神経細胞死、アポトーシス Neuronal Death and Apoptosis
P2-2-72 マウス大脳皮質神経細胞におけるATP誘発性神経細胞死のP2X7受容体の関与について Involvement of P2X7 receptors in ATP-induced neuronal cell death in mouse cultured cortical neurons ○藤本真知子1, 宇田裕香子1, 松田衣代1, 森田由樹1, 毛野祐花1, 須藤悠悟1, 丸宮彩香1, 西田健太朗1, 長澤一樹1 ○Machiko Fujimoto1, Yukako Uda1, Kinuyo Matsuda1, Yuki Morita1, Yuka Keno1, Yugo Sudo1, Ayaka Marumiya1, Kentaro Nishida1, Kazuki Nagasawa1 京都薬科大学　生命薬科学系　衛生化学1 Dept of Environ. Biochem., Univ of Kyoto Pharm, Kyoto, Japan1 P2 nucleotide receptors play important roles in the ATP signaling pathway in brain neuronal networks. Among P2 nucleotide receptors, P2X7 receptors (P2X7R) exhibit low sensitivity to ATP. Under pathological condition such as ischemia/reperfusion, prolonged activation of P2X7R by high concentration of ATP induces neuronal cell death, it being known to be a death receptor. We have previously demonstrated that P2X7R expressed by cultured astrocytes of mouse brain cortex is activated without any exogenous stimuli, implying its roles in maintenance of cellular homeostasis, However, functional expression of P2X7R in mouse neurons are unclear so far. In this study, we investigated whether P2X7R was involved in ATP-induced neuronal cell death in mouse neurons. Mouse cortical neurons were prepared from ddY-strain E15 mouse embryos and cultured using B-27-supplemented EMEM as reported previously. Neuronal death was quantified by measuring the lactate dehydrogenase (LDH) activity in cellular supernatants and staining with propidium iodide. Western blot and immunocytochemistry were performed P2X7-specific antibodies. Treatment of 5 mM ATP caused increase in LDH release, and their LDH releases were inhibited by P2X7R antagonists, A438079 and KN-62. Western blot analysis revealed that expression of P2X7R was detected in neurons, but the expression levels were lower than that in mouse astrocytes. These results suggest that treatment of 5 mM ATP induces neuronal cell death and their death might be involved in P2X7R activation. Now, additional investigations on P2X7R functionality such as its channel/pore activity assessed by YO-PRO-1 uptake are in progress.	P2-2-73 小胞体ストレス性神経細胞死におけるSUMO化の役割 Involvement of SUMOylation in cell death under ER stress ○松崎伸介1,2,3, 渡部音哉1, 岡村麻美1, 高村明孝1,2, 山田浩平1,2, 遠山正彌1, 片山泰一1 ○Shinsuke Matsuzaki1,2,3, Otoya Watanabe1, Asami Okamura1, Hironori Takamura1,2, Kohei Yamada1,2, Masaya Tohyama1, Taiichi Katayama1 大阪大学大学院　連合小児発達学研究科　分子生物遺伝学研究領域1, 大阪大学大学院　連合小児発達学研究科　附属子どものこころの分子統御機構研究センター2, 大阪大学大学院　医学系研究 神経機能形態学3 Dept. Molecular Brain Science, United Graduate School of Child Development, Osaka University, Suita, Osaka1, Molecular Research Center for Children's Mental Development, United Graduate School of Child Development, Osaka University, Suita, Osaka2, Dept. Anatomy and Neurosccience, Graduate School of Medicine, Osaka University, Suita, Osaka3 Small ubiquitin-like modifier proteins (SUMO) display similarities to ubiquitin in both the structure and biochemistry of their conjugation. SUMOylation is an important post-translational modification that provides a rapid and reversible means for controlling the activity, subcellular localization and stability of target proteins involved in a number of neuronal pathways. Previous reports suggest the importance of SUMOylation in cell viability and SUMOylation plays protective roles against oxidative stress. In addition, induction of SUMOylation has reported in the cerebral infarction. These reports suggest the involvement of SUMOylation in ER stress pathway. To elucidate the importance of SUMOylaion in ER stress pathway, we examined the effect of ER stressers, tunicamycine and thapsigargin, on the induction of SUMOylation and we elucidated that ER stress could induce SUMOylation levels. In addition, we examined the effect of SUMOylation on the indution of GRP78 and CHOP by using UBC9 knockdown cells, which show lower level of SUMOylation comparing to control cells. The UBC9 knockdown cells showed the higher mortality than control cells and accerelated the CHOP expression as well. The data suggest that SUMOylation could regulate the CHOP level under the ER stress resulting in protection against ER stressand it might regulate ER stress pathway.
P2-2-74 PC12細胞を用いたパーキンソン病モデルにおける細胞内マグネシウム濃度変化 Magnesium concentration changes in the cellular model of Parkinson's disease in PC12 cells ○新藤豊1, 山中龍1, 鈴木孝治2, 堀田耕司1, 岡浩太郎1 ○Yutaka Shindo1, Ryu Yamanaka1, Koji Suzuki2, Kohji Hotta1, Kotaro Oka1 慶大・理工・生命情報1, 慶大・理工・応化2 Dept Biosci Info, Fac Sci Tech, Keio Univ, Yokohama, Japan1, Dept Appl Chem, Fuc Sci Tech, Keio Univ, Yokohama, Japan2 Parkinson's disease (PD) is a progressive neurodegenerative disorder that results in degeneration of dopaminergic neurons in the substantia nigra. In many studies, the neuronal disorder has been accompanied with mitochondrial dysfunctions. In the previous studies, we demonstrated that mitochondria play important roles in the regulation of cellular Mg2+ concentration. Mg2+ is one of the most essential cations for maintaining cellular functions, and perturbation of Mg2+ contents in cells and tissues are implicated in many diseases. It has been also reported that Mg2+ has a protective effect in the cellular model of PD using MPP+, which induces PD-like dopaminergic cells death. These studies suggest that changes in cellular Mg2+ concentration was involved in the process of PD. However, it has not been fully elucidated. In this study, we therefore investigated the changes in cellular Mg2+ concentration in the process to neuronal cell death induced by MPP+ in PC12 cells. MPP+ induced increase in cytoplasmic Mg2+ concentration and decrease in mitochondrial Mg2+ concentration immediately after application. MPP+ acts as an inhibitor of mitochondrial electron transfer chain and induces the decrease in the mitochondrial membrane potential, and probably it causes Mg2+ release from mitochondria. After 8 and 16 h exposure to 3 mM MPP+, cytoplasmic free Mg2+ concentration was higher than that in non-treated cells. Total Mg2+ contents were also increased in the cells after 16 h exposure to 3 mM MPP+. These results indicate that MPP+ induced Mg2+ influx in PC12 cells. Actually, in the Mg2+-free medium, increase in intracellular free Mg2+ concentration was not observed. Moreover, while 24 h exposure to 3 mM MPP+ induced 50% cell death in normal medium, the toxicity of MPP+ was significantly increased inMg2+-free medium. These results suggests that increase in intracellular Mg2+ concentration has protective effect in the process to dopaminergic cell death induced by MPP+.	P2-2-75 抗うつ薬関連遺伝子Prg1は神経細胞の生存維持に重要な因子である Prg1, an antidepressant-related gene, is an important factor for survival of neurons derived from neural stem cells ○橋本富男1, 山田美佐1, 岩井孝志1, 斎藤顕宜1, 橋本恵理2, 鵜飼渉2, 齋藤利和2, 山田光彦1 ○Tomio Hashimoto1, Misa Yamada1, Takashi Iwai1, Akiyoshi Saitoh1, Eri Hashimoto2, Wataru Ukai2, Toshikazu Saito2, Mitsuhiko Yamada1 国立精神セ・精神薬理1, 札幌医科大学・医・ 神経精神医学2 Dept Neuropsychopharmacol, NCNP, Tokyo1, Dept Neuropsychi, Sapporo Med Univ Sch Med, Sapporo2 Plasticity-related gene 1 (Prg1) is a membrane-associated lipid phosphate phosphatase. Recently, we reported that the expression levels of Math2, a transcription factor regulated Prg1 expression, and Prg1 were induced after chronic treatment with SSRI in mouse hippocampus. However, little is known about physiological functions of Prg1. In this study, we first investigated the distribution of Prg1 in mouse hippocampus. Immunohistochemistry revealed that Prg1 expressed in the mouse dentate gyrus and colocalized with NeuN (neuron marker). Then, we studied the role in neuronal survival by knock-down of Prg1 gene with small interfering RNA (siRNA) in neurons derived from neural stem cells. Prg1 knock-down reduced cell number and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity in neurons derived from neural stem cells. Further, Prg1 knock-down increased genomic DNA fragmentation, suggesting induction of apoptosis. On the other hand, co-transfection with Prg1 siRNA and Prg1-4-silent vector which contains 4 silent mutations in the siRNA target regions, rescued cell number and MTT activity. In addition, co-transfection with Prg1 siRNA and Prg1-4-silent-H253A vector which contains 4 silent mutations in the siRNA target regions and changed catalytic domain of histidine (His253) to alanine did not rescue cell number and MTT activity. The 253th amino acid histidine of Prg1 has been reported to be a key of lipid phosphate phosphatase activity. These results suggest that Prg1 is important for survival of neurons through dephosphorylation activity.
P2-2-76 多様化膜分子群クラスター型プロトカドヘリン欠損マウスの機能解析 Functional analyses of the clustered-protcadherin families using their deletion mice ○長谷川園子1, 岡山厚1, 松下健一郎1, 平林敬浩1, 八木健1 ○Sonoko Hasegawa1, Atsushi Okayama1, Kenichiro Matsushita1, Takahiro Hirabayashi1, Takeshi Yagi1 大阪大院・生命機能・心生物学-JST-CREST1 KOKORO-Biology-JST-CREST,FBS, Oaka Univ, Osaka1 The brain is a complex system which contains an enormous number of diversified neurons to establish functional neural networks. The clustered protocadherins (cPcdhs) are transmembrane proteins belonging to a large subfamily within the diverse cadherin superfamily. The cPcdh genes are classified into three subfamilies: Pcdh-α (14 genes), Pcdh-β (22 genes), and Pcdh-γ (22 genes). As these cPcdh isoforms are expressed in a stochastic and combinatorial manner in individual neurons, diverse set of possible molecules are presented on their cell surface at the single cell level. Thus, the cPcdhs are candidate molecules for conferring potential neuronal diversity and specificity on individual neurons. Although it has already demonstrated that cPcdh proteins are required for correct axonal projections, synaptic formation, and neuronal survival in many brain regions, molecular function of the diverse cPcdhs and their critical role in the nervous system are not understood. In this study, to investigate critical role of cPcdh proteins for the formation of functional neural circuits during development, we have generated mice lacking all cPcdh gene clusters (Pcdh-α, Pcdh-β, and Pcdh-γ). Surprisingly, the mutant mice could not move and died shortly after birth. These phenotypes were not detected from those of each Pcdh-α, Pcdh-β, or Pcdh-γ deficient mutant. We would like to discuss the novel phenotype in the cPcdh deficient mice and the functional significance of the diverse molecules encoded by the three gene clusters.	P2-2-77 Th17はIL-23刺激によってRGMaを発現し、神経細胞死を誘導する RGMa expressed on Th17 cells is enhanced by IL-23 and induces neuronal cell death ○田辺章悟1,2, 石井宏史1,2, 山下俊英1,2 ○Shogo Tanabe1,2, Hiroshi Ishii1,2, Toshihide Yamashita1,2 大阪大院・医・分子神経1, 科学技術振興機構 戦略的創造研究推進事業2 Dept Mol Neurosci, Osaka Univ, Osaka, Japan1, JST-CREST2 Repulsive guidance molecule a (RGMa) is a GPI-anchored protein. RGMa is involved in inhibition of neurite outgrowth and growth cone collapse. After RGMa binds to its receptor, neogenin, RhoA and its effector, Rho kinase, are activated, leading to inhibition of axon outgrowth. On the other hand, it has recently been reported that neuronal guidance molecules such as semaphorin and RGMa play important roles in immune responses. We previously identified that neutralization of RGMa in blood samples from multiple sclerosis (MS) patients containing encepalitogenic T cells leads to downregulation of interleukin-17 (IL-17). Here, we examined RGMa expression on IL-17-producing helper T cells (Th17) and investigated their roles. We found that Th17 cells expressed RGMa in greater extent than other types of helper T cells, as assessed by real time PCR as well as western blot analysis. In addition, RGMa expression was enhanced in Th17 cells by stimulation with pro-inflammatory IL-23, which is known to maintain Th17 functions. Because Th17 cells were reported to induce neuronal cell death, we hypothesized that RGMa expressed on Th17 cells is associated with neuronal cell death. To clarify the hypothesis, we co-cultured Th17 cells and cortical neurons with or without RGMa-neutralizing antibody and performed TUNEL staining to detect apoptosis. As a result, RGMa-neutralizing antibody inhibited neuronal cell death induced by Th17 cells. Th17 cells cultured without IL-23 induced attenuated neuronal cell death compared with IL-23 treatment. Thus, RGMa is an important mediator of neuronal cell death induced by Th17 cells.
P2-2-78 胎児脳におけるNMDA受容体の過剰活性化は、細胞死と発達異常を引き起こす Overstimulation of NMDA receptors in embryonic brain causes multiple brain defects including cell death and neurodevelopmental abnormalities ○相田知海1,2, 澤田祐美1,2, 伊東義真1,2, 高橋祐子1,2, 三品昌美3, 田中光一1,2 ○Tomomi Aida1,2, Yumi Sawada1,2, Yoshimasa Ito1,2, Yuko K Takahashi1,2, Masayoshi Mishina3, Kohichi Tanaka1,2 東医歯大・難研・分子神経1, 東医歯大・脳統合機能センター2, 立命館大・総合理工3 Mol Neurosci, Med Res Inst, Tokyo Med Dent Univ, Tokyo1, Cent Brain Integ Res, Tokyo Med Dent Univ, Tokyo2, Brain Sci, Res Org Sci Tech, Ritsumeikan Univ, Shiga3 In the prevailing view of brain development, neuronal activity is required for late developmental events including neural connection refinement, whereas early events are genetically programmed. We previously showed overstimulation of glutamate receptors by knocking out glutamate transporters GLAST and GLT1 (double knockout, DKO), which are essential for maintaining low extracellular glutamate levels, induced abnormal brain development, including cortical, hippocampal, and olfactory bulb disorganization and defective corticothalamic and thalamocortical axonal projections. In contrast, cell deaths were not observed in the telencephalon of DKO mice. Here, we show that cell deaths occurred in thalamus, hypothalamus and spinal cord of DKO mice. Inactivation of NR1, an essential subunit of NMDA receptor, in DKO mice almost completely rescued both cell deaths and abnormal brain development. These results suggest that overstimulation of NMDA receptors induced different effects on brain development depending on brain regions. NR2 subunits (NR2A–D) are the major determinants of the functional properties of NMDA receptors. In embryonic brain, NR2B is globally expressed, whereas NR2D is localized in regions where cell deaths were extensive in DKO. To determine NR2 subunit responsible for multiple brain defects, we inactivated NR2B or NR2D in DKO mice and found that deletion of NR2B almost completely rescued all brain defects. This work provides compelling in vivo evidence that glutamate is vital, and excess NR2B-containing NMDA receptor activation is detrimental to the developing brain. Further study is required to dissect molecular mechanisms that determine cell death or abnormal brain development.	P2-2-79 Sigma 1 受容体によるミトコンドリアにおける 細胞保護機構 Cytoprotective effects of sigma 1 receptor chaperone at mitochondria ○森友久1,2, 林輝男2, 大屋順平1, 芝崎真裕1 ○Tomohisa Mori1,2, Teruo Hayashi2, Junpei Ohya1, Masahiro Shibasaki1, Tsung-Ping Su2 星薬科大学・薬品毒性学教室1 Department of Toxicology, Hoshi University, School of Pharmacy and Pharmaceutical Sciences1, National Institute on Drug Abuse, Baltimore, U.S.A.2 Sigma 1 receptor (Sig-1R), an endoplasmic reticulum (ER) chaperone protein, exerts its cytoprotective effect against ER stress induced apoptosis. Although Sig-1R also resides at least in part in mitochondria, mechanisms of the anti-apoptotic action by Sig1R at mitochondria remain to be clarified. Bcl-2, an anti-apoptotic protein, resides on the outer membrane of mitochondria, and Bcl-2 has been implicated in a variety of diseases such as Alzheimer's disease, Parkinson's disease, and cancer. Staurosporine, a protein kinase inhibitor, induces the apoptosis by activating the caspase 3 accompanied by the inhibition of Bcl-2. Furthermore, it has been reported that Sig-1R also regulates the Bcl-2 and caspase 3. In this study, we found that staurosporine decreases Bcl-2 level, whereas activates the caspase 3 in Chinese hamster ovary (CHO) cells. Knockdown of Sig-1Rs by siRNA potently enhanced the apoptosis induced by staurosporine, indicating that Sig-1R plays crucial role in the cytoprotective effects against staurosporine-induced apoptosis. We also found that staurosporine increase the cleaved caspase 3 levels in Sig-1R knockdown cells, and this increases of cleaved caspase 3 was supressed by the overexpression of Bcl-2. Overexpression of Bcl-2 also inhibits staurosporine-induced apoptosis in Sig-1R knockdown CHO cells. These findings suggest that Sig-1R regulates apoptosis via the modulation of Bcl-2 and caspase 3.

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