e ポスター

e ポスター 2. グリア (アストロサイト，ミクログリア) e Poster 2. Glia (astrocyte, microglia)

2020/9/10　14:00～15:00　オンデマンドB-1 P1-08 Olig2アストロサイトの抑制性シナプスとの関与 Molecular signatures of the Olig2-astrocytes indicate their specific involvement in GABAergic transmission 辰巳晃子¹、絹川薫^2,3、石西綾美¹、竹村晶子¹、田中達英¹、森英一郎³、和中明生¹ 1. 奈良県立医科大学・医・解剖学第2講座、2. 脳神経内科学講座、3. 未来基礎医学講座 Kouko Tatsumi¹, Kaoru Kinugawa^2,3, Ayami Isonishi¹, Shoko Takemura¹, Tatsuhide Tanaka¹, Eiichiro Mori³, Akio Wanaka¹ 1. Dept. Anat. Neurosci. Nara. Med. Univ., 2. Dept. Neurol., 3. Dept. Future Basic Med. Astrocytes are the most abundant glia in the central nervous system and are known to constitute heterogeneous populations that differ in their morphologies, gene expression patterns, and functions. Although the concept of astrocyte heterogeneity has been established, little is known about how many astrocytic subpopulations exist and how they interact with neurons in the adult brain. We have reported that the transcription factor Olig2 labels a subpopulation of astrocytes (Olig2-astrocytes). The Olig2-astrocytes were distinct from GFAP-expressing astrocytes (GFAP-astrocytes); the two types occupied mutually exclusive territories in regions such as the external globus pallidus (GPe). The distribution pattern of the Olig2-astrocytes suggested their involvement in GABAergic transmission. In this study, to compare the molecular signatures of the two astrocytic populations, we analyzed published single-cell RNA-seq databases of adult mouse brains. Unbiased classification of gene expression profiles and subsequent gene ontology analyses revealed that the majority of Olig2-astrocytes belonged to an astrocytic cluster that is rich in transporter-related genes, especially GABA transporters. To complement the in silico data analyses, we differentially isolated Olig2- and GFAP-astrocytes from the GPe using laser microdissection and compared their gene expression profiles. The Olig2-astrocytes expressed higher levels of glial GABA transporters (GAT-3 and GAT-1) than the GFAP-astrocytes, consistent with the results of the in silico and previous histological analyses. We propose that Olig2-astrocytes specifically modulate GABAergic transmission in the adult brain.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-09 腹側海馬アストロサイトにおけるGqPCRシグナルがマウス行動に与える影響 Role of GqPCR signaling of astrocytes in the ventral hippocampus on animal behaviors 繁冨英治¹、小泉修一¹ 1. 山梨大・院医・薬理 Eiji Shigetomi¹, Schuichi Koizumi¹ 1. Dept Neuropharmacol, Interdiscipl Grad Sch of Med Emerging evidence suggests that astrocytes, one of glial cells in the brain, receive information from neurons to alter their activities and in turn modulate neuronal information by releasing neuroactive substances. Astrocytes express a plethora of Gq-protein coupled receptor (GqPCR) to receive information from neurons to modulate neuronal function and brain function such as learning and memory. It has been shown that impairment of GqPCR-mediated Ca²⁺ signaling in astrocytes and/or gliotransmitter release (e.g. ATP, glutamate) causes depression-like behavior in mice. However, it is not clear whether astrocytic GqPCR signaling can drive behavior related to stress and motivation. We reasoned if we can alter behavior of mice by enhancing astrocytic GqPCR signaling selectively. To this end, we took a chemogenetic approach using Gq-DREADD. We focused on the ventral hippocampus (vHIP), a brain region contributing to stress responses and depression, where we expressed hM3Dq selectively in astrocytes by injecting AAV bilaterally. Activation of hM3Dq by DREADD agonist 21, a hM3Dq agonist, caused nice Ca²⁺ elevation in acute brain slices. Administration of the hM3Dq agonist did not affect place preference assessed by open filed test, suggesting that GqPCR in the vHIP astrocytes do not induce fear-related behaviors. Four days but not one day of administration of hM3Dq agonist decreased immobility time and the number of immobile episodes in the tail suspension test, suggesting that chronic stimulation of GqPCR signaling in astrocytes enhance escape-oriented behaviors. These data suggest that manipulation of GqPCR-mediated Ca²⁺ signaling in the vHIP astrocytes by chemogenetics activation may enhance resilience to stress.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-10 脊髄のアストロサイトにおけるグリシン除去システムの発達 The glycine-removal system in the astrocytes during development in the spinal cord 清水千草¹、小橋川晃広¹、小俣大輔¹、友寄竜司¹、岡野貴江¹、髙山千利¹ 1. 琉球大学・医学部・分子解剖学 Chigusa Shimizu-Okabe¹, Akihiro Kobashikawa¹, Daisuke Omata¹, Ryuji Tomoyose¹, Kie Okano¹, Chitoshi Takayama¹ 1. Department of Molecular Anatomy, School of Medicine, University of the Ryukyus Glycine and GABA are inhibitory neurotransmitters in the adult spinal cord. Both are removed from the synaptic cleft by glycine transporter1 (GlyT1) and GABA transporter 3 (GAT-3). We have previously investigated the developmental changes in GABA-removal system (Kim et al. 2014). However, the development of glycine-removal system in astrocytes remains unclear. To reveal the ontogeny of the glycine-removal system in the astrocytes, we developed an antibody of GlyT1 and examined the immunohistochemical localization of GlyT1 in the embryonic and postnatal mouse cervical spinal cord. It was confirmed that GlyT1 was expressed in the gray matter of the adult spinal cord by immunohistochemistry. Using a transmission electron microscopy, GlyT1 was located in the astrocytes around synapses and axons. On embryonic day 12 (E12), GlyT1 was localized in the mantle layer. On the other hand, weak GAT-3 immunolabeling was expressed to several radial fibers extending from the central canal to the pial surface. On E14, GlyT1 and GAT-3 was expressed in the ventral horn. GlyT1 was localized in the process of radial glia, whereas GAT-3 was expressed in the radial processes.After E18, GlyT1 was co-localized with GAT-3 in astrocytes in the dorsal and ventral horn. In our previous study, GlyT2, which uptakes glycine in presynaptic terminal, was first detected in the ventral horn on E16 (Sunagawa et al. 2017). The expression of GlyT1 was preceded than one of GlyT2. These results suggested that glycine and GABA were uptaken in different region of the radial glia in E12-14, but after that, both neurotransmitters were removed by the same astrocytes.Before the formation of glycinergic terminal, glycine-removal system in astrocytes might be ready.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-11 ミクログリア貪食能不全はアストロサイトにより補完される Astrocytic phagocytosis compensates for microglial dysfunction 小西博之¹、岡本峻幸¹、佐藤克明²、木山博資¹ 1. 名古屋大学、2. 宮崎大学 Hiroyuki Konishi¹, Takayuki Okamoto¹, Katsuaki Sato², Hiroshi Kiyama¹ 1. Nagoya Univ., 2. Univ. Miyazaki Microglia are the main phagocytes in the CNS. We recently established microglia ablation model without affecting non-microglial mononuclear cells, using microglia-specific diphtheria toxin receptor-knockin mice. In this ablation model, microglial debris were rapidly cleared even under the absence of functional microglia, raising a question how the debris were removed. The microglial ablation did not cause infiltration of non-microglial mononuclear phagocytes, such as perivascular macrophages and circulating monocytes, in CNS parenchyma, suggesting that the other cell population cleared microglial debris. We found by RNA-seq that astrocytes became pro-inflammatory activated upon microglial ablation. The activated astrocytes extended their processes to phagocytose microglial debris. Besides this ablation model, phagocytic astrocytes were also observed in Irf8 knockout mice, in which microglia were present but their phagocytic activity was impaired. Gene knockdown experiment of cultured astrocytes revealed that TAM family of phagocytic receptors expressed by astrocytes play central roles in the clearance. TAM phagocytic receptors were expressed by astrocytes even in the physiological condition, suggesting that astrocytes possess phagocytic property although it is normally hidden by microglia. This compensatory function of astrocytes may contribute to the maintenance of the CNS homeostasis.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-12* ミクログリア炎症反応に対するLysophosphatidylinositolの抑制効果 Lysophosphatidylinositol suppressed inflammatory responses by cultured microglia 南畑朋輝¹、森山光章¹、高野桂¹、中村洋一¹ 1. 大阪府立大学統合生理学教室 Tomoki Minamihata¹, Mitsuaki Moriyama¹, Katsura Takano¹, Yoichi Nakamura¹ 1. Laboratory of Integrative Physiology in Veterinary Sciences, Osaka Prefecture University Lysophosphatidylinositol (LysoPI) has been known to have variety of functions in several tissues and cells. Although LysoPI has been reported to be an endogenous ligand for G protein-coupled receptor (GPR) 55 which is predominantly expressed by microglia in the central nervous system (CNS), the functions of LysoPI and GPR55 in the CNS, in particular, the detailed effect of LysoPI on microglial inflammation is not fully understood. Here, we examined the effects of LysoPI on inflammatory responses and the involvement of GPR55 in its effects in microglia. We used the mouse microglial cell line BV-2 treated with lipopolysaccharide (LPS) and rat primary microglia to confirm some results in BV-2 cells. In BV-2 cells, LysoPI decreased LPS-induced nitric oxide (NO) production, the expression of inducible NO synthase protein, pro-inflammatory cytokine release, intracellular reactive oxygen species (ROS) generation and phagocytosis with no effect on cell viability. Also in primary microglia, LPS-induced intracellular ROS generation and phagocytic activity were attenuated by LysoPI. In addition, the decreased phagocytosis by LysoPI in BV-2 microglia was completely reversed by the treatment with CID16020046, a GPR55 antagonist, but the LysoPI-induced decrease in NO production was not affected. These findings suggest that LysoPI decreases microglial phagocytosis through GPR55-dependent pathway and NO production through non-GPR55 pathway. Such changes in microglial inflammatory responses may contribute to neuroprotection and LysoPI could be a therapeutic target for the treatment of neurodegenerative disorders including Parkinson's disease.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-13* グリホサート曝露ラット由来ミクログリアの生理的特性 Physiological properties of cultured microglia from glyphosate-exposed rat cerebellum 梅村清¹、岩永美咲¹、仲田しずか¹、トーマスティオンクオンスン¹、エドバグスプラスティカ¹、小林和人²、穂積直裕¹、野村洋子³、諫田泰成⁴、吉田祥子¹ 1. 豊橋技術科学大学、2. 本田電子株式会社、3. クイーンズカレッジニューヨーク市立大学、4. 国立医薬品食品衛生研 Kiyoshi Umemura¹, Misaki Iwanaga¹, Shizuka Nakada¹, Thomas Tiong Kwong Soon¹, Edo Bagus Prastika¹, Kazuto Kobayashi², Naohiro Hozumi¹, Yoko Nomura³, Yasunari Kanda⁴, Sachiko Yoshida¹ 1. Toyohashi University of Technology, 2. Honda Electronics Co., Ltd, Toyohashi, Japan, 3. Queens College, the City University of New York, New York, USA, 4. National Institute of Health Sciences, Kawasaki, Japan Glyphosate (GLY), the main compound of a broadly applied herbicide, is the negative substance on the Organization for Economic Cooperation and Development (OECD) Test Guidelines; however, the neurodevelopmental safeties of GLY are unknown. Previously we have shown that acute exposure of 100 or 250 mg/kg-GLY, or 250 mg/kg-AMPA to pregnant rats, leads to Purkinje cell death and behavioral disorder in developing cerebellum. In the nervous system composed of neurons, astrocytes, oligodendrocytes, and microglia, each cell is responsible for information transmission and homeostasis. Microglia overactivity is due to the neuronal disease ant the pathogenesis of autism spectrum syndrome (ASD). Observation of microglia exposed chemicals in vitro gives us information on toxicity, inflammation, and physiological activity of them. In this study, we investigated primary glial culture derived from the cerebellum of 250 mg/kg-GLY-exposed offspring at postnatal two days. Under the glia-conditioned medium, much microglia were observed in the culture derived from GLY-exposed pups at DIV 5, while major astrocytes and minor microglia were observed derived from control pups. We observed the phagocytotic activity of cultured microglia from GLY-exposed pups using the acoustic microscopy and their cytological features using the confocal microscopy. Microglia in the culture from GLY- exposed pups showed the ameboid form and migrated active. Despite that there are less microglia in the cerebellar cortex of GLY-exposed pups at postnatal two days, we suggest some microglia activation factors would be expressed in vitro.