs Syx5, reduction of Aβ peptide secretion, and the caspase3-dependent apoptosis. We have been utilizing 4PBA to see whether the compound have protective effect on altered processing of βAPP under ER stress and also on the subsequent apoptotic cell death caused by sustained ER stress. However, we did not know whether 4PBA is also effective in cells treated with Golgi stress inducers. In order to understand the protective mechanism of 4PBA on the changes in the processing of βAPP under Golgi stress condition, the amount of secreted Aβ, intracellular Aβ, and the ratio of Aβ peptides were examined in the presence of 4PBA. We performed time lapse imaging, western blotting, and sandwich ELISA analyses in Golgi stress-induced cells. We will present these data and would like to discuss the results.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P85 【演題取消】		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P86 胎生期慢性グリホサート曝露がもたらす二相性の発達神経毒性 Biphasic Neurodevelopmental toxicity of low-dose chronic Glyphosate exposure in utero ^○佐竹繁寿¹, Ken Futagami¹, Thomas Tiong Kwong Soon¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.生命機能科学研究室, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences ^○Shigehisa Satake¹, Ken Futagami¹, Thomas Tiong Kwong Soon¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1.Toyohashi University of Technology, 2.Queens College, the City University of New York, 3.National Institute of Health Sciences Glyphosate, an active ingredient in the roundup, is mainly detected in genetically modified food which humans are exposed to on a daily basis. It’s function as a herbicide is to inhibit 5-enolpyruvylsikimate-3-phospate synthase (EPSPS) in the shikimic acid pathway, which inhibits aromatic amino acids in plants. This shikimic acid pathway is also present in intestinal bacteria in animals, and in recent years, ASD and neurological disorders have been reported due to glyphosate exposure in the embryonic stage. Prior studies have confirmed defects and inconsistencies in Purkinje cells by acute exposure of glyphosate to pregnant rats at a concentration of 250 mg/kg. In addition, glyphosate-administrated offspring showed ASD-like behavior. In this study, we investigated the effects of several dosages of chronic GLY exposure on the cerebellar cortex in immature offspring. After exposing pregnant rats to GLY-water 0.1 mg/kg/day（total dosage is 2.2 mg/kg, dose A) , 1 mg/kg/day（total dosage is 22 mg/kg, dose B) and 9.1 mg/kg/day（total dosage is 200 mg/kg, dose C) from gestational day 2 (G2) to G23, we observed cerebellar development of the male offspring. Offspring of dose C showed inconsistencies in Purkinje cells as similar as acute high-dose exposure one. Additionally, the growth of offspring was more suppressed than the acute-exposed one so that the GLY-chronic-exposure would have remarkable developmental neurotoxicity. Offspring of dose B showed, conversely, depression of programmed Purkinje cell death. The lowest dose of them, dose A offspring, were observed normally. GLY-chronic exposure caused significant changes in the number of Purkinje cells in the developing cerebellum. These data suggest glyphosate would have biphasic developmental neurotoxicity with dosage.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P87 ブラジル産グリーンプロポリスの主要成分アルテピリンCによる小胞体ストレス及びタンパク質凝集の阻害 Selective inhibition of endoplasmic reticulum stress and protein aggregation by artepillin C, a major component of Brazilian green propolis ^○本山将成, Shintaro Kimura, Madoka Takashima, Takanori Ikawa, Kentaro Oh-hashi, Yuji O Kamatari, Yoko Hirata Department of Life Science and Technology, Graduate School of Natural Science and Technology, Gifu University ^○Masanari Motoyama, Shintaro Kimura, Madoka Takashima, Takanori Ikawa, Kentaro Oh-hashi, Yuji O Kamatari, Yoko Hirata Department of Life Science and Technology, Graduate School of Natural Science and Technology, Gifu University Propolis is a sticky substance made by honeybees. Propolis has been used in many foods and beverages to improve health and prevent diseases by its antibacterial activity. We previously reported that the ethanol extracts of Brazilian green propolis, and its constituents artepillin C, kaempferide, and kaempferol, mitigate oxidative stress-induced cell death via oxytosis/ferroptosis. The flavonoid derivatives kaempferide and kaempferol are antioxidants with radical-scavenging abilities that additionally induce antioxidant response element (ARE)-mediated transcriptional activity. In contrast, phenylated cinnamic acid derivative artepillin C attenuates reactive oxygen species production in a Nrf2/AREindependent manner. Here, we investigated the potential of Brazilian green propolis and its constituents to protect against endoplasmic reticulum (ER) stress in the mouse hippocampal cell line HT22. Ethanol extracts of Brazilian green propolis, artepillin C, and kaempferol attenuated tunicamycininduced cell death and expression of ER stress markers including GADD153 and the spliced form of XBP1. We also found that artepillin C and kaempferol prevented tunicamycin-induced protein aggregation in HT22 cells. Further, artepillin C inhibited the aggregation of mutant canine superoxide dismutase 1 expressed in Neuro2a cells, suggesting that artepillin C possesses a chemical chaperone-like function. These findings indicate that, in addition to oxidative stress, the ethanol extracts of Brazilian green propolis might help to prevent ER stress-related neuronal cell death proposed to be involved in the pathologies of several neurodegenerative diseases. Thus, these results confirm the beneficial characteristics of Brazilian green propolis as a valuable botanical resource.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P88 GM1結合性環状ペプチドによるアミロイドβ凝集阻害メカニズムの解析 The analysis of inhibition mechanism of Aβ assembly by a GM1-binding cyclic peptide ^○宮本恵里花, Mako Nakai, Masaya Nishihara, Teruhiko Matsubara, Toshinori Sato Faculty of Science and Technology, Keio University ^○Erika Miyamoto, Mako Nakai, Masaya Nishihara, Teruhiko Matsubara, Toshinori Sato Faculty of Science and Technology, Keio University One of the characteristics found in the Alzheimer’s disease (AD) patient's brain is a senile plaque formed by the abnormal assembly of amyloid β protein (Aβ). It has been reported that neurotoxic Aβ assemblies are induced by the monosialoganglioside GM1-cluster formed on neuronal membranes. Therefore, we focused on the GM1-cluster as a novel target for inhibition of Aβ aggregation. We have investigated a GM1-binding peptide by a phage-displayed library. However, peptide has low metabolic stability and need to be restricted its conformational space. Therefore, we designed GM1-binding cyclic peptide and it was found that the peptide can inhibit the formation of Aβ assembly induced on GM1-containing lipid bilayer. In this study, we aimed to further clarify the detail of the inhibition mechanism. A GM1-containing lipid membrane (GM1/sphingomyelin/cholesterol) was prepared as a model of neuronal membrane, and the surface topography of the membrane incubated with Aβ to observe the Aβ assembly was analyzed by atomic force microscopy (AFM). The GM1-binding cyclic peptide showed effective inhibitory activity for GM1-induced Aβ assemblies compared to the liner one. Kinetic study by surface plasmon resonance (SPR) method indicated that affinity of the cyclic peptide was same as the liner one. These results suggest that the GM1-binding cyclic peptide can inhibit the formation of Aβ assemblies by not only binding to GM1 cluster but also some other interaction. The GM1-cluster on neuronal membrane can be a novel target for the development of therapeutic agents against AD.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P89 ミトコンドリア局在性タンパク質p13欠損マウスの生後早期の致死性 Early postnatal mortality in mice lacking mitochondria-localized protein p13 ^○松尾若奈¹, 植野寛貴¹, 原さとみ¹, 馬場優志¹, 岩田圭子², 松崎秀夫², 橋本均^1,3,4,5,6, 新谷紀人¹ 1.大阪大学薬学部　神経薬理学分野, 2.United Graduate School of Child Development, Fukui University., 3.United Graduate School of Child Development, Osaka University., 4.Institute of Datability Sciences, Osaka University., 5.Open Tarnsdisciplinary Research Initiative, Osaka University., 6.Department of Molecular Pharmaceutical Sciences, Graduate School of Medicice, Osaka University ^○Wakana Matsuo¹, 寛貴植野¹, 原さとみ¹, 馬場優志¹, 岩田圭子², 秀夫松崎², 橋本均^1,3,4,5,6, 新谷紀人¹ 1.大阪大学薬学部神経薬理学分野, 2.United Graduate School of Child Development, Fukui University., 3.United Graduate School of Child Development, Osaka University., 4.Institute of Datability Sciences, Osaka University., 5.Open Tarnsdisciplinary Research Initiative, Osaka University., 6.Department of Molecular Pharmaceutical Sciences, Graduate School of Medicice, Osaka University Mitochondria-localized protein with molecular weight 13 kDa (p13) is widely expressed in central and peripheral tissues. We previously generated mice lacking p13 with C57/BL6 genetic background (p13-KO/B6 mice) and found a significant loss of p13-KO/B6 pups during the early postnatal period. Here, we investigated the possible mechanisms underlying the postnatal mortality of p13-KO/B6 mice. Kaplan-Meier survival analysis demonstrated that more than half of p13-KO/B6 mice died during the first 2 postnatal days. At P0, we observed presence of milk in the stomachs of p13-KO/B6 pups. However, their body weight and blood glucose levels were significantly lower than those of wild-type littermates. Meanwhile, we observed that more than half of p13-KO mice survived the first two days under an ICR/CD1 genetic background. Taken together, these results suggest that p13 contributes to early postnatal survival and maintenance of the normal blood glucose level, which could be modulated by their genetic background.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P90 Pin1 KO増加するアミロイド陽性沈着物の特性 Characterization of amyloid positive deposit in Pin1 KO mice ^○大滝博和¹, Takafumi Uchida², Akira Yoshikawa^3,4, Akiyoshi Kakita⁵, Kenjiro Ono⁶, Kazuho Honda¹ 1.昭和大学・医・顕微解剖学, 2.Tokyo Kasei University, 3.Department of Physiology, Showa University School of Medicine, 4.Showa University School of Nursing and Rehabilitation Sciences, 5.Departments of Pathology, Brain Research Institute, Niigata University, 6.Department of Neurology, Showa University School of Medicine ^○Hirokazu Ohtaki¹, Takafumi Uchida², Akira Yoshikawa^3,4, Akiyoshi Kakita⁵, Kenjiro Ono⁶, Kazuho Honda¹ 1.Department of Anatomy, Showa University School of Medicine, 2.Tokyo Kasei University, 3.Department of Physiology, Showa University School of Medicine, 4.Showa University School of Nursing and Rehabilitation Sciences, 5.Departments of Pathology, Brain Research Institute, Niigata University, 6.Department of Neurology, Showa University School of Medicine Pin1 is a ubiquitous peptidyl-prolyl cis/trans isomerase (PPIase) and has been shown to contribute to amyloid and tau metabolism. We determined recently the Pin1 (-/-) mice increased amyloid immunoreaction in the thalamus. However, the characteristics of them are still unclear. In present study, we determined the thalamic amyloid deposit in detail. Aged pin1 (-/-) and the wild type mice were obtained by breeding from the heterozygous gene deficient (+/-) mice. The brains (150 – 600 days) were removed after 10% formalin perfusion and was prepared paraffin embedding sections. The brain sections were observed morphological staining, and immunostaining against antibodies for beta-amyloid and some neural markers. Moreover, the brain was observed by transmission electron microscopy (TEM). Staining with a beta-amyloid antibody against 1-14 aa, the immunepositive reactions stains were recognized in thalamic nucleus, but not in hippocampus and cortex. The positive reactions were located next to nucleus and were merged with neural marker, NeuN. The positive reactions were recognized with both wild and Pin1 (-/-) mice. Quantified the positive number and volume under microscopy, the positive number increased with aging although the number was not different in both animals. However, the positive volume was significantly larger in the Pin1 (-/-) mice. With TEM observation, there were the regularly arranged fibers in cytoplasm. The inclusion were eosinophilic with HE, no toluidine blue, no cresyl violet, and no representative amyloid staining such as Congo red and Direct Fast Scarlet 4BS. However, they weakly stained with Luxol fast blue. These were consisted with eosinophilic thalamic inclusion in myotonic dystrophy patient.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P91 ニュートラルレッド色素によるマウス末梢神経系脱髄部位の肉眼的検出 Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye ^○山崎礼二, Yasuyuki Osanai, Tom Kouki, Yoshiaki Shinohara, Nobuhiko Ohno 自治医科大学医学部解剖学講座組織学部門 ^○Reiji Yamazaki, Yasuyuki Osanai, Tom Kouki, Yoshiaki Shinohara, Nobuhiko Ohno Department of Anatomy, Division of Histology and Cell Biology, School of Medicine, Jichi Medical University Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, we have developed a novel method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal (i.p) injection of neutral red (NR) dye. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. During subsequent remyelination between 7 dpl to 21 dpl, NR labeled lesion was gradually decreased. NR labeled activated macrophages and Schwann cells in the PNS lesions. Furthermore, electron microscopy (EM) analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. In EM analysis, Schwann cells occasionally contained myelin debris, suggesting Schwann cells involved in phagocytosis and clearance of myelin debris. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection. We propose that this method could be applied to clarify the molecular mechanisms of PNS remyelination and used in future drug screens for promoting PNS remyelination.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P92 微小管結合蛋白質Tauは、成熟オリゴデンドロサイトで発現し脱髄でその発現が亢進する。 Microtubule-binding protein Tau is expressed in mature oligodendrocytes and is up-regulated during demyelination ^○鳥居知宏¹, Yuki Miyamoto², Tomohiro Miyasaka³, Rinaho Nakata¹, Hiroaki Misonou¹ 1.Graduate School of Brain Science, Doshisha University, 2.Department of Pharmacology, National Research Institute for Child Health and Development., 3.Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University ^○Tomohiro Torii¹, Yuki Miyamoto², Tomohiro Miyasaka³, Rinaho Nakata¹, Hiroaki Misonou¹ 1.Graduate School of Brain Science, Doshisha University, 2.Department of Pharmacology, National Research Institute for Child Health and Development., 3.Department of Neuropathology, Faculty of Life and Medical Sciences, Doshisha University Microtubule binding protein Tau is mostly expressed and localized in the axons of neurons. Since we have reported Tau is expressed in Olig2-positive oligodendrocytes (OLs) as well as neurons, but not astrocytes and microglia, we investigated the expression of Tau in oligodendrocytes during their development with immunostaining using markers of oligodendrocyte, PDGFR alpha, SOX10, GPR17, CC1(APC), and MBP. This study showed that Tau was not detectable in PDGFR alpha-positive oligodendrocyte progenitor cells (OPCs) and GPR17-positive pre-oligodendrocytes. However, Tau antibody labeled CC1-positive oligodendrocytes in adult mouse brain. These results indicate that Tau is expressed in mature oligodendrocytes and may serve as a new oligodendrocyte cell lineage marker. However, Tau is dispensable for myelin formation, because Tau knock-out mice exhibited normal myelination. Furthermore, we performed immunostaining for Tau in Pelizaeus-Merzbacher disease (PMD) model mice, in which proteolipid protein is overexpressed (heterozygous Plp1 transgenic mouse (Plp1-tg+/-). Interestingly, the number of Tau and Olig2 double positive cells were significantly increased in chronic demyelinating lesions in Plp1-tg mice compared with wild type. To confirm whether Tau expression is changed in another demyelination model mouse, we generated cuprizone (CPZ) induced-demyelination mice. Surprisingly, the expression level of Tau in oligodendrocyte was dramatically decreased in CPZ-induced demyelination mice compared with control mice. Taken together our data and previous studies, Tau may be involved in the PMD specific molecular mechanism of pathology in demyelination and/or the part of mechanism in repair process.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P93 疾患特異的iPS細胞を用いたPelizaeus-Merzbacher病のモデル構築と表現型解析 Modeling and Phenotypic analysis of Pelizaeus-Merzbacher disease using patient-derived iPS cells ^○鈴木禎史, Heng Li, Yu-ichi Goto, Ken Inoue 国立精神・神経医療研究センター ^○Sadafumi Suzuki, Heng Li, Yu-ichi Goto, Ken Inoue Department of Mental Retardation and Birth Defect Research National Institute of Neuroscience, National Center of Neurology & Psychiatry Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive leukodystrophy characterized by diffuse hypomyelination in the central nervous system (CNS). PMD is caused by mutations in the proteolipid protein gene PLP1, which encodes the major myelin protein of the CNS. Genomic duplications encompassing the entire PLP1 gene are the most frequent causative mutation. Although, the cellular consequences of how an extra-copy of wild-type PLP1 causes severe hypomyelination remains poorly understood, final differentiation and maturation of oligodendrocytes (OLs) have been thought to be primarily affected. Plp1-duplication model mice were generated, but they do not recapitulate the phenotype and pathophysiology of human PMD. To address cellular pathology of PMD more precisely, we have been generating human induced pluripotent stem cells (hiPSCs) from patients with PMD harboring PLP1 duplication and aim to differentiate them into oligodendrocytes to recapitulate the cellular pathology of PMD in culture. In the early differentiation phase of OL, we observed increase of reactive oxygen species (ROS) accumulation and a partially depolarized inner mitochondrial membrane in OLs in a PMD-derived hiPSC line comparison with a normal control. Also we observed ROS accumulation in oligodendrocyte precursor cells (OPCs). We found abnormalities of mobilization, process number and length of OPCs. These findings preliminarily suggest that the cellular phenotypes of PMD-derived hiPSCs may occur at early stage of OL differentiation, which is earlier than previously thought. We further seek to clarify if the pathophysiology underlying these early events is associated with PLP1 genotypes and gene dosage by analyzing other PMD-derived hiPSC lines.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P94 ミクログリア特異的SIRPα欠損マウスにおける加齢性運動学習能低下の改善 Improvement of age-related decline in motor learning ability by microglia-specific gene targeting of SIRPα ^○今井武史¹, Asuka Tomiyama¹, Sayoko Matsushita¹, Rui Mizutani¹, Daisuke Yokota¹, Eriko Urano¹, Ayako Morita¹, Yuriko Hayashi², Takashi Matozaki³, Hiroshi Ohnishi¹ 1.生体情報検査科学講座, 2.Department of Medical Technology, Gunma Paz University Graduate School of Health Sciences, 3.Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine ^○Takeshi Imai¹, Asuka Tomiyama¹, Sayoko Matsushita¹, Rui Mizutani¹, Daisuke Yokota¹, Eriko Urano¹, Ayako Morita¹, Yuriko Hayashi², Takashi Matozaki³, Hiroshi Ohnishi¹ 1.Department of Laboratory Sciences, Gunma University Graduate School of Health Sciences, 2.Department of Medical Technology, Gunma Paz University Graduate School of Health Sciences, 3.Division of Molecular and Cellular Signaling, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine Microglia, macrophage-like cells in the brain, are important for removal and repair of damaged brain tissue. Microglia-specific gene targeting of a membrane protein SIRPα induces the emergence of CD11c-positive (activated) microglia in healthy normal mouse brain and ameliorates tissue damage in an experimental model of demyelination. Here, we investigated the effect of microglia-specific SIRPα gene targeting on the age-associated decline of the brain functions. In the aged control mice, the motor learning ability evaluated by the rotarod test was completely abolished. On the other hand, learning ability was well conserved in age-matched microglial-specific SIRPα conditional knockout (cKO) mice. Examination of the cerebellum revealed that aging significantly induced CD11c-positive microglia in control mice, while such a phenotype was more pronounced in SIRPα cKO mice. The aging-induced loss of cerebellar neurons, as well as the accumulation of DNA breaks, were comparable between two genotypes. Unexpectedly, the accumulation of lipofuscin, an aging marker, was significantly increased in Purkinje cells of SIRPα cKO mice. Transcriptome analysis revealed that a neuroprotective factor Gpnmb (glycoprotein nonmetastatic melanoma protein B/osteoactivin) was significantly increased in the cerebellum of SIRPα cKO mice. Although the reduction of age-related brain damage by SIRPα gene targeting has not yet been confirmed, SIRPα-deficient microglia may have a protective effect against age-related decline of brain functions.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P95 オリゴデンドロサイトへのAMPA受容体シグナリングは運動スキル学習を刺激する AMPA receptor signalling to oligodendrocytes stimulates motor skill learning ^○清水崇弘¹, Matthew Swire¹, Stuart Nayar¹, Alexander Fudge¹, Eleni Kougioumtzidou¹, Huiliang Li¹, Koujiro Tohyama², David Attwell³, William D Richardson¹ 1.University College London, 2.Centre for Electron Microscopy and Bioimaging Research and Dept of Physiology, Iwate Medical University, 3.Dept of Neuroscience, Physiology and Pharmacology, University College London ^○Takahiro Shimizu¹, Matthew Swire¹, Stuart Nayar¹, Alexander Fudge¹, Eleni Kougioumtzidou¹, Huiliang Li¹, Koujiro Tohyama², David Attwell³, William D Richardson¹ 1.Facalty of Medicine, Wolfson Institute for Biomedical Research, University College London, 2.Centre for Electron Microscopy and Bioimaging Research and Dept of Physiology, Iwate Medical University, 3.Dept of Neuroscience, Physiology and Pharmacology, University College London Oligodendrocyte precursor cells (OPs) form synapses with unmyelinated axons and receive glutamatergic input from passing action potentials. We showed previously that AMPAR–mediated signalling in OPs increases myelin production in developing white matter by enhancing oligodendrocyte (OL) survival. We also reported that new OL generation enhances motor skill learning in adult mice (ability to run on a “complex wheel” with unevenly spaced rungs). However, how new OLs contribute to learning is still not understood. In this study, we asked if AMPAR-mediated signalling in OPs/OLs is required for adaptive myelination and motor learning. In the constitutive absence of AMPAR-mediated signalling — using Sox10-Cre to delete GluA2/4 on a GluA3-null background (GluA2/3/4 KOs) — numbers of OLs and myelin internodes were reduced by ~25% during development (P28) and by ~15% in adulthood (P90+), relative to littermate controls. New OL generation in adult GluA2/3/4 KOs was also reduced, judging by ISH for Pcdh17it, which specifically labels newly-differentiating OLs. GluA2/3/4 KOs ran less well than control littermates on a simple running wheel. Therefore, to examine more closely the role of AMPAR signalling in motor skill learning we generated tamoxifen-inducible, OL lineage-specific GluA2/3/4 cKOs using Pdgfra-CreERT2 (for OPs), Opalin-CreERT2 (for OLs) or Sox10-CreERT2 (for OPs+OLs). Unexpectedly, OP-specific cKOs were relatively unimpaired, but GluA2/3/4 cKO in either OLs or OPs+OLs strongly inhibited motor learning, suggesting that AMPAR signalling from axons to differentiated OLs is critical, possibly by stimulating myelin synthesis and increasing myelin thickness around electrically active axons.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P96 オリゴデンドロサイト分化・髄鞘化における新規分子メカニズムの探索 Towards understanding of novel molecular mechanisms regulating oligodendrocyte differentiation and myelination ^○石野雄吾¹, Shoko Shimizu¹, Masaya Tohyama^1,2, Shingo Miyata¹ 1.近畿大学東洋医学研究所, 2.Osaka Prefectural Hospital Organization ^○Yugo Ishino¹, Shoko Shimizu¹, Masaya Tohyama^1,2, Shingo Miyata¹ 1.Division of Molecular Brain Science, Research Institute of Traditional Asian Medicine, Kindai University, 2.Osaka Prefectural Hospital Organization Glial cells are recently recognized as essential components with numerous key roles within the nervous system. Among them, oligodendrocytes and Schwann cells have strong interactions with axons by wrapping them to form myelin sheath in the central nervous system and peripheral nervous system, respectively. Myelin provides support and insulation to the nervous system and is prerequisite for the smooth and high-speed nerve conduction. Therefore, defects and malfunctions of those glial cells have broad impacts within the nervous system. While structural alterations of myelin forming oligodendrocytes in several pathological situation have been illustrated, genetical aspects that account for those defective events remain to be fully understood. It is well known that post-translational modifications of transcription factors regulate oligodendrocyte differentiation and myelination. While large amount data have been accumulated about phosphorylations, the function of arginine methylation of transcription factors, which is mediated by protein arginine methyltransferases (PRMTs), is still poorly understood. To elucidate a novel molecular mechanism controlling oligodendrocyte differentiation and myelination, we screened protein-protein interactions among PRMTs and transcription factors and identified several partners. We’d like to propose arginine methylation as a novel and essential post-translational modification to understand more detail of normal development and pathological condition of oligodendrocytes.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P97 中枢神経系における髄鞘形成不全後の小径軸索のメンテナンス機構 The maintenance of small diameter axons after myelination defect in the CNS ^○高橋りこ, Hinako Saito, Yukina Hosoda, Chikako Hayashi, Nobuharu Suzuki 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野 ^○Riko Takahashi, Hinako Saito, Yukina Hosoda, Chikako Hayashi, Nobuharu Suzuki Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University In the CNS, oligodendrocytes form myelin that has an important function in maintaining axons. In demyelinating diseases such as multiple sclerosis, axonal loss is eventually induced with defects of the energy metabolism. Previous studies elucidate that the axonal loss in the diseases is dominantly observed in small diameter axons in the spinal cord. Last time, we reported that small axons were damaged in aged teneurin-4 deficient (Ten-4 -/-) mice, which displayed hypomyelination of the small axons. In this study, we attempted to elucidate the mechanism of the axonal damage in the mutant mice. First, we performed an immunohistochemical analysis using 1-year-old mouse spinal cords. Neurofilament (NF) was diffusely stained in the small axon areas of Ten-4 -/- mice, though wild-type (WT) NF signal was dotted. The Ten-4 -/- small axon areas were positive for SMI-32 and amyloid precursor protein. In addition, activated microglia and astrocytes were increased in the Ten-4 -/- white matter. Further, we performed the same analyses of immunohistochemistry of 7-week-old mouse tissues. As a result, there were no significant differences between WT and Ten-4 -/- tissues, suggesting that the damage/degeneration of small axons in the aged Ten-4 -/- mice occurred after the myelination defect. From these observations, we expected axonal loss in the damaged areas. However, surprisingly, an electron microscopy analysis showed that the small axons did not disappear but were maintained in the Ten-4 -/- tissue even though these axons were not myelinated. Finally, we found that the expression of several proteins related to the metabolism for energy production was increased in the small axon regions of the Ten-4 -/- spinal cord. These molecules may be key for maintaining the small axons.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P98 甲状腺機能低下症モデルマウスにおける性および週齢に依存したミクログリアの形態 Sex- and age-dependent morphology of microglia in a mouse model of hypothyroidism ^○新山哲士, Mami Noda 九州大学 ^○Tetsushi Niiyama, Mami Noda Laboratory of Pathophysiology, Graduate School of Pharmaceutical Sciences, Kyushu University There is a close relationship between the nervous system and the endocrine system. Recently, it has become clear that glial cells also have an important role in bridging the endocrine system and the nervous system. Thyroid hormone, one of the endocrine substances, is essential for the development and function of the central nervous system (CNS). Thyroxine (T4), a thyroid hormone precursor circulating in the bloodstream, passes through cerebrovascular endothelial cells and is taken up by astrocytes, where it is converted to triiodothyronine (T3), an activated thyroid hormone. T3 produced by astrocytes is released into the extracellular space where it exerts its effects on microglia and other cells. It has been reported that the prevalence of hypothyroidism in adults is higher in women than in men and increases with age. Hypothyroidism can affect mental health conditions such as depression and increase the risk of cognitive impairment and neurodegeneration such as Alzheimer's disease. Microglia are involved in the removal of degenerated neurons and axons and maintain CNS homeostasis. Various CNS diseases and neurodegenerative disorders are known to be caused by microglial cell dysfunction. We have found that there is a difference in behavioral tests and glial cell fluorescence intensity in male and female hypothyroidism models and at different ages. These results suggest that hypothyroidism alters the function of glial cells and affects behavior and mental status. Microglia are known to undergo morphological changes under different conditions. Therefore, in this study, we will quantitatively compare the morphology of microglia in different sexes and ages of a mouse model of hypothyroidism.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P99 髄鞘形成初期におけるアクチン骨格制御分子 Arpc1aの発現様式と機能解析 The expression pattern and functional analysis of Arpc1a controlling actin cytoskeleton at the initial stage of myelination ^○岩瀬未帆, Momona Yamada, Chikako Hayashi, Nobuharu Suzuki 東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野 ^○Miho Iwase, Momona Yamada, Chikako Hayashi, Nobuharu Suzuki Dept. of Mol. and Cell Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University In the CNS, oligodendrocytes (OLs) form myelin around axons, which enables the rapid conduction of action potential. When OLs extend their processes, ensheathe axons, and form compacted spirals of myelin, a balance between assembly and disassembly of actin cytoskeleton is precisely controlled. At the initial myelination stage, the assembly of actin filaments is regulated by Arp2/3 complex, consisting of seven subunits, whose functions are still incompletely understood. One of them, Arpc1a exhibits the highest mRNA expression level among the subunits during OL differentiation and myelination. In this study, we analyzed the expression pattern and function of Arpc1a in CNS myelination. In immunohistochemistry of mouse spinal cords, at postnatal day (P) 3 and 7, the intense expression signal of Arpc1a was observed surrounding axons, while the signal disappeared at P14. The postnatal expression of Arpc1a was also detected by RT-PCR and Western blotting. In immunocytochemistry of mouse primary cell culture, Arpc1a was expressed in both oligodendrocyte progenitor cells (OPCs) and OLs, and colocalized with F-actin along cell processes and in the tips of the processes. When primary OLs were well-differentiated and formed the ring/lamellar morphology, Arpc1a was eliminated from the inner part of the membrane sheet region and remained only at the outer edge of the ring/lamellar, similar to F-actin. The colocalization of Arpc1a with F-actin was also observed in the OPC line CG-4. Finally, overexpression and knockdown experiments revealed that Arpc1a regulated actin cytoskeletal organization in OL lineage cells. These results suggested that Arpc1a plays an important role in the formation of actin cytoskeleton in OLs at the initial stage of CNS myelination.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P100 脳出血モデルラットにおける超早期運動介入がミクログリア活性化に及ぼす影響 Effects of very early exercise intervention on motor function recovery and microglial activation in a rat model of cerebral hemorrhage ^○玉越敬悟¹, Madoka Maeda², Nae Murohashi³, Ami Saito¹ 1.Department of Physical Therapy, Niigata University of Health and Welfare, 2.Kuwana Hospital, 3.Niigata Seiro Hospital ^○Keigo Tamakoshi¹, Madoka Maeda², Nae Murohashi³, Ami Saito¹ 1.Department of Physical Therapy, Niigata University of Health and Welfare, 2.Kuwana Hospital, 3.Niigata Seiro Hospital We found that exercise intervention within 24 hours after intracranial hemorrhage (ICH) promotes the expression of proinflammatory factors in the injured area and worsens motor dysfunctions. Since microglia may be involved in the increased expression of proinflammatory factors, we examined the activation of microglia by very early exercise intervention after ICH, in this study. Male Wistar rats were randomly divided into three groups: sham surgery group (SHAM), ICH+non-exercise group (ICH+Cont), and ICH+very early exercise group (ICH+VET). The ICH+VET group underwent treadmill running exercise starting at 6 hours after surgery and lasting until 6 days after surgery. Horizontal ladder and Rotarod tests were used to evaluate motor functions. Brain tissue samples from the injured striatum were collected on day 7 after ICH. The protein expression levels of CD80, CD163, NeuN, PSD95, and GFAP were analyzed by Western blotting. At 25 hours after ICH, motor dysfunctions in the ICH+VET group were significantly worse than those of the SHAM and ICH+Cont groups. At 6 days after ICH, there was no significant difference between the groups. Brain tissue analysis showed that CD80 protein expression was significantly increased in the ICH+VET group compared to the SHAM group at 27 hours after ICH. The CD163 protein expression level in the ICH+VET group was significantly lower than that in the ICH+Cont group at day 6 after ICH onset. The present results suggest that exercise intervention within 24 hours after ICH activates M1 microglia, and that activation of M1 microglia may be involved in the worsening of motor dysfunctions associated with ICH. In addition, the activation of M2 microglia was inhibited by continuous exercise intervention from 24 hours to 6 days after ICH.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P101 末梢ミエリンタンパク質L-MPZのPKCリン酸化による細胞接着活性の解析 Analysis of PKC-dependent cell adhesion activity of peripheral myelin protein L-MPZ ^○後藤雅裕¹, Yuna Watanabe¹, Tetsuhiro Nakajima¹, Yoshinori Otani², Hiroko Baba^1,3, Yoshihide Yamaguchi¹ 1.東京薬科大学大学院　薬学研究科　機能形態学教室, 2.Department of Anatomy and Neuroscience ,Faculty of Medicine, Shimane University, 3.Department of Occupational Therapy, Faculty of Rehabilitation, Niigata University of Health and Welfare ^○Masahiro Goto¹, Yuna Watanabe¹, Tetsuhiro Nakajima¹, Yoshinori Otani², Hiroko Baba^1,3, Yoshihide Yamaguchi¹ 1.Department of Molecular Neurobiology. School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 2.Department of Anatomy and Neuroscience ,Faculty of Medicine, Shimane University, 3.Department of Occupational Therapy, Faculty of Rehabilitation, Niigata University of Health and Welfare Translational readthrough is an important mechanism of gene translation regulation. Large myelin protein zero (L-MPZ), an isoform of peripheral myelin protein zero (MPZ, P0), is the first reported common mammalian protein synthesized by this mechanism under physiological conditions. L-MPZ is localized in myelin in the peripheral nervous system. Recently, we reported that abnormally increased L-MPZ in L-MPZ mice, which synthesize L-MPZ without P0, results in a phenotype like Charcot-Marie-Tooth (CMT) disease, including motor disturbance and morphological abnormalities in myelin and axon. However, the physiological function of L-MPZ in peripheral myelin is still unknown. P0 is involved in adhesion of myelin layers and a protein kinase C (PKC) phosphorylation site in the intracellular C-terminal region (P0 PKC) is known to regulate its activity. L-MPZ has an additional PKC phosphorylation site in the L-MPZ-specific region (L-MPZ PKC). Therefore, to investigate the relationship between PKC-mediated L-MPZ-specific phosphorylation and membrane adhesion function, we constructed a bicistronic expression vector containing P0 or L-MPZ cDNAs with/without mutations in the PKC phosphorylation sites (Ser was replaced by Ala at P0 PKC, L-MPZ PKC, or both sites). The expressions of these proteins in transfected HeLa cells were confirmed by immunostaining. To clarify adhesion activities of L-MPZ and PKC phosphorylation site mutants, we performed the cell adhesion assay using these transfected HeLa cells. P0 and L-MPZ exhibited cell adhesion activities, but mutant proteins showed reduction of cell adhesion activity. These results suggest that PKC-dependent phosphorylation of L-MPZ may regulate membrane adhesion activity in myelin, which is similar to P0.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P102 外傷性脳損傷における生理活性脂質(2ccPA)のアストロサイト由来テネイシンCを介した神経保護効果 2-carba-cyclic phosphatidic acid suppresses the apoptosis of neuron mediated by tenascin-C from astrocyte in traumatic brain injury ^○中島麻里¹, Mari Gotoh^2,3, Kei Hashimoto¹, Misaki Endo¹, Kimiko Murakami-Murofushi¹, Hiroko Ikeshima-Kataoka¹, Yasunori Miyamoto¹ 1.お茶の水女子大学大学院人間文化創成科学研究科ライフサイエンス専攻, 2.Institute for Human Life Innovation, Ochanomizu University, 3.Research division of human welfare science, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University ^○Mari Nakashima¹, Mari Gotoh^2,3, Kei Hashimoto¹, Misaki Endo¹, Kimiko Murakami-Murofushi¹, Hiroko Ikeshima-Kataoka¹, Yasunori Miyamoto¹ 1.Graduate School of Humanities and Sciences, Ochanomizu University, 2.Institute for Human Life Innovation, Ochanomizu University, 3.Research division of human welfare science, Ochanomizu University, 4.Faculty of Science and Engineering, Waseda University 2-carba-cyclic phosphatidic acid (2ccPA), a lipid mediator, suppresses inflammation in the vicinity of the stab wound as a model of traumatic brain injury (TBI). We have previously reported that 2ccPA regulates microglial polarization towards a neuroprotective phenotype. However, the regulatory mechanism of 2ccPA in the repair of stab wounds via astrocytes remains elusive. In this study, we examined the neuroprotective effects of 2ccPA via tenascin-C (TN-C), an extracellular matrix protein, in astrocytes after the infliction of a stab wound. First, the administration of 2ccPA suppressed neuronal apoptosis in the lesions within 1 h and 1 d after the injury. In this acute phase, 2ccPA remarkably increased the expression levels of glial fibrillary acidic protein, which is a marker of astrocytes; and significantly reduced the percentage of activated astrocytes on day 3 after the injury. Simultaneously, 2ccPA increased the ratio of TN-C + astrocytes in the vicinity of the lesion. To analyze the involvement of TN-C via astrocytes in the suppression of apoptosis by 2ccPA, 2ccPA was added to primary cultured astrocytes and the astrocyte conditioned media was added to primary cultured cortical neurons. This addition of conditioned media suppressed neuronal apoptosis. Furthermore, the neuroprotective effect was reduced by the knockdown of TN-C in astrocytes, while the addition of TN-C protein rescued this effect. Therefore, our study indicates that the increase in TN-C expression in astrocytes as promoted by 2ccPA treatment suppresses the neuronal apoptosis; therefore, 2ccPA could be a therapeutic agent for the repair of traumatic brain injury.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P103 結節性硬化症モデルマウスにおける、アストロサイトの活性化に起因した記憶障害 Reactive astrocyte-mediated memory disorder in tubular sclerosis complex model mouse ^○島田忠之, Hiroko Sugiura, Kanato Yamagata Child brain project, Tokyo Metropolitan Institute of Medical Science ^○Tadayuki Shimada, Hiroko Sugiura, Kanato Yamagata Child brain project, Tokyo Metropolitan Institute of Medical Science Tuberous sclerosis complex (TSC) patients harbor hamartomas in the brain and other organs. The neuropsychiatric symptoms of TSC patients include refractory epilepsy, autism spectral disorders and mental retardation. One of the responsible genes for TSC is Tsc1. To investigate if the brain astrocytes contribute to the neuropsychiatric symptoms of TSC patients, we developed astrocyte-specific Tsc1 knockout mice (cKO mouse) as a TSC model mouse and examined their phenotypes. Immunohistochemical analyses showed that GFAP-highly-positive cells, reactive astorsytes, were increased in the brain, especially in the hippocampus, piriform cortex, and amygdala in cKO mice. In addition, the malformed dendritic spines ware observed in dentate gyrus in hippocampi, where one of the most severe gliosis was observed. Moreover, three-chamber test analysis revealed that the cKO mice showed impaired social memory. Because Tsc1 deletion activates downstream small G-protein, Rheb, we tried pharmacological inhibition of Rheb in cKO mice. Treatment with Rheb inhibitors restored gliosis and spine malformation in cKO mice. Abnormal social memory behaviors were also improved by Rheb inhibitor administration. We found that expression of syntenin, one of the downstream proteins of Rheb, is upregulated in the hippocampus of cKO mice, and Tsc1/syntenin double cKO mice showed improved social memory. These results suggested that activation of the Tsc/Rheb/syntenin signaling in the astrocytes could induce astrogliosis, affecting the spine morphology by the crosstalk between the astrocytes and neurons. Resulting neural dysfunction would develop abnormal social behaviors observed in a mouse model of TSC.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P104 紀伊半島に多発する筋萎縮性側索硬化症/パーキンソン認知症複合の病態にはアストロサイトにおけるミトコンドリア障害が関与している Astrogliopathy is caused by mitochondrial abnormalities in amyotrophic lateral sclerosis and Parkinsonism-dementia complex of the Kii peninsula ^○Leventoux Nicolas¹, Satoru Morimoto¹, Mitsuru Ishikawa¹, Fumito Endo², Shinsuke Shibata¹, Koji Yamanaka², Shigeki Kuzuhara³, Yasumasa Kokubo⁴, Hideyuki Okano¹ 1.慶應義塾大学医学部生理学, 2.Department of Neuroscience & Pathobiology, Research Institute of Environmental Medicine (RIEM), Nagoya University, 3.Faculty of Health Science, Suzuka University of Medical Science, 4.Kii ALS/PDC Research Center, Mie University Graduate School of Regional Innovation Studies ^○Nicolas Leventoux¹, Satoru Morimoto¹, Mitsuru Ishikawa¹, Fumito Endo², Shinsuke Shibata¹, Koji Yamanaka², Shigeki Kuzuhara³, Yasumasa Kokubo⁴, Hideyuki Okano¹ 1.Department of Physiology, Keio University School of Medicine, 2.Department of Neuroscience & Pathobiology, Research Institute of Environmental Medicine (RIEM), Nagoya University, 3.Faculty of Health Science, Suzuka University of Medical Science, 4.Kii ALS/PDC Research Center, Mie University Graduate School of Regional Innovation Studies Amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) of the Kii peninsula (Kii ALS/PDC) is an intractable disease causing multiple system neurodegeneration, and endemic to natives of the southern coast area of the Kii peninsula in Japan. Although the cause remains unknown, astrocytic abnormalities such as reactive astrocytes and aging-related tau astrogliopathy constitute characteristic neuropathological features. To investigate this disease, we first developed and published a homemade protocol to derive functional astrocytes from induced pluripotent stem cells (iPSCs) that we called iPasts. Thereafter, we established iPSCs cell lines derived from five patients with Kii ALS/PDC and generated iPasts from them (Kii iPasts). We succeeded to recapitulate in vitro, in Kii iPasts, several pathological phenotypes such as reactivation, generation of reactive oxygen species, and reduced glutamate uptake capacity, compare to control iPasts. This technology we developped enabled to compare Kii iPasts and healthy control iPasts transcriptomes by RNA sequencing analysis. Here, we focus on one gene drastically downregulated, involved in mitochondrial metabolism, and which has also been reported as ALS- and PD-linked in a few reports. By lentiviral overexpression of this target gene as well as using a small molecule to activate mitochondria, we confirmed that astrocytic functions such as glutamate uptake and mitochondria respiration could be rescued in Kii iPasts. These results open the path to explore mitochondria as a therapeutic target in Kii ALS/PDC.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P105 Hedgehogシグナルは神経因性疼痛マウスモデルにおいて痛覚過敏形成に関与する Hedgehog signaling plays a crucial role in hyperalgesia associated with neuropathic pain in mice ^○奥田洋明, Nichakarn Kwankaew, Tatsuya Ishikawa, Kiyomi Hori, Noriyuki Ozaki 金沢大学医薬保健研究域医学系機能解剖学 ^○Hiroaki Okuda, Nichakarn Kwankaew, Tatsuya Ishikawa, Kiyomi Hori, Noriyuki Ozaki Department of Functional Anatomy, Graduate School of Medical Science, Kanazawa University Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. The Hedgehog (Hh) signaling pathway is related to hyperalgesia in Drosophila but does not affect baseline nociceptive threshold. In invertebrates, the contribution of the Hh signaling pathway to neuropathic pain is a highly debated issue. In this study, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve transection (SNT) surgery, activated microglia increased in ipsilateral spinal dorsal horn compared to that in sham group, but decreased 21 days after surgery. In contrast, activated astrocytes in the spinal cord did not differ between the groups. On day 21 postsurgery, SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in DRG neurons. Intrathecal administration of Hh signaling inhibitor vismodegib attenuated mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment of Hh signaling activator SAG in naïve mice induced mechanical allodynia, which was abolished by ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling pathway by pretreatment with vismodegib led to a significant reduction in ATP secretion and frequency/number of spontaneous elevations of intracellular calcium ion levels in DRG cultured cells. Thus, the Hh signaling pathway seems to modulate neural activity of DRG neurons via ATP release and plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P106 発達期白質のMac2陽性細胞の分布 Distribution of Mac2-positive cells in developing white matter ^○Ishibashi Tomoko¹, Yuika Takei¹, Shiho Ohshima¹, Kouichi Hashimoto², Hiroyuki Konishi³, Hiroshi Kiyama³, Hiroko Baba^1,4 1.東京薬科大学薬学部機能形態学, 2.Department of Neurophysiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 3.Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, 4.Department of Occupational Therapy, Faculty of Rehabilitation, Niigata University of Health and Welfare ^○Ishibashi Tomoko¹, Yuika Takei¹, Shiho Ohshima¹, Kouichi Hashimoto², Hiroyuki Konishi³, Hiroshi Kiyama³, Hiroko Baba^1,4 1.Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences, 2.Department of Neurophysiology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 3.Department of Functional Anatomy and Neuroscience, Nagoya University Graduate School of Medicine, 4.Department of Occupational Therapy, Faculty of Rehabilitation, Niigata University of Health and Welfare There is growing evidence of heterogeneity among microglia during different stages of development and adulthood in specific brain regions. Especially in the developing white-matter (wm) regions, a distinct amoeboid microglia subset characterized by high expression of several genes, such as Clec7a and Spp1, has been reported. The Clec7a subset of microglia populated the wm by P4, peaking around P7, and seems to phagocytize newly formed oligodendrocytes. Previously we reported a restricted distribution of amoeboid Mac2+ cells in the myelinating distal wm, but not in the already myelinated area. About 60% of Mac2+ cells were Iba1+ microglia, but in these myelinating regions in P7 mice, about 40% were GFAP+ astrocytes instead. The aim of the present study is to understand the physiological roles of Mac2+ cells in developing wm. In the mouse brain, Mac2+ cells showed a rather restricted distribution and a transient appearance. Mac2+ cells were observed only in the cerebellum (cb) wm regions and corpus callosum (cc) by P3, at which time either Clec7a+ microglia or immature oligodendrocytes were rarely seen. Mac2+ cells continued to increase and peaked around P5 to P7, after which their number decreased again – they were no longer detected by P14. Although the timing of myelination is earlier in the cb than in the cc, Mac2+ cells started to appear slightly earlier in the cc than in the cb. Furthermore, there was no significant change in the number of Mac2+ cells in Csf1r-cKO mice that showed microglia-selective deletion or in Siglech^{dtr/+^{mice that showed specific ablation of microglia. These results suggest that the Mac2+ subset of microglia could be a different population and might not be required for Csf1r signaling to survive in the developing wm.}}

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P107 アルツハイマー病病態におけるApoE機能を解析するヒト多能性幹細胞モデルの作出 Generation of human induced pluripotent stem cell derived models analyzing the functions of Apolipoprotein E in Alzheimer’s disease pathology ^○村上玲¹, Watanabe Hirotaka¹, Morimoto Satoru¹, Sonn Iki¹, Hashimoto Tadafumi², Iwatsubo Takeshi², Okano Hideyuki¹ 1.Department of Physiology, Keio University School of Medicine, 2.Department of Neuropathology, Graduate School of Medicine, The University of Tokyo ^○Rei Murakami¹, Watanabe Hirotaka¹, Morimoto Satoru¹, Sonn Iki¹, Hashimoto Tadafumi², Iwatsubo Takeshi², Okano Hideyuki¹ 1.Department of Physiology, Keio University School of Medicine, 2.Department of Neuropathology, Graduate School of Medicine, The University of Tokyo [Purpose] The number of patients suffered from Alzheimer’s Disease (AD) is increasing. The detailed mechanism of AD onset is not yet elucidated, and it is necessary to clarify the pathogenic mechanism for developing the effective therapy. Among the three alleles of Apolipoprotein E (APOE)gene including (E2, E3, E4), APOE4 is the most well-known risk factor for sporadic AD that occupies almost all AD. However, some issues have remained unclear concerning on sporadic AD and ApoE, such as the physiological functions of each ApoE protein isoform and the cell types ApoE affected. As the purpose of this study, we plan to develop the in vitro cellular models analyzing the functions of ApoE isoforms and analyze the ApoE functions in sporadic AD pathology. [Method and Result] We converted APOE3 human induced pluripotent stem cells (hiPSCs) to APOE4 genotype by CRISPR/CAS9-mediated genome editing. Astrocytes were induced from APOE3 and APOE4 hiPSCs. Then we analyzed gene expression of APOE3 and APOE4 astrocytes and identified some differentially expressed genes. We evaluated the function of lipid transport of APOE3 and APOE4 astrocytes. While the molecular weights of lipoproteins ApoE3 or ApoE4 are quite similar, intracellular lipid accumulation of APOE4 astrocytes was different from APOE3 cells. [Conclusion] The present iPSC-derived astrocytes models are the promising tool for elucidating the functions of ApoE isoforms in AD pathology.		2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P108 細胞外グルタミン濃度によるグルタミン合成酵素の発現変化 The role of extracellular glutamine concentration in regulating astrocytic glutamine synthetase expression ^○那須優介, Miho Terunuma 新潟大学医歯学総合研究科口腔生化学分野 ^○Yusuke Nasu, Miho Terunuma Division of Oral Biochemistry, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University Glutamine synthetase (GS) catalyzes the reaction that synthesizes glutamine from ammonia and glutamate, and plays an important role in their metabolism. Astrocytes, one of the major glial cells in the central nervous system, is the only cell types that express GS to protect neurons from the neurotoxicity of ammonia and glutamate. Interestingly, an increasing number of studies have indicated that GS is associated with several brain disorders including Alzheimer’s disease, schizophrenia, mesial temporal lobe epilepsy and depression. According to a recent report, GS expression in the liver is regulated by extracellular glutamine levels (Nguyen TV et al. Mol Cell. 2016). However, the detailed mechanism regulating GS expression in astrocytes remains unclear. In this study, we investigated how GS expression in astrocytes is regulated in response to the extracellular glutamine concentration. We found that GS expression was decreased in astrocytes exposed to high glutamine. Since glutamine has previously been reported to induce the ubiquitination of GS and promotes its degradation in liver (Nguyen TV et al. Mol Cell. 2016), we are currently investigating these signaling in astrocytes. In contrast, glutamine-deprived astrocytes dramatically increased GS expression. The application of glutamate and NH4Cl, the sources of glutamine, in glutamine-deprived astrocytes reduced the expression of GS suggesting that the amount of intracellular glutamine may be the determinant of GS levels in astrocytes. However, the measurement of intracellular glutamine indicated that the pool of intracellular glutamine is not significantly increased even after glutamate and NH4Cl stimuli. We are currently investigating the mechanism of GS up-regulation in glutamine-deprived astrocytes.

2021/9/30　9:00～22:30，10/1　9:00～18:00　オンデマンド eポスター会場 P109 細胞外マトリックスタンパク質Fibulin-7は中枢神経系の髄鞘形成において細胞間相互作用を制御する The extracellular matrix protein fibulin-7 regulates cellular interaction for CNS myelination ^○山田桃奈¹, Miho Iwase¹, Chikako Hayashi¹, de Vega Susana², Nobuharu Suzuki¹ 1.東京医科歯科大学大学院医歯学総合研究科遺伝子細胞検査学分野, 2.Dept. of Med. for Orthop. and Motor Organ, Juntendo University Grad. Sch. of Med ^○Momona Yamada¹, Miho Iwase¹, Chikako Hayashi¹, de Vega Susana², Nobuharu Suzuki¹ 1.Dept. of Mol. and Cell. Biol., Grad. Sch. of Med. and Dent. Sci., Tokyo Medical and Dental University, 2.Dept. of Med. for Orthop. and Motor Organ, Juntendo University Grad. Sch. of Med Fibulin-7 (Fbln7) is an extracellular matrix (ECM) protein consisting of an N-terminal sushi domain, three EGF-like motifs, and a C-terminal fibulin-type module. Fbln7 is overexpressed in glioblastoma and induces the formation of aberrant vessels. Integrin β1 is one of the cell surface receptors for Fbln7. However, the expression pattern and functions of Fbln7 in normal CNS development has not been elucidated. A recent comprehensive RNA-Seq analysis study showed Fbln7 is specifically expressed in oligodendrocyte (OL)-lineage cells, including OL precursor cells (OPCs), among all of the CNS cell types. Here we analyzed the expression pattern and function of Fbln7 in oligodendroglial cells. First, we performed RT-PCR and Western blotting of Fbln7 from CNS tissues from late embryonic stage to adult stage. The mRNA and protein expression of Fbln7 was detected in the spinal cord from late embryonic to early postnatal stage. By immunohistochemistry in the postnatal day (P) 3 mouse spinal cord, Fbln7 was localized surrounding the neuronal axons and glial cells, including OPCs. However, at P7, its expression was more localized between axons and myelin. In primary cell culture, Fbln7 was expressed in the branching points of OLs protrusions and around the tips of the protrusions. To analyze the function of Fbln7, the OPC line CG-4 was seeded on the recombinant Fbln7 (rFbln7)-coated wells. We found that rFbln7 promoted protrusion formation with actin polymerization. Additionally, soluble rFbln7 significantly increased the formation of cell aggregates through cell-cell adhesion. In conclusion, Fbln7 is a novel ECM protein that regulates the cellular interaction through the formation of cell protrusions and adhesions during CNS myelination.