Poster

Poster 3 Glia ポスター 3 グリア細胞

P3-1 Insulin regulates expression of neprilysin and IDE in cultured astrocytes インスリンは初代培養アストロサイトのネプリライシンとインスリン分解酵素発現を調節する Yamamoto Naoki（山本直樹）¹，石黒凌¹，谷田守²，鈴木健二³，祖父江和哉⁴ ¹Fac. Pharm., Sci., Hokuriku Univ. ²Dept. Physiology 2, Kanazawa Med. Univ. ³Dept. Pharmacy, Ritsumeikan Univ. ⁴Dept. Anesthesiol., Nagoya City Univ. Alzheimer's disease (AD) has been considered as a metabolic dysfunction disease, and is associated with impaired insulin signaling. Determining the potential mechanisms of insulin signaling and resistance is important for AD treatment. The impaired clearance of amyloid-β peptide (Aβ) significantly contributes to amyloid accumulation, typical in AD brains. Loss of expression of important Aβ degrading enzymes in the brain such as, neprilysin (NEP) and insulin degrading enzyme (IDE) can promote Aβ deposition in sporadic late-onset AD patients. We investigated whether insulin regulates the degradation of Aβ by inducing the expression of NEP and IDE, and then elucidated the relationship for activation of intracellular signal transduction in astrocytes. Treatment with insulin significantly induced the decrease of NEP expression and the increase of IDE expression in cultured astrocytes. Insulin-related effects on the expression of NEP and IDE were induced by the activation of ERK. NEP reduction and IDE induction were achieved by the release into the cultured medium and the translocation into cellular membranes of cultured astrocytes, respectively. Moreover, insulin-treated astrocytes significantly facilitated the degradation of exogenous Aβ. Interestingly, insulin-treated astrocytes pre-treated with ERK inhibitor markedly increased the levels of exogenous Aβ. These results suggest that insulin induced Aβ degradation via an increase in NEP secretion and IDE expression in astrocytes, through the activation of the ERK-mediated pathway. The inhibition of insulin-signaling pathway delayed Aβ degradation through inhibitions of these inductions for NEP and IDE, and competition with insulin and Aβ.		P3-2 Intracellular labile zinc level is a determinant of vulnerability of cultured astrocytes to oxidative stress 酸化ストレスに対するマウスアストロサイトの脆弱性は細胞内遊離型亜鉛レベルに依存する Nagasawa Kazuki（長澤一樹），古田能裕，大石晃弘 Dept. of Environ. Biochem., Kyoto Pharm. Univ. Recently, we found that treatment of cultured mouse astrocytes of ddY-strain mice (ddY-astrocytes) with 400 μM of H₂O₂ for 24 h increased intracellular labile zinc levels without cell toxicity. In contrast, 170 μM of H₂O₂ for 12 h is reported to kill mouse astrocytes obtained from C57BL/6-strain mice (C57BL/6-astrocytes) with increase of intracellular labile zinc. To clarify this discrepancy, we compared intracellular zinc levels and cell toxicity between H₂O₂-treated ddY- and C57BL/6-astrocytes. Exposure of C57BL/6-astrocytes to 170 or 400 μM of H₂O₂ for 12 h dose-dependently decreased cell viability and administration of plasma membrane-permeable zinc chelator TPEN prevented the 170 μM H₂O₂-induced astrocyte death, while neither concentration of H₂O₂ killed ddY-astrocytes. By absolute quantification of intracellular zinc levels with a zinc specific probe ZnAF-2, the level in H₂O₂-treated C57BL/6-astrocytes was higher than that in H₂O₂-treated ddY-astrocytes, and these increases were suppressed by the TPEN. There was no apparent difference in expression levels of zinc transporters ZIPs and ZnTs between the two astrocytes, while expression of a zinc releasable channel TRPM7 was found at the plasma membrane in ddY-astrocytes, but not in C57BL/6-astrocytes, although its total cellular expression levels were almost the same in both astrocytes. In addition, a TRPM7 blocker, 2-aminoethoxydiphenyl borate, increased the intracellular zinc levels in H₂O₂-treated ddY-, but not C57BL/6-, astrocytes. Collectively, it is suggested that vulnerability of astrocytes to oxidative stress depends on the increased levels of intracellular labile zinc, and TRPM7 localized at the plasma membrane contributes, at least in part, to ameliorate the cell injury by decreasing the zinc levels.

P3-3 Mechanism of thymidine incorporation into acid insoluble fraction via nucleoside transporters on oxidative stress DNA injury in Cultred Astrocytes 培養アストロサイトの酸化ストレスDNA障害におけるヌクレオシド輸送体を介した酸不溶性画分へのチミジン取り込み機構 Tanaka Koh-Ichi（田中康一）^1,2,3，富田和男^1,3，北中順惠²，北中純一²，佐藤友昭³，竹村基彦²，西山信好¹ ¹Division of Pharmacology, Department of Pharmacy, School of Pharmacy, Hyogo University of Health Sciences, Hyogo, Japan ²Department of Pharmacology, Hyogo College of Medicine, Hyogo, Japan ³Department of Applied Pharmacology, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan We have found that cultured astrocytes pretreated with N₆, 2’-O-dibutyryladenosine 3’,5’-cyclic monophosphate (DBcAMP), a permeable analogue of cAMP, but not astrocytes pretreated without DBcAMP and neurons, have the ability to incorporate thymidine into acid insoluble fraction via nucleoside transporters at an early time for repair on hydrogen peroxide (H₂O₂)-induced DNA injury. We studied expression of equilibrative nucleoside transporter (ENT) and concentrative nucleoside transporter (CNT) and the relation between thymidine incorporation into intracellular spaces (membrane transport) and acid insoluble fraction (DNA repair) on cultured astrocytes pretreated with DBcAMP in the presence and absence of H₂O₂ for oxidative stress. We concerned that astrocytes express ENT1, ENT2, and CNT2, CNT3, but not CNT1, by western blot, immunocytochemistry and RT-PCR, and H₂O₂ caused decrease in membrane transport of thymidine from extracellular spaces to intracellular spaces and increase in incorporation of thymidine into acid insoluble fraction to cultured astrocytes pretreated with DBcAMP via ENT2 and CNT3 by radioactive tracer analisis of [³H]-tymidine. These finding indicate that cultured differentiated astrocytes could incorporate thymidine effectively into acid insoluble fraction for repair on H₂O₂-induced DNA injury, although the function of ENT2 and CNT3 might be impaired.		P3-4 Mechanism of memory impairment in Astrocyte specific Tsc1 knockout mouse アストロサイト特異的Tsc1ノックアウトマウスにおける記憶障害のメカニズム Shimada Tadayuki（島田忠之），杉浦弘子，山形要人 Synaptic plasticity project, Tokyo Metropolitan Institute of Medical Science Mutations in the Tsc1 gene cause tuberous sclerosis complex (TSC). TSC patients harbor hamartomas in the brain and other organs. The neuropsychiatric symptoms of TSC patients include refractory epilepsy, autism spectral disorder and mental retardation. To investigate if the brain astrocytes contribute to the neuropsychiatric symptoms of TSC patients, we developed astrocyte-specific Tsc1 knockout mice (GFAP-Tsc1 cKO mice) and examined their phenotypes. Sociability was evaluated by 3 chamber test and contextual memory task was monitored by contextual fear discrimination test. GFAP-Tsc1 cKO mice showed abnormal social memory and impaired contextual memory. Immunohistochemical analyses revealed that GFAP-positive cells were increased in the brain, especially in the hippocampus, piriform cortex and amygdala. Moreover, the aberrant dendritic spines ware observed in dentate gyri of the hippocampi, where one of the most severe gliosis was observed.Because Tsc1 protein makes a complex with Tsc2 and inactivates Rheb protein together, Rheb could be activated in the astrocytes in GFAP-Tsc1 cKO mice. Treatment with Rheb inhibitors improved abnormal behaviors of the cKO mice and spine malformation. Gliosis in the dentate gyri was also decreased by Rheb inhibitor administration. Thus, activation of the Tsc/Rheb signaling in the astrocytes could induce astrogliosis, resulting in the change of spine morphology. The spine deformity may cause deterioration of neural function and would develop abnormal behaviors observed in a mouse model of TSC.

P3-5 Establishment of differentiation methods and its functional analysis of microglia-like cells from mice hematopoietic stem cells in peripheral blood マウス末梢血から採取した造血幹細胞を用いたミクログリア様細胞への分化誘導とその機能解析 Kuroda Eriko（黒田絵莉子）¹，戸田侑紀¹，芦原英司¹，高田和幸² ¹Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto, Japan ²Division of Integrated Pharmaceutical Sciences, Kyoto Pharmaceutical University, Kyoto, Japan [Introduction] Microglia are the resident immune cells in the brain and implicated in the development of age-related neurodegenerative disorders, such as Alzheimer’s disease (AD). The primary cause of AD is thought to be amyloid-β (Aβ) deposition in the brain, called “amyloid hypothesis”. We previously found microglia possess the ability of Aβ phagocytosis, and have hypothesized that the transplantation of microglia into the brain is a novel therapeutic strategy against AD. However, preparing human primary microglia is ethically and technically difficult. In this study, we examined the potential of hematopoietic stem cells in peripheral blood (PBSCs) for the replacement of microglia.[Methods] C57BL/4 mice were intraperitoneally injected with granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist (AMD3100) for 5 days. PBSCs were isolated as lineage-negative/c-kit-positive cells, which enrich the population of HSCs, by magnetic cell sorting. PBSCs were then treated with macrophage colony-stimulating factor (M-CSF) and interleukin-34 (IL-34) to induce microglia-like (ML) cells. We analyzed the characteristics of the ML cells by flow cytometry and immunocytochemical staining.[Results and Discussion] M-CSF and IL-34 treatment induced PBSCs to the cells that expressed microglia markers, such as ionized calcium binding adapter molecule 1, CD11b, F4/80, triggering receptor expressed on myeloid cells 2, TMEM119, and P2Y12R. They had the same phagocytic ability of Aβ compared to microglia. Furthermore, ML cells from aged mice also showed the phagocytic ability of Aβ. Thus, we establish a culture system to differentiate PBSCs to ML cells. We propose PBSCs as a promising cell source for the low-invasive and safer cell therapy against AD.		P3-6 Donepezil suppresses intracellular Ca2+ mobilization through the PI3K pathway in rodent microglia 齧歯類ミクログリアにおいて、ドネペジルはPI3K経路を介して細胞内カルシウムイオン上昇を抑制する Haraguchi Yoshinori（原口祥典），溝口義人，今村義臣，扇谷昌弘，門司晃 Department of Psychiatry, Faculty of Medicine, Saga University, Saga, Japan Microglia are resident innate immune cells which release many factors including proinflammatory cytokines or nitric oxide (NO) when they are activated in response to immunological stimuli. Pathophysiology of Alzheimer’s disease (AD) is related to the inflammatory responses mediated by microglia. Intracellular Ca²⁺ signaling is important for microglial functions such as release of NO and cytokines. In addition, alteration of intracellular Ca²⁺ signaling underlies the pathophysiology of AD, while it remains unclear how donepezil, an acetylcholinesterase inhibitor, affects intracellular Ca²⁺ mobilization in microglial cells. We examined whether pretreatment with donepezil affects the intracellular Ca²⁺ mobilization using fura-2 imaging and tested the effects of donepezil on phagocytic activity by phagocytosis Assay in rodent microglial cells.In this study, we observed that pretreatment with donepezil suppressed the TNFα-induced sustained intracellular Ca²⁺ elevation in both rat HAPI and mouse primary microglial cells. Pretreatment with donepezil suppressed the TNFα-induced intracellular Ca²⁺ elevation through the PI3K pathway in rodent microglial cells. Using DAF-2 imaging, we also found that pretreatment with donepezil suppressed the production of NO induced byTNFα treatment and the PI3K pathway could be important for the donepezil-induced suppression of NO production in rodent microglial cells. Finally, phagocytosis assay showed that pretreatment with donepezil promoted phagocytic activity of rodent microglial cells through the PI3K but not MAPK/ERK pathway. These suggest that donepezil could directly modulate the microglial function through the PI3K pathway in the rodent brain, which might be important to understand the effect of donepezil in the brain.

P3-7 Microglia enhance the functional maturation of blood-brain barrier by regulating the cytokine/chemokine dynamics ミクログリアはサイトカインケモカインの濃度を調整し、血液脳関門機能発達を制御している Shigemoto-Mogami Yukari（最上（重本）由香里）¹，干川和枝¹，諫田泰成¹，佐藤薫¹ ¹Lab. Neuropharmacol., Div. Pharmacol., NIHS ²Dept. Neuropharmacol., Univ. of Yamanashi The blood-brain barrier (BBB) function is regulated by various cells comprised of neurovascular unit (NVU). Microglia are present around the capillaries in the developmental brain, however, their role on the functional maturation of the BBB is still unclear. In this study, we investigated the roles of microglia in the BBB maturation using in vitro BBB model comprised of endothelial cells, pericytes, and astrocytes. When we added non-stimulated microglia (Non-MG) to astrocytes in the brain side during the maturation period, trans-endothelial electrical resistance (TEER) and the expression level of claudin-5, a member of tight junction proteins (TJs) were significantly increased. On the other hand, when we added LPS-activated microglia (LPS-MG), significant decrease in the TEER and the expression levels of tight junction proteins (ZO-1, occludin, and claudine-5) were detected. We next investigated involvement of cytokines/chemokines in the effects of microglia. Among cytokines/chemokines, the concentrations of VEGF showed a correlation with changes of BBB barrier functions, while the concentration Fractalkine showed an inverse correlation with the changes of BBB functions. We clarified that non-MG-induced increase in TEER was induced by VEGF, while non-MG-induced increase in TJ protein expressions were induced by inhibition of fractalkine signal. These results suggest that microglia regulate BBB maturation by controlling the concentration of cytokines/chemokines.		P3-8 Functional expression of TRP channels and their roles in oligodendrocyte precursor cells オリゴデンドロサイト前駆細胞におけるTRPチャネルの機能的発現と細胞機能における役割 Shirakawa Hisashi（白川久志）¹，大橋佳奈¹，出屋敷綾音¹，永安一樹¹，柴崎貢志²，金子周司¹ ¹Dept. Mol. Pharmcol., Grad. Sch. Pharm. Sci., Kyoto Univ. ²Dept. Mol. Cell. Neurobiol., Grad. Sch. Med., Gunma Univ. Oligodendrocytes, which differentiate from oligodendrocyte precursor cells (OPCs), ensheath axons with myelin, play an essential role in rapid conduction of action potentials, and metabolically support neurons. Elucidation of the mechanisms underlying the proliferation, migration and differentiation of OPCs is considered indispensable for determining the causes of CNS diseases. However, the relationship between these functions of OPCs and their intracellular Ca²⁺ signaling has not been fully elucidated. Here, we focused on transient receptor potential (TRP) channels, especially TRP vanilloid 4 (TRPV4) and TRP melastatin 3 (TRPM3). In the experiments with TRPV4, a Ca²⁺-permeable channel that responds to hypo-osmolarity, mild temperature and endogenous arachidonic acid metabolites, Trpv4 mRNA was detected in OPCs in vivo and in primary cultured rat OPCs. In Ca²⁺ imaging experiments, treatment with the selective TRPV4 agonist GSK1016790A induced Ca²⁺ responses in OPCs in a concentration-dependent manner, which was almost completely suppressed by the selective TRPV4 antagonist HC067047. Stimulation of TRPV4 by GSK1016790A augmented OPC proliferation, which was abolished by HC067047, the intracellular Ca²⁺ chelator BAPTA-AM, and the protein kinase C inhibitor bisindolylmaleimide II. As to TRPM3, Trpm3 ⁺/Pdgfra⁺ OPCs were detected in vivo and in vitro. Ca²⁺ imaging showed that PS (30-100 μM) increased the Ca²⁺ concentration in cultured OPCs, which was inhibited by the selective TRPM3 inhibitor isosakuranetin. Taken together, these results suggest that TRPV4 and TRPM3 is functionally expressed in OPCs in vivo and in vitro, and TRPV4 increases the proliferation of these cells without affecting their ability to differentiate into oligodendrocytes.

P3-9 Electroconvulsive stimulation transiently enhances the permeability of the rat blood-brain barrier and induces astrocytic changes 電気痙攣刺激（ECS）による血液脳関門の透過性変化に関する研究 Ito Masanobu（伊藤賢伸），金城智也，鈴木利人，新井平伊 Department of Psychiatry, Juntendo University Faculty of Medicine, Tokyo 背景：血液脳関門（BBB）は、病原体が脳に侵入することを防ぐなど、生体防御にとって重要な働きを持っている。一方で精神神経薬理学的には、BBBが多くの薬剤の通過を阻害することで使用できる薬剤が限定されている。BBBの透過性を自在に変化させることができれば、これまで使用が難しいとされていた薬剤も使用可能となる可能性がある。電気痙攣刺激（ECS）は、本来通過しない高分子を一過性に通過する可能性が指摘されていたが、その変動は血圧変動に伴うものと考えられてきた。しかしながら近年様々な研究が進むにつれて、BBBが単に血管内皮細胞のタイトジャンクションによって形成されているのではなく、その周囲を包む周囲細胞やアストロサイトの終末足などにより複合的に形成されたneurovascular unitを形成していることが明らかとなっている。我々はその透過性変化を定量化し明確化するために、ラットを用いて研究を行った。方法：Wistar rat maleにECSを施行し、蛍光色素であるsodium fluoresceinを血管内に投与した。投与後120分循環させた。麻酔後灌流固定し、脳内の蛍光をin vivo imagine systemを使用して可視化し、さらに定量した。結果：ECS直後にsodium fluoresceinを投与すると、コントロールと比較し、ECS群では有意に脳の蛍光発光は増加していた。しかし、この有意差は、ECS後15分後にsodium fluoresceinを投与しても認められなかった。さらに脳内から分泌されるS100Bの血中濃度を測定すると、ECS群ではコントロールと比較して有意に上昇していた。電顕写真で確認すると血管内皮細胞の明らかな違いは検出されなかったが、毛細血管を取り巻くアストロサイトの終末足は、ECS群では腫大していた。結語：ECSによりBBB透過性は一過性に双方向性に亢進するが、可逆性であることが示された。ECSによるタイトジャンクションの破壊は確認できず、別の経路での透過性亢進が示唆された。今後はECSによる物質の透過がどのような経路で行われるのか明らかにして行きたい。		P3-10 The effect of carba-derivative of cyclic phosphatidic acid on microglial and astrocyte cells during the repair of a stab-wounded mouse cerebral cortex マウス大脳皮質穿刺損傷の修復過程における2ccPAの効果～ミクログリア及びアストロサイトに着目して～ Nakashima Mari（中島麻里）^1,2，橋本恵^1,2,4，濱野文菜^1,2，池島（片岡）宏子^5,6，後藤真里^2,3，室伏きみ子³，宮本泰則^1,2 ¹Div. of Life Sci., Grad. Sch. of Humani. and Sci., Ochanomizu Univ. ²Inst. for Human Life Innov., Ochanomizu Univ. ³Research Division of Human Welfare Science, Ochanomizu Univ. ⁴Research Fellow of Japan Society for the Promotion of Science. Kojimachi, Chiyoda-ku, Tokyo, Japan. ⁵Faculty of Science and Engineering, Waseda University, Okubo, Shinjuku-ku, Tokyo, Japan. ⁶Department of Pharmacology and Neuroscience, Keio University School of Medicine, Shinanomachi, Shinjuku-ku, Tokyo, Japan. Traumatic brain injury (TBI) induces inflammation around the injury site. We previously revealed that hemorrhage and inflammation during the repair of a stab-wounded cerebral cortex are attenuated by carba-derivative of cyclic phosphatidic acid (2ccPA).2ccPA is one of the compounds in which phosphate oxygen is replaced with a methylene group at the sn-2 position, and a metabolically stabilized derivative of cPA. In this study, we aimed to characterize the effects of 2ccPA on microglial and astrocyte cells during the repair of stab-wounded mouse cerebral cortex. First, the effect of 2ccPA on microglial cells in the stab-wounded regions were examined. The administration of 2ccPA to brain injured mice decreased a microglia marker, Iba1 mRNA expression level, suggesting that 2ccPA attenuates the inflammation after the stab wound via microglial cells. Activated microglial cells can be categorized into two opposite types. M1 phenotype, which promotes inflammation, and M2 phenotype, which produces the anti-inflammatory cytokine. In the present study, the administration of 2ccPA decreased the number of a M1 marker, CD86-positive cell, and increased the number of a M2 marker, CD206-positive cell, indicating that 2ccPA modulates the microglial polarization toward M2 phenotype. In addition, we analyzed the mRNA and protein expression levels of astrocyte marker, GFAP. 2ccPA down-regulated both of the levels. These data suggest that the suppressive effect of 2ccPA to inflammation after the stab wound occurs not only via microglial activation but also via astrocyte activation. Taken together, these results suggest that 2ccPA affects the properties of microglial cells and astrocytes after the stab wound and thereby contributes to the suppression of inflammation.