グリア、グリア-ニューロン相互作用 Glia and Glia-Neuron Interaction
P3-1-33 ATF6αの欠損はマウス脳虚血モデルにおいてグリア細胞死及び遷延性神経障害を引き起こす Deletion of ATF6α causes glial death and delayed neuronal death after middle cerebral artery occlusion in mice ○北尾康子1, 上出智也2, 稲畠有規1, 橋田耕治1, 寳田-家又美佳1森和俊3, 高橋良輔4, 松山知弘5, 吉川陽文2, 濱田潤一郎2, 堀修1 ○Yasuko Kitao1, Tomoya Kamide2, Yuki Inahata1, Koji Hashida1, Mika Takarada-Iemata1, Ta Minh Hieu1, Kazutoshi Mori3, Ryosuke Takahashi4, Tomohiro Matsuyama5, Akifumi Yoshikawa2, Jun-ichiro Hamada2, Osamu Hori11 金沢大学院　医　神経分子標的学1, 金沢大院　医　脳神経外科2, 京都大院　理　生物物理3, 京都大　医　臨床神経4, 兵庫医大　神経再生5 Dept Neuroanat, Kanazawa Univ, Kanazawa1, Dept Neurosurg, Kanazawa Univ, Kanazawa2, Dept Biophysics, Kyoto Univ, Kyoto3, Dept Neurol Kyoto Univ, Kyoto4, Inst Adv Med, Hyogo Med Sch, Kobe5 Accumulating evidence suggests a crucial role for the unfolded protein response (UPR) in brain ischemia. In this study, we investigated the relevance of ATF6α, a master transcriptional factor in the UPR, after permanent middle cerebral artery occlusion (MCAO) in mice. Enhanced level of expression of GRP78, a downstream molecule of ATF6α, was observed in both of neurons and glial cells in wild-type mice after MCAO. Analysis using wild-type and ATF6α -/- mice revealed that deletion of ATF6α gene enhanced infarct volume in a relatively late period (5 days) after MCAO, suggesting occurrence of delayed neuronal damage in ATF6α -/- mice. Consistent with these data, reduced level of glial activation (glial scar formation) and enhanced level of glial death both in astrocytes and microglia were observed in peri-infarct area of ATF6α -/- mice 3 days after MCAO. Furthermore, the size of 3-nitrotyrosine-positive area, which is indicative of oxidative stress-exposed region, was much larger in ATF6α -/- mice than in wild-type mice 5 days after MCAO. Analysis also revealed that expression of a potent astrocyte-activating factor IL-6 and astrocyte-induced neuroprotective molecules such as brain-derived neurotrophic factor (BDNF) and heme oxygenase-1 (HO-1) were reduced in in ATF6α -/- mice after MCAO. These results suggest that ATF6α may contribute to neuronal survival by promoting glial activation and survival after brain ischemia.	P3-1-34 脳損傷に誘発されるアストロサイトCa2+シグナルの反応性アストロサイト形成と神経保護作用における役割 Calcium-dependent N-cadherin upregulation mediates reactive astrogliosis and neuroprotection after brain injury ○金丸和典1, 久保田淳1, 関谷敬1, 廣瀬謙造2, 大久保洋平1, 飯野正光1 ○Kazunori Kanemaru1, Jun Kubota1, Hiroshi Sekiya1, Kenzo Hirose2, Yohei Okubo1, Masamitsu Iino1 東京大院・医・細胞分子薬理1, 東京大院・医・神経生物2 Dept Pharmacol, Grad Sch Med, Univ of Tokyo, Tokyo1, Dept Neurobiol, Grad Sch Med, Univ of Tokyo, Tokyo2 Brain injury induces phenotypic changes in astrocytes called reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling in astrocytes, and that the Ca2+ signaling is required for astrogliosis. We found that type 2 IP3 receptor knockout mice deficient in astrocytic Ca2+ signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified that N-cadherin and pumilio 2 (Pum2) function as downstream signaling molecules, and show that brain injury induces upregulation of N-cadherin around the injured site. This effect is mediated by Ca2+-dependent downregulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca2+ signaling and the downstream function of N-cadherin play an indispensable role in the cellular responses to brain injury. These findings define a new signaling axis required for reactive astrogliosis and neuroprotection following brain injury.
P3-1-35 ミクログリアの抗炎症作用機序-神経損傷反応性に発現するSiglec-Hについて Siglec-H is an anti-inflammatory molecule expressed in microglia in response to nerve injury ○小林正明1, 小西博之2, 木山博資2 ○Masaaki Kobayashi1, Hiroyuki Konishi2, Hiroshi Kiyama2 名古屋大学大学院 医学系研究科 機能組織学1, 名古屋大学大学院 医学系研究科 機能組織学2 Department of Functional Anatomy & Neuroscience, Nagoya University, Graduate School of Medicine, Nagoya, Japan.1, Department of Functional Anatomy & Neuroscience, *CREST, JST, Nagoya University, Graduate School of Medicine, Nagoya, Japan.2 In response to neuronal injury microglia is activated and influence on the injured neurons. Although regulatory mechanisms underlying the microglial activation have not been fully elucidated, molecules expressed on microglial surfaces are assumed to play crucial role. In this study, we initially focused on a membrane protein, DNAX activation protein of 12 kDa (Dap12), which is known to regulate activation of osteoclasts and macrophages. DAP12 forms complexes with several other membrane proteins and transduce signals of these associated molecules into cytoplasm by functioning as an adaptor protein. We used the mouse model of hypoglossal motor nerve injury, and revealed that mRNAs for DAP12 and two of its known associated molecules, sialic acid binding Ig-like lectin H (Siglec-H) and triggering receptor expressed on myeloid cells-2 (Trem2), were prominently induced in microglia. Since functional consequences of Siglec-H remain elusive, we further examined consequence of Siglec-H expression. Immunohistochemical double-staining confirmed that Siglec-H was specifically expressed in Iba1-positive microglia and the immunoreactivity was markedly increased in activated microglia which were surrounding soma of injured motor neurons. Next we prepared mouse primary microglia and stimulated them with an agonistic antibody raised against the extracellular domain of Siglec-H. Pretreatment of microglia with the antibody significantly suppressed the LPS-induced expression of mRNAs for iNOS and pro-inflammatory cytokines such as TNF-alpha and IL-6. These results suggest that Siglec-H-DAP12 signaling may suppress toxicity of activated microglia and thereby protect injured motor neurons from death.	P3-1-36 ミクログリアにおけるTRPV1チャネルの機能的解析 Physiological implications of TRPV1 in microglia ○三宅崇仁1, 白川久志1, 宮之原遵1, 中川貴之1, 金子周司1 ○Takahito Miyake1, Hisashi Shirakawa1, Jun Miyanohara1, Takayuki Nakagawa1, Shuji Kaneko1 京都大院・薬・生体機能解析学分野1 Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ., Kyoto, Japan.1 Microglia, resident immunocompetent cells in the CNS, are known to survey the environment in healthy brain. In response to the brain injury or dysfunction, microglia are activated, and then accumulate at the damaged site and remove the cellular debris. Some microglia exert a neuroprotective action, but others are excessively activated and secrete a large amount of inflammatory cytokines, which sometimes exacerbate the neurological damage or dysfunction. Transient receptor potential (TRP) channels are known as one of the sensor proteins to translate the external cues into some neurological signals. Besides the existence of some TRP channels in the neuron, immune cells are also widely known as the cells that functionally possess some kinds of TRP channels. Although microglia also have some members of TRP channel family, there is little knowledge in the physiological roles of TRP channels in microglia. Here we focused on the physiological roles of microglial TRPV1, which is opened by capsaicin, endocannabinoid, heat and acid. In this study, we examined the effects of capsaicin on the microglial physiological function, such as migration and phagocytosis. Application of capsaicin induced microglial migration in a concentration-dependent manner, which was completely inhibited by co-application of SB366791, a specific TRPV1 antagonist. Moreover, microglial phagocytotic activity was significantly reduced by capsaicin, which was also recovered by co-application of SB366791. Furthermore, capsaicin-induced microglial activation were not observed in microglia from TRPV1 knockout mice. These results show that microglial TRPV1 has a regulatory role in migration and phagocytosis, which imply that stimulation of TRPV1 may have some physiological roles at the lesion site of the brain.
P3-1-37 齧歯類とコモンマーモセットにおける大脳皮質アストロサイトの解剖学的比較 Anatomical comparison of rodent and common marmoset cerebral cortical astrocytes ○大江祐樹1, 松永英治2, 入來篤史2, 平瀬肇1 ○Yuki Oe1, Eiji Matsunaga2, Atsushi Iriki2, Hajime Hirase1 理化学研究所　脳科学総合センター　神経グリア回路研究チーム1, 理化学研究所　脳科学総合センター　象徴概念発達研究チーム2 RIKEN, Brain Science Institute Laboratory for Neuron-Glia Circuitry, Saitama1, RIKEN, Brain Science Institute Laboratory for Symbolic Cognitive Development, Saitama2 Astrocyte morphology and distribution have changed during evolution from rodents to primates. Especially in the cortex which is arguably the most evolved region in the primate brain, For instance, GFAP - a maker protein specific to astrocytes - in primates has been reported to show more complex branching processes and delineate primate specific astrocyte subtypes. Interlaminar astrocytes are primate specific astrocytes which have long processes originating from layer 1 and extending to layer 3 or 4. As most primate anatomical studies of astrocytes have been performed with the catarrhines (e.g., macaque, chimpanzee, and human) which evolutionarily diverged relatively later period, the time point at which these primate specific astrocytes emerged remains uncertain. We investigated the cortical expression of GFAP in common marmosets (Callithrix jacchus) which evolutionarily diverged before catarrhine primates. Interlaminar astrocytes of marmosets showed shorter processes than those of the catarrhines. In fact, many of the processes terminated within layer 1, although the processes were more intricate than the rodents. Moreover, whereas the astrocyte density in rodent cortex is higher in layer 1 than in other layers, we found that the astrocyte density in marmoset cortex is less in layer 1 than in other layers. These results suggest that signs of histological diversification of cortical astrocytes are present in primitive primates, although the degree of heterogeneity is less extensive.	P3-1-38 アストロサイト分泌因子Follistatin like-1の自然免疫活性化による神経発達障害への関与 Follistatin like-1 released from astrocyte is involved in the impairment of neuronal development by innate immune activation ○山田真之亮1, 永井拓1, 衣斐大祐2, 山田清文1 ○Shinnosuke Yamada1, Taku Nagai1, Daisuke Ibi2, Kiyofumi Yamada1 名古屋大院・医・医療薬学1, マウントサイナイ医科大学2 Dept Neuropsychopharmacol & Hosp Pharm, Nagoya Univ Grad Sch Med, Nagoya, Japan1, Dept Psychiatr, Mt Sinai Sch Med, New York, USA2 Astrocytes regulate neuronal function through the action of gliotransmitters and humoral factors. Polyinosinic-polycytidylic acid (polyI:C) is known to induce strong innate immune responses via toll-like receptor 3 (TLR3). Our previous studies demonstrated that the astrocyte-conditioned medium (ACM) from murine astrocyte cultures treated with polyI:C (polyI:C-ACM) impaired neurite outgrowth and spine formation of primary cultured hippocampal neurons. The expression level of interferon-induced transmembrane protein 3 (Ifitm3) was increased in polyI:C treated astrocytes and polyI:C-ACM derived from Ifitm3 knockout (KO) astrocytes showed no impairment of neuronal development. To identify the humoral factors that affect neuronal development, we conducted a secretome analysis in polyI:C-ACM. The levels of follistatin like-1 (Fstl1) were significantly up-regulated in polyI:C-ACM and astrocytes. PolyI:C-ACM derived from Fstl1 knockdown astrocytes attenuated the impairment of neurite outgrowth. The level of Fstl1 in polyI:C-ACM was reduced in Ifitm3 KO astrocytes, while expression level in astrocytes was not affected. Extracellular level of Fstl1 was significantly increased in COS7 cells co-transfected with Ifitm3 and Fstl1 than the level in COS7 cells transfected Fstl1 alone. Fstl1 expression was evident in astrocytes after polyI:C administration in vivo. Our findings suggest that Fstl1 is one of candidate molecules that affect neuronal development after innate immune activation by polyI:C and Ifitm3 controls the extracellular level of Fstl1. Further analysis is needed to clarify the mechanisms by which Fstl1 induces the impairment of neuronal development.
P3-1-39 成体海馬から分離したNG2培養細胞のAMPA受容体を介したグルタミン酸シグナルの役割 Roles for AMPA receptor-mediated glutamate signaling in cultured NG2 cells from adult rat hippocampus ○二本松 菊池尚美1, 于秀軍1, 楯林義孝1 ○Naomi Nihonmatsu-Kikuchi1, Xiujun Yu1, Yoshitaka Tatebayashi1 東京都医学総合研究所1 Tokyo Metropolitan Institute of Medical Science, Tokyo1 The NG2 (nerve/glial antigen 2) –expressing cells (NG2 cells) are a subpopulation of glial cells that are different from oligodendrocytes, astrocytes and microglias by a characteristic set of markers including NG2 and platelet–derived growth factor α receptor (PDGFRA). NG2 cells are ubiquitously distributed in both gray and white matter areas. Some of these are dividing cells and there is some evidence that they can differentiate into oligodendrocytes during adulthood. NG2 cells express glutamate (Glu) receptors and form synapses with glutamatergic neurons throughout the adult CNS. Although Glu influences the proliferation and maturation of perinatal NG2cells in vitro, Glu signaling in adult NG2 cells is still unclear. However, only an in vitro model of perinatal NG2 cells, but it is not clear whether the characteristics and functions of adult NG2 cells are same with, was used for investigations. Here we developed a novel method to culture adult NG2 cells from the diverse regions of rat brain (<3-month-old) using a step gradient and a Glu –free defined medium containing fibroblast growth factor 2 (FGF2) to better understand the molecular mechanisms governing NG2 cells. The cells, isolated in a lower buoyancy density (<1.041 g/ml) of the gradient, rapidly proliferate and become nearly 100% NG2 +, PDGFRA +, and olig2 + within a few passages. Antioxidants, especially catalase, were essentially required for their initial expansion. Glu was not toxic or anti-proliferative but rather trophic for these cells via α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors without affecting the NG2 cells properties. These findings will open new avenue for the study of functional properties of excitatory synapses on adult NG2 cells in the CNS.	P3-1-40 成熟マウス黒質網様部単一細胞におけるD1ドーパミン受容体の発現解析 D1-dopamine receptor expression in astrocytes in the substantia nigra pars reticulata ○長友克広1, 菅世智子2, 山田勝也1 ○Katsuhiro Nagatomo1, Sechiko Suga2, Katsuya Yamada1 弘前大院・医・統合機能生理1, 弘前医療福祉大2 Dept. Physiol., Hirosaki Univ. Grad. Sch. Med., Hirosaki, Japan1, Hirosaki Univ. Health & Welfare, Hirosaki, Japan2 Dopaminergic neurons in the midbrain nucleus substantia nigra pars compacta (SNc) release dopamine from their dendrites extending deeply into the adjacent substantia nigra pars reticulata (SNr) which is comprised mostly of GABAergic neurons. With immunostainings and real time RT-PCR, we confirmed that SNr shows abundant D1-dopamine receptor (D1R) expression. However, a majority of GABAergic neurons acutely dissociated from the SNr did not respond to DA in perforated patch experiments. This has been further supported by single-cell real time RT-PCR of SNr neurons. D1R mRNA has been detected in only 1 out of 21 acutely dissociated SNr GABAergic neurons expressing GAD mRNA prepared from vesicular GABA transporter-Venus (VGAT-Venus) mice. No D1R mRNA was detected in TH mRNA-expressing, SNr dopaminergic neurons obtained from TH-GFP mice tested (0 out of 9). In contrast, D1R mRNA was detected in 6 out of 8 striatal D1R-YFP expressing neurons obtained from D1R-YFP transgenic mice. Interestingly, studies using the D1R-YFP mice showed that SNr glial cells express strong D1R (i.e. YFP) signal in their process, whereas SNr neurons and cortical neurons/glial cells showed no such expression. Double immunostainings of SNr and cortical cells acutely dissociated from adult mice with anti-D1R and anti-GFAP antibodies revealed strong D1R expression in SNr astrocytes (n=261), but not in SNr neurons (n=127) and cortical astrocytes (n=71). Possible significance in the expression of D1R in SNr astrocytes will be discussed.
P3-1-41 損傷脳における炎症は必要か？ Is inflammatory response essential for injured mouse brain? ○池島（片岡）宏子1,2, 乾さやか1, 松井裕3, 上出利光4, 安井正人1 ○Hiroko Ikeshima-Kataoka1,2, Sayaka Inui1, Yutaka Matsui3, Toshimitsu Uede4, Masato Yasui1 慶應大・医・薬理1, 早稲田大・理工学術院・創造理工2, 北光記念病院循環器内科3, 北海道大学遺伝子病制御研究所マトリックスメディスン研究部門4 Dept. Pharmacol. & Neurosci., Keio Univ. Sch. Med., Tokyo1, Facul. Sci. & Engin., Waseda Univ., Tokyo2, Div. Cardiovasc. Med., Hokko Memorial Hosp., Hokkaido3, Dept. Matrix Med., Inst. Genet. Med., Hokkaido Univ., Hokkaido4 Reactive glial cells such as microglia and astrocytes proliferate, migrate and secrete some pro-inflammatory cytokines at pathological condition induced by the injury or ischemia in the central nervous system (CNS). To analyze the molecular mechanisms for activated glial cells, we made a stab wound to mouse cerebral cortex. Microarray analysis was performed for RNA extracted from the tissue around stab wound injury compared among day after 0, 1, 3 and 7. Most of the genes in top 20 genes for high expression level around lesion site were concerned in immunological or inflammatory function. We successfully identified and focused on to osteopontin (OPN), which is an inducer of pro-inflammatory cytokine production expressed in reactive microglial cells and reactive astrocytes in the CNS. However, functional role of OPN in reactive astrocytes is unknown. For the functional analysis of OPN in reactive astrocytes, we examined stab wound brain of OPN-deficient (OPN/KO) mice, and found that reactivity of astrocytes was altered. Furthermore, the cell shape of primary culture of astrocytes from OPN/KO mice was unusual. By the stimulation with endotoxin, lipopolysaccharide, to those cells some of the pro-inflammatory cytokine expression was altered. Taken together, these findings indicate that OPN plays an important role in the activation of astrocytes under pathological conditions in the CNS.	P3-1-42 セロトニン1Aアゴニストによる神経-アストロサイト連関の修飾 Modulating effects of serotonin 1A agonist on neuron-astrocyte interaction ○宮崎育子1, 村上真樹1, 竹島美香1, 鳥越菜央2, 三好耕1, 北村佳久2, 浅沼幹人1 ○Ikuko Miyazaki1, Shinki Murakami1, Mika Takeshima1, Nao Torigoe2, Ko Miyoshi1, Yoshihisa Kitamura2, Masato Asanuma1 岡山大院・医歯薬・神経情報学1, 岡山大院・医歯薬・臨床薬剤学2 Dept. of Brain Sci., Okayama Univ. Grad. Sch. of Med., Dent. and Pharmaceut. Sci.1, Dept. of Clinical Pharmacy, Okayama Univ. Grad. Sch. of Med., Dent. & Pharmaceut. Sci.2 Astrocytes are abundant neuron-supporting glial cells that harbor a powerful arsenal of neuroprotective antioxidative molecules and neurotrophic factors. Indeed, astrocyte-derived antioxidants reduce oxidative stress in surrounding neurons. It is known that stimulation of astrocyte serotonin 1A (5-HT1A) receptor results in the release of S100β protein from astrocytes, and that extracellular S100β exerts autocrine effects to promote astrocyte proliferation. Here we examined whether enrichment with healthy astrocytes can provide neuroprotection against dopaminergic neurotoxicity. Serotonin 1A agonist 8-OH-DPAT induced astrocyte proliferation and increased antioxidative molecules such as metallothionein (MT) in cultured astrocytes. The treatment with 8-OH-DPAT significantly ameliorated the reduction of dopamine neurons in neuron-astrocyte coculture after exposure to H2O2 or 6-hydroxydopamine (6-OHDA), but not in enriched neuronal culture alone. Reduction of mesencephalic dopamine neurons after H2O2- or 6-OHDA-exposure were ameliorated by preincubation with conditioned media from 8-OH-DPAT-treated astrocytes. These protective effects were cancelled by 5-HT1A antagonist or MT-1/-2-specific antibody. These results suggest that stimulation of 5-HT1A receptor in astrocytes can protect dopaminergic neurons against oxidative stress, and that MT secreted from astrocytes may be a major contributor to the 8-OH-DPAT-induced neuroprotective effects.

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