e ポスター

e ポスター 3. 神経免疫、神経内分泌、神経栄養因子 e Poster 3. Neuroimmunity, Neuroendocrine, Neurotrophic factor

2020/9/10　14:00～15:00　オンデマンドB-1 P1-14 視床下部腹内側核に発現する下垂体アデニル酸シクラーゼ活性化ポリペプチドは夜間において状態依存的に摂食を促進する． Pituitary adenylate cyclase-activating polypeptide in the ventromedial hypothalamus state-dependently increased appetite in the nocturnal period. 神戸悠輝¹、グエンチュン¹、栗原崇¹、中町智哉²、新谷紀人³、橋本均³、宮田篤郎¹ 1. 鹿児島大学、2. 富山大学、3. 大阪大学 Yuki Kambe¹, Thanh Trung Nguyen¹, Takashi Kurihara¹, Tomoya Nakamachi², Norihito Shintani³, Hitoshi Hashimoto³, Atsuro Miyata¹ 1. Kagoshima Univ., 2. Toyama Univ., 3. Osaka Univ. Pituitary adenylate cyclase-activating polypeptide (PACAP) is abundantly expressed in the hypothalamus among the central nervous system and contributes to the hypothalamic functions including appetite regulation. Although it is suggested that food intake is decreased in PACAP (-/-) mice, the detailed mechanism is still under discussion. To address this issue, we attempted to manipulate PACAP signaling, and investigated food consumption and peptide expression which are known to be involved in the appetite regulation in the hypothalamus. The food consumption of the (-/-) mice at 8 h refeeding after 2 days fasting was significantly lower than that of the PACAP (+/+) mice. The nocturnal and the daily food intake of (-/-) mice was significantly lower than those of (+/+) mice, but the diurnal food intake was unchanged. mRNA expression level of agouti-related peptide (AgRP) was decreased, but that of proopiomelanocortin (POMC) was increased in the hypothalamus of (-/-) mice at 4 h refeeding. Furthermore, intracerebroventricular administration of PACAP antagonist, PACAP_6-38, decreased food intake and body weight gain in (+/+) mice 1, 2, 4 h after refeeding, and decreased the expression level of AgRP at 4 h refeeding. Among the subregions of hypothalamus, the PACAP mRNA expression of the ventromedial hypothalamus (VMH) was significantly increased by fasting as compared with that of the paraventricular hypothalamus (PVH). The selective overexpression of PACAP by the infection of adeno-associated virus (AAV) harboring PACAP cDNA in the VMH region resulted in an increment of the food intake in the nocturnal period and at 8 h refeeding, and increased the AgRP expression in the hypothalamus in the nocturnal period. Taken together, the present results suggest that the appetite is enhanced by the increment of PACAP expression in the VMH via modulation of AgRP expression.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-15 Sorting nexin 25の視交叉上核における日内発現変化とBDNF-TrkBシグナリング調節への関与 Circadian rhythms of sorting nexin 25 in the suprachiasmatic nucleus and contribution to the regulation of BDNF-TrkB signaling 竹村晶子¹、石西綾美¹、田中達英¹、辰巳晃子¹、山野眞利子¹、奥田洋明²、長野護³、南陽一³、重吉康史³、和中明生¹ 1. 奈良県立医科大学、2. 金沢大学、3. 近畿大学 Shoko Takemura¹, Ayami Isonishi¹, Tatsuhide Tanaka¹, Kouko Tatsumi¹, Mariko Yamano¹, Hiroaki Okuda², Mamoru Nagano³, Yoichi Minami³, Yasufumi Shigeyoshi³, Akio Wanaka¹ 1. Nara Medical University, 2. Kanazawa University, 3. Kindai University In the hypothalamic suprachiasmatic nucleus (SCN), receptor-mediated signaling is important in communication among individual neurons to entrain mammalian circadian rhythms. Both brain derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyocin-related receptor tyrosine kinase B (TrkB) exhibit circadian rhythms in the SCN, suggesting that the changes in the TrkB endocytosis and subsequent degradation may be involved in the circadian rhythm regulation. In the present study, we focused on the sorting nexin 25 (SNX25). SNX25 belongs to the sorting nexin superfamily, whose members regulate intracellular organelle trafficking and membrane recycling. By co-immunoprecipitation assay, we found that SNX25 bound to the full-length TrkB, but not with its C-terminal-truncated isoforms. In the in vitro system, TrkB was internalized and degraded in response to BDNF stimulation. Overexpression of SNX25 accelerated degradation of full-length TrkB in this system. In the mouse SCN, Snx25 mRNA and SNX25 protein exhibited circadian rhythm-dependent expressions; both were maximal at about zeitgeber time (ZT) 16 in the subjective night and minimal at ZT8 in the subjective day. Prominent SNX25 immunoreactivity was found in the arginine vasopressin-positive neurons of the SCN. Given the fact that the BDNF exhibits peak expression in the subjective night, our data suggest that SNX25 promotes the trafficking of TrkB for lysosomal degradation and thereby contributes to the circadian rhythm regulation.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-16 血液脳関門機能成熟におけるミクログリアの役割 The role of microglia in the functional maturation of blood brain barrier 最上（重本）由香里^1,2、干川和枝¹、北村貴美子¹、佐藤薫¹ 1. 国立医薬品食品衛生研究所薬理部、2. 山梨大学医学部薬理 Yukari Shigemoto-Mogami^1,2, Kazue Hoshikawa¹, Kimiko Kitamura¹, Kaoru Sato¹ 1. Div. Pharmacol., National Institute of Health Sciences, 2. Dept. Pharmacol., Yamanashi Univ. The blood-brain barrier (BBB) restricts the transport of substances between vasculature and brain. Recent studies have clarified that various kinds of cells in neurovascular unit are related to the BBB functions. We are investigating the roles of microglia in BBB functional maturation. We first used commercially available in vitro BBB model. When we added primary microglia to the brain side of the in vitro BBB model during the maturation period, trans-endothelial electrical resistance (TEER) and the expression level of claudin-5, the essential member of tight junction proteins (TJs), were significantly increased. On the other hand, when we added LPS-activated microglia, the TEER and the expression levels of TJs were significantly decreased. We examined the involvement of cytokines/chemokines in the effects of microglia on the BBB functions. We comprehensively compared the concentrations of cytokines/chemokines in the above two conditions with totally opposite outcomes. The results suggested that the VEGF and fractalkine are involved in the pro-maturational effects of microglia. We therefore examined the effects of VEGF and fractalkine on the BBB functional maturation respectively, and clarified that microglia-induced increase in TEER was mediated by VEGF, while the increases in TJs were mediated by the inhibition of fractalkine signaling in the presence of VEGF. We further attempted to clarify whether the mechanisms including VEGF and fractalkine are active in in vivo BBB maturation. We first examined the time course of BBB maturation from P1-30. We observed that the capillaries in the white matter were getting wrapped by a lot of microglia in the process of BBB maturation. We are currently determining the correlation between BBB maturation, microglial wrapping of the capillaries, and the concentration changes in cytokines/chemokines.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-17* 全身性炎症時の髄膜-CNSにおける免疫細胞の挙動の可視化 Visualizing the dynamic motility of the immune cells in meninges and CNS with pathological conditions in vivo 進藤麻理子¹、辻貴宏^1,3、加藤大輔²、和氣弘明^1,2 1. 神戸大学大学院医学研究科、2. 名古屋大学大学院医学系研究科、3. 京都大学大学院医学研究科 Mariko Shindo¹, Takahiro Tsuji^1,3, Kato Daisuke², Hiroaki Wake^1,2 1. Kobe University Graduate School of Medicine , 2. Nagoya University Graduate School of Medicine , 3. Kyoto University Graduate School of Medicine CNS have immune privilege due to the blood-brain barrier and blood-meningeal barrier. Previous studies have shown that the meninges contain various immune cells that invade to CNS with pathological condition in CNS such as multiple sclerosis and Alzheimer's disease. Although meninges can act as an interface between the CNS and systemic immune system, little has been known about its immune functions and how immune cells affect on CNS under systemic inflammatory conditions. In this study, we used in vivo two-photon microscopy and Cx3cr1^gfp/+ mice express EGFP in monocytes or macrophages, dendritic cells, NK cells, and microglia to visualize CX3CR1-positive cells in meninges and brain parenchyma, and observed the interaction between parenchymal microglia and systemic immune cells that express CX3CR1 to elucidate the role of physiological and pathological functions of CX3CR1-positive cells in meninges. We visualized the various type of the dynamic motile CX3CR1-positive cells in meninges to measure their activation with systemic inflammation induced by intraperitoneal injection of lipopolysaccharide (LPS). The density of CX3CR1-positive cells at the boundary between meninges and parenchyma was increased as compared with pre-injection of LPS. In particular, the number of CX3CR1-positive cells was increased in the subarachnoid space area fluorescently labeled with dextran administered directly into the subarachnoid. The development of this experimental system will allow to assessment of the meningeal-parenchymal immune responses in various disease processes. Furthermore, the spatial and temporal observation of interactions with other glial cells, neurons, and immune cells, and integration with previous findings, is expected to uncover new roles for the immune system in CNS.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-18* インターロイキン17Aの大脳皮質構築への影響とASD病態への関与 Effects of interleukin 17A on cortical architecture and the involvement in ASD pathology 鮑培毅^1,2、佐々木哲也^1,2、高田拓弥¹、武井陽介^1,2 1. 筑波大医生命医科学・解剖学・神経科学、2. 筑波大院人間総合ニューロサイエンス学位プロ BAO PEIYI^1,2, Tetsuya Sasaki^1,2, Takuya Takada¹, Yosuke Takei^1,2 1. Dept Anat & Neurosci, Fac Med, Univ of Tsukuba, Ibaraki, Japan, 2. 2.Doctorate Program Neurosci, Grad Sch Comp Human Sci, Univ of Tsukuba, Ibaraki, Japan Th17 cells, which produce the proinflammatory cytokine IL-17, play important roles in the induction of inflammation. A previous study has reported that increase in IL-17A contribute to disruption of blood brain barrier integrity, which leads to memory disturbance in mice. Furthermore, elevated levels of IL-17A have been detected in the serum of a subset of ASD patients. However, to date, the role of IL-17A in pathogenesis of neuropsychiatric disorders remains to be determined. To evaluate the effect of long-term upregulation of IL-17A to function and development of CNS in vivo, and elucidate the significance of IL-17A in pathogenesis of neuropsychiatric diseases, we used transgenic mice with C57BL/6 background in which retinoic acid related orphan receptor gamma-t (RORγt) is overexpressed under the control of human CD2 gene (RORγt Tg mice). RORγt is known to be a transcription factor required for differentiation of Th17 cells. In this study, we confirmed that serum IL-17A level is markedly upregulated in the RORγt Tg mice compared to WT mice. To obtain a first clue of the effect of IL-17A upregulation in CNS, we performed immunohistochemical analysis of RORγt Tg mice using various neuronal and glial markers, and analyzed behaviors that could relate with higher brain functions. In order to examine the direct effects of IL17 on cortical formation, intracerebroventricular administration of IL17A during mid-fetal period was used to investigate abnormalities in cortical structure and its effects on neuronal and glial production. In this presentation, we discuss their significance in pathogenesis of neuropsychiatric disorders such as ASD and schizophrenia.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-19* GTPシクロヒドロラーゼI遺伝子のエンハンサー領域におけるマイクロサテライト多型とパーキンソン病の関連 Microsatellite polymorphism in the enhancer region of the human GTP cyclohydrolase I gene and its application in an association study of Parkinson's disease. 宮木詩織¹、佐竹渉²、戸田達史²、原怜¹、一瀬宏¹ 1. 東京工業大学生命理工学院、2. 東京大学神経内科学 Shiori Miyaki¹, Wataru Satake², Tatsushi Toda², Satoshi Hara¹, Hiroshi Ichinose¹ 1. School of Life Science and Technology, Tokyo Institute of Technology, 2. Department of Neurology, The University of Tokyo Introduction Tetrahydrobiopterin (BH4) is a cofactor for the biosynthesis of monoamine neurotransmitters and nitric oxide. BH4 is synthesized from GTP through three enzymatic reactions, and the first and rate-limiting enzyme is GTP cyclohydrolase I (GCH). Previously we have identified an enhancer region in Intron 1 of the GCH gene responding to immune stimulation by LPS. There is a microsatellite of GT repeats very close to the enhancer region. In this study, we measured the transcriptional activity of the GCH gene with the different number of the GT repeats using a luciferase reporter assay and examined the allele frequency of the number of the GT repeats in patients with Parkinson's disease (PD). Methods We constructed the luciferase reporter vectors consisting of 5.2-kb of the promoter region of the human GCH gene with the part of intron1 contains enhancer region and the different number of the GT repeats from 15 to 30. Transcription activity were examined in human neuroblastoma SH-SY5Y cells and mouse macrophage RAW264.7 cells. The number of GT repeats in the human genome from controls (n = 233) and PD patients (n = 242) were determined with fluorescent labelled primers. Results and Discussion The transcriptional activities measured by the luciferase assay were altered depending on the number of the GT repeats in SH-SY5Y cells. We also assayed in LPS-stimulated and non-stimulated RAW264.7 cells. While non-stimulated cells did not exhibit a significant difference, there were significant differences among the repeat lengths in LPS-stimulated cells. Then, we investigated the number of the GT repeats in PD patients and compared the allele frequency with that in the control individuals. Most frequent allele was the 20 repeats with 43%, and the number of the GT repeats varied from 17 to 29. The frequency of 21 repeats were slightly (p = 0.089) less in PD patients compared with controls. Our results suggest that the GCH transcriptional activity should be affected by the number of the GT repeats in the GCH gene and the differences in the number of repeats may have some effects in the development of PD.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-20* 胎生期グリホサート曝露による遅発性神経毒性及びミクログリア活性 Delayed neurotoxicity of prenatal glyphosate exposure and microglial activation 二神健¹、Thomas Tiong Kwong Soon¹、Christine Lee Li Mei¹、大塚晴子¹、野村洋子²、諫田泰成³、吉田祥子¹ 1. 豊橋技術科学大学、2. ニューヨーク市立大学クイーンズ校、3. 国立医薬品食品衛生研究所 Ken Futagami¹, Thomas Tiong Kwong Soon¹, Christine Lee Li Mei¹, Haruko Ohtsuka¹, Yoko Nomura², Yasunari Kanda³, Sachiko Yoshida¹ 1. Toyohashi Univ. of Technology, 2. Queens College, the City Univ. of New York, 3. National Institute of Health Sciences Prenatal chemical exposure is a growing health concern as it can cause a lifelong lasting effect on the psychological and physiological health in both pregnant mother and offspring, which supports the Developmental Origins of Health and Disease (DOHaD) hypothesis. 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 or aminomethylphosphonic acid (AMPA), a metabolite in the soil, are unknown. The primary herbicidal function of GLY is to inhibit a key plant enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which participates in the biosynthesis of aromatic amino acids via the shikimate pathway in bacteria, fungi, and plants, so that GLY may have the potential to modify the animal and human gut microbiota. Previously we have shown that acute exposure of 100 or 250 mg/kg- GLY, or 250 mg/kg-AMPA to pregnant rats at G15 lead to Purkinje cell death and behavioral disorder in developing cerebellum of the offspring. In this study, we investigated the mechanism of neurodevelopmental toxicity by prenatal GLY exposure. In treated offspring, their growth was depressed, not a first week but two weeks after birth. Additionally, the expression of IL-1 beta, an inflammatory cytokine, was increased at P5 transiently, whereas iNOS expression was increased after P16 in the GLY-exposed cerebellar cortex. Microglia were activated in the Purkinje layer both in acute and chronic GLY-exposed offspring. We suggest GLY would induce delayed inflammation in developing neuronal systems.		2020/9/10　14:00～15:00　オンデマンドB-1 P1-21* 発生期LPS神経毒性における細胞学的メカニズム Cytological mechanism of developmental neurotoxicity embryonic LPS exposure 大塚暖子¹、シャルマディヴェール¹、常本和伸²、トーマスティオンクオンスン¹、諫田泰成²、吉田祥子¹ 1. 豊橋技術科学大学、2. 国立医薬品食品衛生研究所 Haruko Otsuka¹, Sharumadhi Veloo¹, Kazunobu Tsunemoto², Kwong Soon Thomas Tiong¹, Yasunari Kanda², Sachiko Yoshida¹ 1. Toyohashi University of Technology, 2. National Institute of Health Sciences Autism Spectrum Disorders (ASD) and psychiatric disorders are problems that have been rising in recent years, and maternal stress during pregnancy is one of the candidates causing neurological disorders in children. On the other hand, innate immunity changes due to infection and antibiotics may cause some neurological disorders. Intestinal environment and inflammation play important roles in immune homeostasis, and neurodevelopmental health would be maintained intestinal microbiota and resistance to inflammation. We have reported neurological, cytological, and behavioral alterations of ASD model rats exposed valproic acid (VPA), chlorpyrifos (CPF), and lipopolysaccharide (LPS). LPS, which is in the outer membrane of Gram-negative bacteria, is a proinflammatory factor via binding to Toll-like receptor 4 and may cause mental disorders. Cerebellar degeneration is one of the pathological focal points in observing communication disorders and behavior patterning of autism and mental disorders. In this study, we investigated the relationship between cell death and endoplasmic reticulum stress. LPS-treated pups, which were exposed to 100 µg / kg of LPS on embryonic day15, showed increased inflammatory cytokine expression such as TNFalpha and IL-1beta compared to control rats. Additionally, the LPS-treated pups show out of misalignment and lacking of the Purkinje cells and an increase of microglia, whereas excessive folds in cerebellar V and Ⅵ lobules were confirmed. They seem inconsistent because the Purkinje cells were decreased, but granule cell-related lobules were extended. We hypothesize the balance between protective and apoptotic effects of endoplasmic reticulum stress would relate to LPS-induced mental disorder.

2020/9/10　14:00～15:00　オンデマンドB-1 P1-22 非侵襲的生体イメージングを用いたマウス脳内における活動依存的なBDNF遺伝子発現誘導の可視化 Visualizing activity-regulated BDNF expression in living mouse brain using non-invasive in vivo imaging 福地守¹、萩原なみ¹、三反崎聖¹、齊藤亮平²、牧昌次郎²、中島夢奈¹、森寿³ 1. 高崎健康福祉大学、2. 電気通信大学、3. 富山大学 Mamoru Fukuchi¹, Nam Hagiwara¹, Mitazaki Satoru¹, Ryohei Saito², Shojiro Maki², Yumena Nakajima¹, Hisashi Mori³ 1. Takasaki Univ. of Hlth. & Welfare, 2. The Univ. of Electro-Communications, 3. Univ. of Toyama BDNF is a neurotrophic factor that plays a fundamental role in the expression of higher brain functions such as memory and learning. Recently, it has been suggested the correlation between brain BDNF levels and cognitive function, as well as Alzheimer's disease. For these reasons, we are focusing on the temporal and spatial changes in BDNF expression in the brain to elucidate mechanisms underlying age-related cognitive decline and Alzheimer's disease. We have been working on visualizing changes in BDNF expression in living mouse brains by using a transgenic mouse strain termed "BDNF-Luciferase (BDNF-Luc) mouse". However, D-luciferin, which is a substrate of firefly luciferase, has poor BBB permeation. Therefore, it was quite difficult to detect bioluminescence signals in the brain region. To solve this problem, a new luciferase substrate, AkaLumine hydrochloride, also known as “TokeOni”, which can permeate BBB and emits bioluminescence light in the near-infrared wavelength, was administered to BDNF-Luc mice. We successfully detected bioluminescence signals in the brain region. Furthermore, using TokeOni, we succeeded in visualizing the induction of BDNF expression in the hippocampus after administration of kainic acid and the induction of BDNF expression in the visual cortex after visual stimulation, in living mice. Therefore, it was strongly suggested that TokeOni is a useful tool that makes it possible to visualize BDNF expression changes in the living brain in a non-invasive manner.