グリア、グリア-ニューロン相互作用 Glia and Glia-Neuron Interaction
P2-1-49 α 7ニコチン受容体を介したミクログリアの活性調節機構 Functional regulation of LPS-activated microglia via α 7-nicotinic acetylcholine receptor ○田中佐知子1, 米田至宏1, 沼澤聡1 ○Sachiko Tanaka1, Michihiro Yoneda1, Satoshi Numazawa1 昭和大学薬学部生体制御機能薬学講座毒物学部門1 Division of Toxicol, Dept Pharmacol, Toxicol and Therapeut, Sch Pharmacy,Showa Univ, Tokyo1 Microglia works as immune cells in the central nervous system and it shows opsonic action by activation, and it thus contributes to neuronal degeneration. Activated microglia in these processes produces and releases inflammatory cytokines such as interleukin-1β(IL-1β) and tumor necrosis factor alpha (TNFα), and results in production of ROS including nitric oxide (NO). These actions produced by activated microglia, are thought to be a cause of neurodegenerative disorders through damage to normal cells. α7- nicotinic acetylcholine receptor (α7-nAChR) is identified in microglia and monocyte and α7-nAChR-mediated nicotine inhibits the release of TNFα from macrophages. We examined the effects of nicotine and α7-nAChR agonist, GTS-21 on lipopolysaccharide (LPS)-stimulated ability of phagocytosis and gene expressions of IL-1β and TNFα in order to elucidate whether α7-nAChR is involved in functional regulation of microglia. Microglia was prepared from 4 weeks old Balb/c mice. Microglia was treated with LPS for 3 hr and then the gene expressions of IL-1β and TNFα were increased. Microglia was also treated with LPS for 2 days and then the CD11b immunopositive cells showed activated phenotype (amoeboid form) revealing round and blunt shape. These cells showed phagocytosis using fluoro-labeled beads. Nicotine and GTS-21 inhibits LPS-stimulated phagocytosis. This result suggests that α7-nAChR agonist is able to inhibit LPS-stimulated phagocytosis. It means that α7-nAChR agonist has functional regulation of microglia.	P2-1-50 ドパミンによる培養アストロサイトのEC-SOD発現増加 Dopamine increased the expression of extracellular superoxide dismutase in cultured astrocytes ○高野桂1, 田中信行1, 河辺憲司1, 森山光章1, 中村洋一1 ○Katsura Takano1, Nobuyuki Tanaka1, Kenji Kawabe1, Mitsuaki Moriyama1, Yoichi Nakamura1 大阪府立大院・生命環境科学・獣医・統合生理1 Lab. Integrative Physiology, Vet. Sci., Osaka Pref. Univ., Osaka1 Oxidative stress is thought to play a role in dopaminergic neuron-selective degeneration in several pathological conditions such as Parkinson's disease. Although dopamine (DA) stored in the synaptic vesicle is stable, the excess leaked DA is spontaneously oxidized to yield superoxide anion (O2-) and reactive DA quinones, causing damages of dopaminergic neurons. In the CNS, superoxide dismutase (SOD) isozymes are expressed and play important roles in defense against brain injury and neurodegenerative diseases. Among three isozymes of SOD, extracellular (EC)-SOD should play a role to detoxify O2- in extracellular space; however, a little is known about EC-SOD in brain. In the present study, we examined the effects of DA on SOD expression in cultured rat cortical astrocytes. By means of RT-PCR, all mRNA of three isozymes of SOD could be detected; however, only EC-SOD was increased by DA exposure for 24 h, dose-dependently. The expression of EC-SOD protein and the cell-surface SOD activity in astrocytes also increased with 100 μM DA exposure. The increase of EC-SOD mRNA by DA was inhibited by a DA transporter inhibitor, GBR12909, whereas it was not changed by DA receptor antagonists, SKF-83566 (for D1) and haloperidol (D2). Furthermore, a monoamine oxidase inhibitor, pargyline, and antioxidants, N-acetyl-L-cysteine and glutathione, also did not affect the DA-induced expression of EC-SOD mRNA. On the other hand, an inhibitor of nuclear factor kappaB (NF-κB), ammonium pyrrolidine-1-carbodithioate, suppressed the DA-induced expression of EC-SOD mRNA. These results suggest that DA incorporated into the cells caused the induction of EC-SOD mRNA followed by the enhancements of EC-SOD protein level and the enzyme activity, and that NF-κB activation is involved in the mechanisms of the EC-SOD induction. The regulation of EC-SOD in astrocytes surrounding dopaminergic neurons may contribute to the defensive mechanism against oxidative stress in brain.
P2-1-51 脱髄疾患における髄鞘再生機能分子の動態解析 Establishment of molecular imaging method to analyze molecular dynamics of remyelination in multiple sclerosis ○植木孝俊1,2, , 間賀田泰寛4, 尾内康臣3 ○Takatoshi Ueki1,2, Gandhervin Kesavamoorthy1, Yasuhiro Magata4, Yasuomi Ouchi3 浜松医科大学医学部解剖学講座神経機能学分野1, 米国立衛生研究所・国立糖尿病消化器腎臓疾患研究所発生細胞生物学研究部2, 浜松医科大学メディカルフォトニクス研究センター生体機能イメージング研究室3, 浜松医科大学メディカルフォトニクス研究センター分子病態イメージング研究室4 Dept Anatomy and Neurosci, Hamamatsu Univ Sch Med, Hamamatsu, Japan1, Lab Cellular and Developmental Biol, NIH/NIDDK, Bethesda, USA2, Dept Biofunctional Imaging, Med Photonics Res Ctr, Hamamatsu Univ Sch Med, Hamamatsu, Japan3, Dept Mol Imaging, Med Photonics Res Ctr, Hamamatsu Univ Sch Med, Hamamatsu, Japan4 Recent investigation has revealed that glial cell lineage is not simply classified into three types of cells, astrocyte, oligodendrocyte and microglia, as classically described, but another new population, NG2 cell, should be taken into consideration as a member of glial family. Among functions of NG2 cells shown by previous physiological studies, modification of synaptic transmission, generation of new neuron and oligodendrocyte to repair the injured CNS, and regulation neural functions via neuron-NG2 cell synapse are included, but molecular machinery underlying various functions of NG2 cell is still to be solved. Interestingly, prominent evidence demonstrated the high expression level of CD44 in NG2 cell. CD44 is a membrane protein, which is cleaved by &gamma-secretase. Because the involvement of CD44 in NG2 cell's function has not been appreciated well yet, the present study investigated how CD44 works to accomplish multiple roles of NG2 cell in the CNS by developing molecular imaging system on the basis of paramagnetic relaxation, where functional nuclear magnetic resonance (NMR) probe switches on and emits NMR signal upon cleavage of CD44 by &gamma-secretase. The newly developed analytical method enabled monitoring of the generation of intracellular fragment of CD44 under NMR, and the present results demonstrated that the activation of &gamma-secretase induced astrocytic differentiation of NG2 cell, whereas the inhibition of &gamma-secretase arrested NG2 cells in immature status. These data propose the possibility that membranous CD44 of NG2 cell might be a therapeutic target to treat demyelinated disorders such as multiple sclerosis, in which immature NG2 cells are accumulated in the affected brain. Further investigations are required to identify the transcriptional target of the intracellular domain of CD44 in NG2 cell, and also to appreciate the precise implication of &gamma-secretase in glial development and interaction with neuron.	P2-1-52 大脳皮質発達期におけるミクログリアのスパイン形成への関与 Involvement of microglia in dendritic spine formation in immature barrel cortex ○宮本愛喜子1,2, 江藤圭2, 鍋倉淳一1,2 ○Akiko Miyamoto1,2, Kei Eto2, Junichi Nabekura1,2 総研大・生命科学・生理科学1, 生理研・発達生理・生体恒常機能発達機構2 Dept Physiol, Sch Life Sci, SOKENDAI1, Dept Develop Physiol, Natl Inst Physiol Sci, Okazaki2 Understanding the mechanisms of synapse formation is important for understanding how neural circuits build. However the mechanisms of synapse formation are not fully understood. Recently resting (ramified) microglia which are immunocompetent cells in the central nervous systems, have some functions for synapses. For example, ramified microglia contact with synapses and perform surveillance, while resting microglia are involved in synapse elimination at surround area of ischemia where may occur reorganization of neural circuit. Additionally, activated microglia are known to release some molecules related to synapse formation. (e.g. brain derived trophic factor, thrombospondin) In this study, to investigate whether microglia are involved in the synapse formation, we observed microglia-neuron interaction in the barrel cortex at postnatal age 8-10 mice, the period in which major ascending projection from layer 4 to layer 2/3 is formed. To visualize neurons and microglia simultaneously, we injected the construction to express red fluorescent protein in L2/3 pyramidal neurons by in utero electroporation for IbaI-EGFP mice which expressed EGFP at microglia. Using in vivo two photon imaging technique, we observed that filopodia was formed during microglial contact at L2/3 pyramidal cell basal dendrite and this phenomenon is age dependent. At this age, microglia have large cell soma and small process number, processes area compared to those in adult mice microglia. These morphology indicated that neonatal mice microglia have more activated state than adult mice microglia. Since it is known that minocycline suppressed microglial activity, we investigate the effect of minocycline on the spine density. In consequence, spine density in minocycline injected mice was decreased relative to saline injected control mice. These data indicated that microglia are involved in spine formation during cortical development.
P2-1-53 脳虚血傷害後、活性化アストロサイトは貪食性を呈する Phagocytic astrocytes in the ischemic brain ○森澤陽介1, 平山友里1, 芝田晋介2, 岡野栄之2, 小泉修一1 ○Yosuke Morizawa1, Yuri Hirayama1, Shinsuke Shibata2, Hideyuki Okano2, Schuichi Koizumi1 山梨大学大学院医学工学総合研究部薬理学1, 慶應大・医・生理2 Dept. Neuropharmacol., Univ. Yamanashi1, Dept. Physiol, Keio University School of Medicine2 Rapid clearance of cellular debris is thought to be essential for the maintenance of brain functions, and thus disruption of this clean-up process would affect many pathological events. In the CNS, only microglia, professional phagocytes, have been thought to play such a role. However, accumulating evidence have shown that non-professional phagocytes such as epithelial cells and fibroblasts, are also highly involved in the clearance in the peripheral tissues. Astrocytes are highly responsive to various brain pathologies and are transformed into several phenotypes. In the present study, we show that astroytes are also able to phagocytose neuronal materials in vitro and in vivo. In the primary culture, astrocytes engulfed the dead cells as well as fluorescent beads, formed phagocytic cups and ingested them in a temperature- or actin polymerization-dependent manner, suggesting that astrocytes should phagocytose them. Such astrocytic phagocytosis was confirmed by immuno-electron microscopic analysis in vivo. No or few phagocytic astrocytes were observed in naive mice, whereas huge amount of phagocytic astrocytes were seen after a transient middle cerebral artery occlusion. As for a mechanism(s), we found several phagoycytic pathways in astrocytes, some of which were independent of those seen in microglia. Taken together, these results suggest that astrocytes should become phagocytic phenotype after brain ischemia, and are presumably involved in the maintenance of brain homeostasis and remodeling.	P2-1-54 培養オリゴデンドロサイト前駆細胞に発現するTRPチャネルの機能解析 Involvement of TRP channels in the function of oligodendrocyte precursor cells ○景山慶子1, 白川久志1, 中川貴之1, 金子周司1 ○Keiko Kageyama1, Hisashi Shirakawa1, Takayuki Nakagawa1, Shuji Kaneko1 京都大院・薬・生体機能解析1 Dept. Mol. Pharmacol., Grad. Sch. Pharm. Sci., Kyoto Univ, Kyoto1 Myelination of axon is an important process to ensure normal neurotransmission in the vertebrate central nervous system (CNS). Oligodendrocyte precursor cell (OPC), which are widely dispersed throughout the white matter of the CNS, is capable of differentiating into mature, myelin-forming oligodendrocyte. Previous studies indicate that impairment of proliferation, migration, differentiation, and process extension of OPC are major cause of remyelination failure in demyelinating disease including multiple sclelosis (MS). Although Ca2+ signaling has been demonstrated to regulates OPC functions, there is not enough explanation about Ca2+ signaling in OPC. Transient receptor potential (TRP) channel is a nonselective cation channel, which is highly expressed in brain; however, the functional role of TRP channel in OPC remain to be elucidated. Using cultured OPC isolated from rat and mouse neonatal brain, we examined that expression of TRP channels and found that some isoforms of canonical TRP (TRPC), melastatin TRP (TRPM), vanilloid TRP (TRPV) and TRP ankyrin 1 (TRPA1) are expressed in cultured OPC. In this study, we focus on the functionally expressed TRP channels and evaluate the possible association of these channels with the cell function of OPC.
P2-1-55 大脳基底核のアストロサイトは自発運動により形態を変化させる Astrocytic morphologies in the basal ganglionic nuclei are closely related to motor activities in adult mice ○辰巳晃子1, 奥田洋明1, 森田晶子1, 和中明生1 ○Kouko Tatsumi1, Hiroaki Okuda1, Shoko Morita1, Akio Wanaka1 奈良県立医科大学・医・第2解剖学1 Department of Anatomy and Neuroscience, Nara Medical University, Kashihara, Nara1 Olig2 positive cells constitute a subpopulation of oligodendrocyte precursors and are widely distributed in the adult brain. Using the Olig2CreER: ROSA-GAP43-EGFP double transgenic mice, we have demonstrated that the Olig2 cells take region-specific fates in response to cellular environments. This mouse was particularly useful for visualizing the morphology of cells that underwent recombination, because GAP43-EGFP is a fusion protein targeted to cellular membrane. We previously reported that the GFP positive cells with a bushy appearance are differentiated astrocytes and the bushy morphologies became more complex after two-week voluntary wheel-running in some nuclei of the basal ganglia(e.g. the subthalamic nucleus(STN)and the globus pallidus). Such changes in astrocyte morphology may be linked to neuronal activities in the basal ganglia. In the present study, we unilaterally injected 6-hydroxydopamine(6-OHDA) into the substantia nigra compacta of the double transgenic mouse and made a Parkinson's disease model in this mouse. Upon apomorphine injection, the mice exhibited rotation behaviors that reflected imbalance of dopaminergic activities. In this experimental paradigm, we checked the morphologies of the GFP immunoreactive cells and found that they had more complex bushy morphologies in the 6-OHDA-injected side(activated side) of the basal ganglia, especially in the STN than in the contralateral side. These findings suggest that morphologies of astrocytes are closely related to neuronal activities in the basal ganglionic nuclei. Possible functional implication of this relationship is that astrocytes of the STN may modulate neuronal outputs of the nuclei through regulating glutamate metabolism. Such a neuron-astrocyte relationship is under investigation.	P2-1-56 神経活動維持のモノカルボン酸トランスポーター依存性の部位差 Regional difference in dependence of neuronal and synaptic activities on monocarboxylate transporters ○永瀬将志1, 渡部文子1,2, 加藤総夫1 ○Masashi Nagase1, Ayako M. Watabe1,2, Fusao Kato1 慈恵医大・神経生理1, 科学技術機構・さきがけ2 Lab. Neurophysiol. Jikei Univ. Sch. Med., Tokyo1, PRESTO, JST, Kawaguchi2 Monocarboxylate transporters (MCTs) play key roles in lactate transfer from astrocytes to neighboring neurons that consume high levels of ATP molecules to maintain their activities. We have already demonstrated that, in the nucleus of the solitary tract (NTS), a structure relatively resistant to energy deprivation, almost half of excitatory synaptic transmission depends on the lactate transfer via MCTs even in the presence of intracellular ATP and extracellular glucose, in the brainstem slices of young rats. To examine whether this is a common feature of the central synapses, we analyzed effects of pharmacological inhibition of MCTs with a MCT inhibitor, alpha-cyano-4-hydroxycinnamic acid (4-CIN), on neuronal and synaptic activities in the following brain regions: pyramidal cells in the hippocampus (CA1) and the lateral amygdala (LA) and the cerebellum Purkinje cell (PC). In a similar manner to NTS neurons, 4-CIN (1 mM) significantly reduced the amplitude of excitatory postsynaptic currents (EPSCs) in these neurons. However, this effect was accompanied by a significant increase in paired-pulse ratio (PPR) in the PC, unlike in CA1, LA and NTS where the PPR was only slightly affected. Neurons in CA1 and a part of LA neurons displayed an inward shift of the holding current in contrast to PC and NTS neurons, the holding current of which were much less affected. 4-CIN induced a QX-314 sensitive outward current in LA and PC, but not in NTS. Whereas 4-CIN did not markedly affect the membrane potential in NTS neurons, 4-CIN led to hyperpolarization of Purkinje neurons. Those results indicate that, MCT-mediated lactate transfer is not always essential for the maintenance of membrane potential but is indispensable for the integrity of excitatory synaptic transmission.
P2-1-57 アストロサイト不均一性の成因を探る遺伝子発現解析 Gene expression analyses explaining regional astrocyte heterogeneity ○鹿川哲史1, 清水健史2, 荒木喜美3, 竹田直樹4, 中潟直巳5, 信久幾夫1, 田賀哲也1 ○Tetsushi Kagawa1, Takeshi Shimizu2, Kimi Araki3, Naoki Takeda4, Naomi Nakagata5, Ikuo Nobuhisa1, Tetsuya Taga1 東京医科歯科大・難研・幹細胞制御1, 生理研2, 熊本大・生命資源・疾患モデル3, 熊本大・生命資源・技術開発4, 熊本大・生命資源・資源開発5 Dept Stem Cell Reg, Med Res Inst, Tokyo Med and Dent Univ, Tokyo1, Div Neurobiol and Bioinfo, Natl Inst Physiol Sci, Okazaki2, Div Dev Genet, Cent Anim Resr Dev, Kumamoto Univ, Kumamoto3, Div Transgenic Tech, Cent Anim Resr Dev, Kumamoto Univ, Kumamoto4, Div Reprod Engn, Cent Anim Resr Dev, Kumamoto Univ, Kumamoto5 Astrocytes exhibit various functions to support neural activities: nutrition delivery, blood-brain barrier formation, neurotransmitter recycling, and synapse formation. However, it is poorly understood that such diverged functions are accounted for by different sub-lineage populations or by cellular plasticity made in response to microenvironment. As a first step to understand heterogeneity of astrocytes across brain regions, we compared gene expression profiles of cortical and hippocampal astrocytes. These two populations of astrocytes were GFP-labeled by crossing Emx1-Cre KI delta-neo mice (provided by Drs. Iwasato and Itohara) and GFAP-reporter mice that carry the "loxP-nlacZ-loxP-GAP43GFP" cassette in glial fibrillary acidic protein (GFAP) gene locus. Cortical and hippocampal astrocytes isolated from postnatal one-week mouse brains using fluorescence-activated cell sorter displayed largely different gene expression patterns in metabolic and biosynthetic processes, etc. Next, we asked whether such heterogeneity was maintained even after conditioning for one-week in an identical culture environment. GFP-positive cortical astrocytes were recovered from 1:1 mixed culture of primary cortical and hippocampal cells (cortical cells prepared from Emx1-Cre/GFAP-reporter mice and hippocampal cells prepared from wild-type mice). On the other hand, GFP-positive hippocampal astrocytes were recovered from 1:1 mixed culture of primary hippocampal and cortical cells (hippocampal cells prepared from Emx1-Cre/GFAP-reporter mouse and wild-type cortical cells). Although the culture environment drastically changed gene expression profiles of astrocytes, cortical and hippocampal astrocytes retained their regional identity to some extent, including long intergenic non-coding RNAs (lincRNAs). Our results suggest that astrocyte heterogeneity is conducted by both their sub-lineage differences and plastic functional changes in response to cellular environment.	P2-1-58 M-CSF依存性ミクログリアの増殖とMAPキナーゼカスケードの関連性 Relevance of MAP kinase cascade to M-CSF-dependent microglial proliferation ○山本伸一1, 高坂新一2, 中嶋一行1,2 ○Shinichi Yamamoto1, Shinichi Kohsaka2, Kazuyuki Nakajima1,2 創価大学　工学部　生命情報工学科1, 国立精神・神経センター　神経研究所2 Dept. of Bioinformatics, Faculty of Engineering, Soka University, Tokyo1, Dept. of Neurochemistry, National Institute of Neuroscience, Tokyo2 Axotomy of rat facial nerve leads to an increase of microglial cell number in the ipsilateral facial nucleus. In the previous study, we demonstrated that up-regulated macrophage-colony stimulating factor (M-CSF) in the transected facial nucleus triggers the induction of cFms (receptor for M-CSF), proliferating cell nuclear antigen (PCNA) and cell cycle-associated proteins, including cyclins, cyclin-dependent protein kinases (Cdks) and Cdk inhibitors (CdkIs) in microglia and causes the microglia to divide. Furthermore, the microglial proliferation was found to be regulated by the interactions among cyclin A, cyclin D, Cdk2, Cdk4 and p21, and the induction of cyclins/PCNA and cFms, which are requisite for microglial proliferation, was found to be differentially regulated by c-Jun N-terminal kinase (JNK) and p38 in M-CSF-stimulated microglia. However, the knowledge about signaling mechanism for M-CSF-dependent microglial proliferation is largely limited. In the present study, we investigated JNK/p38-related signaling pathway in M-CSF stimulated microglia. A line of experiment for detecting phosphorylated-proteins revealed that mitogen activated protein kinase activated protein kinase-2 (MAPKAPK-2), cyclic AMP responsive element binding protein (CREB) and mitogen-and stress-activated protein kinase-1 (MSK1) are activated by stimulation with M-CSF. These signaling molecules are thought to be located at downstream of JNK/p38. On the other hand, at upstream of JNK/p38 mitogen activated protein kinase kinase 4 (MKK4) and MKK3/6 were activated. Distinct from MAPK cascade, Akt was also phosphorylated by M-CSF stimulation. In conclusion, M-CSF signaling in microglia was found to be transmitted to MAPKAPK-2, CREB and MSK-1 through MKK4-JNK/MKK3/6-p38 cascade. These signaling cascades and/or Akt signaling are suggested to be associated with the induction of cyclins/PCNA and cFms in M-CSF-stimulated microglia.
P2-1-59 TLR4活性化によるミクログリアの生存維持にGM-CSF自己産生とTNF/TNFR2シグナルが関与する Possible involvement of GM-CSF and TNF/TNFR2 signaling in survival of Toll-like receptor 4-activated microglia ○神垣真由美1, 秀和泉1, 柳瀬雄輝2, 田中芳樹1, 原田佳奈1, 関貴弘1, 田中茂1, 秀道広2, 酒井規雄1 ○Mayumi Kamigaki1, Izumi Hide1, Yuhki Yanase2, Yoshiki Tanaka1, Kana Harada1, Takahiro Seki1, Shigeru Tanaka1, Michihiro Hide2, Norio Sakai1 広島大院・医歯薬保・神経薬理1, 広島大院・医歯薬保・皮膚科学2 Dept. of Mol. and Phamacol. Neurosci, Inst of Biomed & Health Sci, Hiroshima Univ1, Dept. of Dermatol, Inst of Biomed & Health Sci, Hiroshima Univ2 We previously reported that the activation of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) induced rapid death in a concentration dependent manner in rat primary cultured microglia, but a certain part of microglia survived for much longer time of period than control cells which gradually underwent death within two days. To clarify the mechanism of TLR4-mediated survival of microglia, we examined whether microglia could produce their critical survival factors such as M-CSF and GM-CSF in response to LPS. Real-time PCR showed that GM-CSF mRNA expression was hardly detected in control cells, but drastically increased in LPS-stimulated microglia. High intensity of GM-CSF immunostaining was observed in LPS-stimulated surviving microglia. P2X7 receptor blocker, A438079 suppressed GM-CSF mRNA expression as well as survival in LPS-stimulated microglia, indicating a potential role of P2X7 receptor in GM-CSF-dependent survival. In contrast, LPS stimulation did not increase M-CSF mRNA expression. In microglia, LPS stimulation causes a marked release of TNF, which could in turn activate TNF receptors, pro-apoptotic TNF type 1 receptor (TNFR1) and anti-apoptotic TNF type 2 receptor (TNFR2). LPS stimulation induced a marked increase in TNFR2 mRNA, but not TNFR1 mRNA in microglia. Finally, an elevated mRNA expression of arginase-1, a marker of protective M2 microglia, was observed in LPS-stimulated surviving microglia. These results suggest that TLR4 may mediate survival of microglia through self-production of GM-CSF and up-regulation of autocrine TNF/TNFR2 signaling. This surviving subpopulation of microglia may exert a protective function.	P2-1-60 ミクログリアによるTNFα産生を促進するアストロサイト由来因子 Astrocyte-derived molecule(s) enhancing the production of tumor necrosis factor alpha (TNFα) in microglia ○増田寿明1, 高坂新一2, 中嶋一行1,2 ○Toshiaki Masuda1, Shinichi Kohsaka2, Kazuyuki Nakajima1,2 創価大・工・生命情報工1, 国立精神・神経医療研究センター、代謝研究部2 Deptpartment of Bioinformatics, Faculty of Engineering, Soka University, Tokyo, Japan1, Department of Neurochemistry, National Institute of Neurosciece, Tokyo, Japan2 Tumor necrosis factor alpha (TNFα) is one of the representative inflammatory cytokines that are produced in the disordered nervous system including brain injuries and brain diseases. The cytokine has been believed to be produced in activated microglia in vivo. However, it is poorly known how the production in activated microglia is modulated in vivo. In this study, we investigated the modulation of TNFα producetion in microglia from the standpoint of intercellular interaction with astrocytes. Microglia and astrocytes were prepared from neonatal rat brain-derived primary cultures. Microglia induced the cytokine when stimulated with lipopolysaccharide (LPS), and the induced amounts were promoted in the presence of astrocytes, suggesting that astrocytes stimulate microglial activation. The effects of astrocytes to promote the production of TNFα in microglia were confirmed by co-culture system using trans-well. Actually, astrocytic conditioned medium (Ast-CM) enhanced the production of TNFα in LPS-stimulated microglia. The TNFα-enhancing activity of Ast-CM was recognized to be stably retained for a few months at 4°C. To estimate molecular size of the active molecule, Ast-CM was dialyzed against DMEM by using Spectra/Pore tube (molecular cut 25 kDa). As a result, the resultant Ast-CM was found to keep the TNFα-enhancing activity, suggesting that the active molecule is not permeable. Furthermore, to obtain the precise information as to molecular size of astroyete-derived active molecule we analyzed Ast-CM by gel-filtration method with Sephacryl S-300. The TNFα-enhancing activity was eluted at 43-67 kDa with a peak. Accordingly, it is likely that astrocytes produce and release a specific molecule that stimulates microglia to enhance the production of TNFα.

上部に戻る

前に戻る