一般ポスター

一般ポスターグリア-1

7月6日（木）　13:20-14:20　ポスター会場① 1P④-1 Exploring the role of astrocyte GPCR signaling in behavioral flexibility Bolati Wulaer 理化学研究所脳神経科学研究センター Bolati Wulaer RIKEN Center for Brain Science The orbitofrontal cortex (OFC) is a crucial brain area for behavioral flexibility, however, the cellular mechanisms that underlie this flexibility remain elusive. Our study demonstrates that the neuromodulator norepinephrine elicits ex vivo astrocyte Ca2+ signaling in the OFC, primarily through the activation of Gq-coupled alpha1-adrenoreceptors. Chemogenetic stimulation of astrocyte-specific Gq/Ca2+ signaling was shown to reduce local neuronal activity and induce behavioral inflexibility in mice, whereas their motor coordination, working memory, or novelty recognition remained intact. We will report data using in vivo imaging and RNA-seq analysis aim to uncover the underlying molecular mechanisms. Our results suggest that the astrocyte Gq/Ca2+ signaling in the OFC is essential for the neural circuits that support behavioral flexibility.		7月6日（木）　13:20-14:20　ポスター会場① 1P④-2 Behaviorally-activated astrocyte ensembles in whole mouse brains. 出羽　健一¹, 加世田　晃大¹, 桑原　葵¹, 笠井　淳司², 長井　淳¹ 1. 理化学研究所　脳神経科学研究センター　グリア-神経回路動態研究チーム, 2. 大阪大学大学院薬学研究科神経薬理学分野 Ken-ichi Dewa¹, Kodai Kaseda¹, Aoi Kuwahara¹, Atsushi Kasai², Jun Nagai¹ 1. Dept. of Glia-Neuron Circuit Dynamics, RIKEN CBS, Wako, Japan, 2. Laboratory of Molecular Neuropharmacology, Graduate School of Pharmaceutical Sciences, Osaka University An open question in astrocyte biology concerns if, when, where and how astrocyte signaling is regulated in a brain region specific manner to influence circuit functions and behavior. Recently, RNA-sequencing revealed that astrocytes differ by brain region with gene expression profiles and mount distinct molecular responses to various brain state transitions (i.e. molecular diversity). In addition, behaviorally-relevant stimuli activate a fraction of astrocytes across brain areas, suggesting functional diversity. Here we tackle the question of where and how specific populations of astrocytes respond to particular behaviorally-relevant stimuli. We aim to capture and manipulate Behaviorally-activated Astrocyte Ensembles (BAE [bei]) to reveal their consequential roles in circuits and behavior. To this end, we constructed a AAV PHP.eB, serotype that can breach the BBB and reach all regions of brain, based new genetic tool for brain-wide activated astrocyte selective tagging and manipulation. Combining this tool with whole-brain block-face serial microscopy tomography, tagging of activated astrocytes in the whole brain was achieved. In addition, we performed whole brain tagging with fear conditioning tests, and found a step dependent activation of astrocytes in the process of fear memory conditioning and recall.

7月6日（木）　13:20-14:20　ポスター会場① 1P④-3 アストロサイトの分枝伸長におけるインテグリン系細胞接着の役割 Roles of integrin-based adhesions in extending branches of astrocytes 生駒　千枝子, 笠井　虹汰, 井上　幸大, 米本　秋, 佃　聡子, 田村　安樹子, 寺崎　朝子千葉大学大学院理学研究院 Chieko Ikoma, Kota Kasai, Kodai Inoue, Shu Yonemoto, Satoko Tsukuda, Akiko Tamura, Asako Terasaki Graduate School of Science , Chiba Univercity Astrocytes in vivo have long branched stems and peripheral astrocyte processes (PAPs) containing actin-rich cytoskeletal structures. Some actin-binding proteins have been reported to affect the morphology of mammalian cultured astrocytes. However, these studies used fibroblast-like astrocytes cultured in DMEM containing 10% fetal bovine serum. Additionally, localization of integrin-based adhesions linking to actin cytoskeleton has yet to be elucidated to date. Previously, we reported that chicken astrocytes in Neurobasal medium containing b-FGF and B27 formed stems and PAPs (Tsukuda et. al., 2019). Here, we used these astrocytes to observe the localization focal adhesion components. Anti-zyxin antibody stained the base of PAPs, which may be cell-substrate adhesions. Zyxin was also found in cell-cell adhesions. Anti-paxillin also stained the base of PAPs, but mainly stained small plaques in protruding areas of branching stems. Vinculin was found only in larger plaques in astrocytes cultured for > 1 week. Live imaging showed their localization did not overlap in some areas, suggesting that adhesion components differed from typical focal adhesions although they also have a similar maturation process. Time-lapse imaging of zyxin and paxillin in extending branches of astrocytes indicated that cell-substrate adhesions might support the active movement of PAPs.		7月6日（木）　13:20-14:20　ポスター会場① 1P④-4 神経活動により放出される細胞外ATPの時空間ダイナミクス Spatiotemporal dynamics of extracellular ATP released by neuronal activity 鈴木　秀明^1,2, 繁冨　英治^1,2, 平山　幸歩¹, 高橋　由香里³, 池中　一裕⁴, 田中　謙二⁵, 加藤　総夫³, 尾藤　晴彦⁶, 小泉　修一^1,2 1. 山梨大　院医　薬理, 2. 山梨大　院医　GLIAセンター, 3. 慈恵医大　神経科学, 4. 生理研　分子神経生理, 5. 慶應大　医　先端研　脳科学, 6. 東京大　院医　神経生化学 Hideaki Suzuki^1,2, Eiji Shigetomi^1,2, Yukiho Hirayama¹, Yukari Takahashi³, Kazuhiro Ikenaka⁴, Kenji Tanaka⁵, Fusao Kato³, Haruhiko Bito⁶, Schuichi Koizumi^1,2 1. Dept Neuropharmacol, Interdiscipl Grad Sch Med, Univ Yamanashi, 2. Yamanashi GLIA Center, 3. Dept Neurosci, Jikei Univ, 4. Div Neurobiol and Bioinfo, NIPS, 5. Div Brain Sci, Inst Adv Med Res, Keio Univ Sch Med, 6. Dept Neurochem, Grad Sch Med, Univ Tokyo ATP acts as a signaling molecule in neurotransmission and neuron-glia signaling and is known to be involved in psychiatric disorders such as depression. Although ATP is known to be released by multiple types of cells in the CNS, it remains unknown how and from which cells ATP is released under physiological conditions due to the lack of a method to detect the spatiotemporal dynamics of extracellular ATP with high sensitivity. We imaged astrocyte extracellular ATP signal using a genetically encoded G protein-coupled receptor activation-based ATP sensor called GRAB_ATP1.0 in the CA1 region of acute hippocampal slices. Electrical stimulation of the Schaffer collateral resulted in ATP rise extracellularly in astrocytes. Pharmacological experiments show that the source of activity-dependent ATP release could be presynaptic sites of neurons. Microglia depletion prolonged the duration of ATP signal, suggesting that microglia, which highly express NTPDase1, rapidly degrade ATP to terminate ATP signaling. We found comparable ATP responses occurred in IP₃ receptor type 2 knock-out mouse model as the wild type mice, suggesting that IP₃R2-signaling in astrocytes has a relatively small contribution to the ATP responses. Overall data show that neuronal activity induces ATP release from axons or axon terminals and its ATP is subsequently negatively regulated by microglia in physiology.

7月6日（木）　13:20-14:20　ポスター会場① 1P④-5 TROY signaling in maintenance of blood-brain barrier integrity 小森　忠祐, 久岡　朋子, 森川　吉博和医大・医・第二解剖 Tadasuke Komori, Tomoko Hisaoka, Yoshihiro Morikawa Dept. of Anat. & Neurobiol. Wakayama Med. Univ. We have reported that TROY, a member of tumor necrosis factor receptor superfamily, is expressed in the neural stem/glial progenitor cells and astrocytes of CNS. Because TROY is an orphan receptor, the localization of TROY ligands and its role in the CNS remain unknown. We first investigated the localization of TROY-ligands using a fusion protein consisting of the extracellular domain of mouse TROY and the Fc portion of human IgG1 (TROY-Fc). The binding of TROY-Fc was histologically detected in GFAP⁺ cells surrounding blood vessels in the brain. To elucidate the role of TROY-ligands, we next generated transgenic (TG) mice expressing TROY-Fc under CAG promoter, in which TROY-Fc neutralize TROY-ligands. When transcardiac injection of a leakage tracer, sulfo-NHS-biotin, was performed in adult mice, dramatic leakage of the tracer across the blood-brain barrier (BBB) was detected in TG mice, compared with WT mice. This data is compatible with those in TROY-knockout mice. Transmission electron microscopy revealed endfoot swelling of perivascular astrocyte in the cerebral cortex and hippocampus of TG mice. These results suggest that TROY signaling in astrocytes is involved in the maintenance of BBB integrity and TROY-Fc TG mice may be useful for investigating the function of TROY-ligands. This work was supported by JSPS KAKENHI Grant Number 18K19756.		7月6日（木）　13:20-14:20　ポスター会場① 1P④-6 パラノーダルジャンクション形成不全におけるプルキンエ細胞の変化 Alteration of Purkinje cells in disruption of paranodal axoglial junctions 早川　翔馬, 下宮　晴, 大滝　博和, 石橋　智子東薬大　薬　機能形態 Shoma Hayakawa, Haru Shimomiya, Hirokazu Ohtaki, Tomoko Ishibashi Dep. of Functional Neurobiol, Tokyo Univ. of Pharmacy and Life Sciences Myelin loops attach to the axonal membrane and form paranodal axoglial junctions (PNJs) at paranodes. The PNJs contribute to axonal homeostasis in addition to their roles as lateral fences that restricted the location of nodal axolemmal proteins. We reported that CST-/- mice that partially lack PNJs, develop Purkinje axonal swellings after myelination and that IP3R1 accumulations were the earliest finding therein. However, it remains elusive how IP3R1 accumulates in the restricted region in PNJ disrupted Purkinje axons and whether elevated Ca2+ concentration occurs in the axonal swellings. We investigated (1) a distribution of Syntaphilin (SNPH) to understand the state of impaired axonal transport, (2) a distribution of two kinds of calcium-activated potassium channel, Slo1 K+ channel and IK, to understand the state of axonal [Ca2+] in CST-/- Purkinje cells. Immunohistochemical analysis showed that dendritic SNPH intrusion in Purkinje cells of PNJ disrupted CST-/- mice with age. The distribution of MAP2 also showed irregular dot-like staining pattern. Both of calcium-activated K+ channels were significantly positive staining in IP3R1-positive swellings, suggesting intracellular Ca2+ is elevated at the swollen regions. These results suggest that the partial disruption of PNJs result in impaired axonal transport and may affect Purkinje dendritic morphology.

7月6日（木）　13:20-14:20　ポスター会場① 1P④-7 プロサポシントランスジェニックマウスにおける網膜視細胞変性の解析 Degeneration of Retinal Photoreceptor Cell in Prosaposin Transgenic Mice 石塚　佑太¹, 鎌尾　浩行², 渡邉　昂¹, 小野　公嗣³, 大塚　正人⁴, 松田　純子¹ 1. 川崎医科大学　病態代謝学, 2. 川崎医科大学　眼科学, 3. 埼玉医科大学　保健医療学部　臨床検査学科, 4. 東海大学　医学部　基礎医学系分子生命科学 Yuta Ishizuka¹, Hiroyuki Kamao², Takashi Watanabe¹, Koji Ono³, Masato Ohtsuka⁴, Junko Matsuda¹ 1. Dept. of Pathophysiol. & Metab., Kawasaki Med. Sch., Okayama, Japan, 2. Dept. of Ophthal., Kawasaki Med. Sch., Okayama, Japan, 3. Sch. of Med. Tech., Fac. of Health & Med care., Saitama Med. Univ., Saitama, Japan, 4. Dept. of Mol Life Sci., Div. of Basic Med Sci. & Mol Med., Tokai Univ. Sch. of Med., Kanagawa, Japan Retinitis pigmentosa (RP) is an intractable disease characterized by retinal photoreceptor degeneration. Prosaposin (PSAP) is a precursor of saposins which are essential for sphingolipid degradation in lysosomes; genetic deficiencies of saposins cause severe lysosomal storage disorders. However, little is known about the function of PSAP itself. Therefore, we generated transgenic mice that systemically overexpress PSAP (PSAP-Tg mice). We found the PSAP dose-dependent selective degeneration of retinal photoreceptor cells with gliosis in PSAP-Tg mice. This phenotype closely resembles the pathology of RP. These results indicate that PSAP may play an important role in the retinal photoreceptor cells and its abnormal expression could be a cause of RP.		7月6日（木）　13:20-14:20　ポスター会場① 1P④-8 神経毒性評価におけるグリア細胞の重要性：マウス由来の株化細胞を用いた基礎的検討 Importance of glial cells in assessing neurotoxicity: a basic study using mouse-derived strain cells. 扇谷　昌宏, 古部　瑛莉子, 吉田　成孝旭川医科大学医学部解剖学講座機能形態学分野 Masahiro Ohgidani, Eriko Furube, Shigetaka Yoshida Department of Functional Anatomy and Neuroscience Microglia, known as immune cells, are sensitive to environmental changes and have acted disruptively and protectively against neurons. In addition, their involvement in various neuropsychiatric disorders has been reported in recent years. Based on these facts, we believe it is essential to include glial cells in the neurotoxicity evaluation system and have been developing the basis of the evaluation system.　We have investigated the neurotoxicity evaluation system using mouse-derived neuronal and microglial cell lines, emphasizing the convenience of the initial screening. A co-culture system of neurons and glial cells was constructed, and toxicity evaluation and gene expression analysis were performed. As test substances, we used metals whose neurotoxicity has been studied extensively.　Interestingly, it was found that some toxicities increased, some decreased, and some remained unchanged in the co-culture system compared to the monoculture of each cell. Furthermore, gene expression analysis results also confirmed that some expression changes were captured only in the co-culture system and that drastic changes occurred between the monoculture and co-culture systems.　These facts suggest that the conventional neurotoxicity evaluation system using neurons alone is inadequate and that an evaluation system including glial cells is essential.

7月6日（木）　13:20-14:20　ポスター会場① 1P④-9 グリア創薬への応用を目指したヒト不死化アストロサイトの活性化プロファイルの解明 HASTR/ci35 cells are promising tools for studies of reactive human astrocytes 根岸　由佳, 長谷川　理歩, 高崎　桃花, 馬場　知代, 森尾　花恵, 降幡　知巳東京薬科大学薬学部医療薬学科個別化薬物治療学教室 Yuka Negishi, Riho Hasegawa, Momoka Takasaki, Tomoyo Baba, Hanae Morio, Tomomi Furihata Lab. Clin. Pham. & Exp. Therapeut., Tokyo Univ. Pharm. Life Sci. Neurotoxic reactive astrocytes have gained significant attention as drug targets for treatment of various central nervous system diseases. To facilitate drug development studies, in vitro reactive human astrocyte models are necessary. Therefore, we aimed to characterize a reactive profile of human astrocytes/conditionally immortalized, clone 35 (HASTR/ci35). After exposed to the cytokine mix (TNFα, IL-1β, 50 ng/mL each) for 96 hr, HASTR/ci35 cells were subjected to RNA-sequencing, quantitative PCR, and cytokine array analyses. To test the neurotoxicity, LUHMES cells were cultured alone or co-cultured with HASTR/ci35 cells in the presence or absence the cytokine mix, followed by MAP2 immunostaining. The results of a series of the gene expression analyses showed that, upon exposure to pro-inflammatory cytokines, HASTR/ci35 cells exhibited significant up-regulation of various inflammation-associated gene expression levels, such as ICAM-1, IL-6, and PTX3. Furthermore, the results of the co-culture experiments showed that exposure to the cytokine mix markedly impaired neurite outgrowth of LUHMES cells only in the presence of HASTR/ci35 cells. HASTR/ci35 cells show an unequivocal reactive response to inflammatory stimuli with particular focus on their neurotoxicity, suggesting that they can serve as a human reactive astrocyte model for use in astrocyte-targeted drug development studies.