ポスター発表　細胞死/神経保護

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細胞死/神経保護

P2-20 GPIアンカー型セルロプラスミンノックダウンは過酸化水素誘導性のアポトーシスを増強する秋丸朋未¹，大橋憲太郎^1,2，木内一壽^1,2，平田洋子^1,2 岐阜大学大学院工学研究科生命工学専攻¹，岐阜大学大学院連合創薬医療情報研究科創薬科学専攻² Iron is an essential metal for organisms, but Fe²⁺ interacts with hydrogen peroxide and leads to cell death via the formation of hydroxyl radicals. Ceruloplasmin（Cp）oxidizes toxic Fe²⁺ to non-toxic Fe³⁺, and consists of two isoforms, a secreted form from the liver and glycosylphosphatidylinositol（GPI）-linked form anchored to the plasma membrane of astrocyte. The accumulation of iron leads to free radical-mediated damage and causes neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Therefore, we examined possible roles of GPI-linked Cp（GPI-Cp）in preventing neurons from oxidative stress-induced cell death. From western blot analysis, the expression of Cp protein was detected in mouse hippocampal cells（HT22）and rat pheochromocytoma cell（PC12）, but not in mouse neuroblastoma cells（Neuro2a）, motor neuron cell line（NSC-34）, and C6 rat glioma cell. Next, mRNA levels of both the isoforms were assessed by RT-PCR. HT22 cells expressed two isoforms, however, PC12 cells only expressed the secreted form. Neither of them were detected in other neuronal cell lines. Therefore, we transfected siRNA constructs for GPI-Cp into HT22 cells, and found the most effective one. Then, siRNA stably expressing cells were established and cloned by the limiting dilution. Both GPI-Cp mRNA and protein were remarkably decreased in these cells. Hydrogen peroxide decreased Bcl-2 levels and increased caspase-3 cleavage in the GPI-Cp stable cell line, suggesting that the hydrogen peroxide-induced apoptosis was facilitated by the GPI-Cp knock-down. These result implied that GPI-Cp protected neurons from oxidative stress in the central nervous system.		P2-21 マウスcation transport regulator homolog 1（Chac1）の発現制御解析野村雄紀¹，大橋憲太郎^1,2，平田洋子^1,2，木内一壽^1,2 岐大院・連創・創薬科学¹，岐大院・工・生命工² Endoplasmic reticulum（ER）is responsible for the folding and modification of newly synthesized proteins. Some pathophysiological conditions cause accumulation of misfolded proteins in the ER. In these conditions, known as ER stress, ER promotes UPR signals to restore folding capacity, but apoptosis is induced when ER stress is too severe. Apoptosis due to UPR is suggested to be partly responsible for neurodegenerative diseases.Cation transport regulator-like protein 1（Chac1）is one of ER stress inducible genes and is reported to degrade glutathione. As intrinsic Chac1 protein is not detectable by western blot analysis, we tried to detect Chac1 using Chac1-overexpressing cells after treatment with ER stress inducible agents（thapsigargin and tunicamycin）and a proteasome inhibitor（MG132）. Consequently, the treatment with MG132 made it possible to detect Chac1 protein. This result suggests that Chac1 protein is probably degraded by the ubiquitin-proteasome system. There is a Kozak-like sequence facilitating translation just before the translation start site of Chac1 mRNA. Therefore, we made Chac1 constructs having full length or deleted 5'UTR and transfected each of them into the cells. As a result, Chac1 translation was increased in parallel with the 5'UTR length. Chac1 is usually detected around 32 kDa by western blot analysis, however another band was emerged when the cells was treated with MG132. As there are two Met codons after the translation start site in the Chac1 coding region, we presume that this 17 kDa protein is Chac1 translated from either of AUGs. Thus, we made Chac1 constructs substituted Met by Ile and transfected into the cells and found that the low-molecular weight protein is the short form of Chac1 translated from the second Met codon.

P2-22 GM-CSF自己産生とJAK2/STAT5シグナル伝達がTLR4活性化ミクログリアの生存に関与する神垣真由美¹，秀和泉¹，柳瀬雄輝²，白榊紘子¹，田中茂¹，白藤俊彦¹，秀道広²，酒井規雄¹ 広島大学医歯薬保健学研究科神経薬理学¹，広島大学医歯薬保健学研究科皮膚科学² The activation of Toll-like receptor 4（TLR4）by lipopolysaccharide（LPS）induced rapid death of rat primary cultured microglia in a concentration dependent manner. However, a subpopulation of microglia escaped from death, and survived much longer than two days, in which all control cells had died. Astrocyte-derived factors, such as macrophage colony-stimulating factor（M-CSF）and granulocyte macrophage colony-stimulating factor（GM-CSF）, are known to support survival of microglia. However, it is not clear how LPS-stimulated microglia could keep surviving by themselves. To clarify the mechanism underlying TLR4-mediated survival, we examined the possibility that microglia may produce survival factors in response to LPS. The mRNA expression levels of M-CSF and GM-CSF in microglia after LPS stimulation were measured by using real-time PCR. The mRNA level of M-CSF was not changed. But, LPS induced a marked increase in GM-CSF mRNA expression, which was very low under unstimulated condition. Moreover, increased mRNA level of GM-CSF receptor α subunit（GM-CSFRα）and common β subunit（βc）were also observed in microglia stimulated by LPS. We then examined the activation of JAK2/STAT5 downstream of GM-CSFR, which controls transcription of survival-related genes. Western blot analysis showed that the LPS stimulation of microglia resulted in the phosphorylation of STAT5. Furthermore, the pre-treatment with a specific JAK2 inhibitor, NVP-BSK805, suppressed both STAT5 phosphorylation and the survival LPS-stimulate of microglia. These results suggest that TLR4 activation may enhance survival of a subpopulation of microglia through self-production of GM-CSF and upregulation of GM-CSFR. This GM-CSF autocrine pathway may activate cytoprotective JAK2/STAT5 signals.		P2-23 培養小脳顆粒細胞の低カリウム誘発細胞死モデルにおけるサイロキシンの細胞生存および神経突起維持効果小柳洸志¹，根岸隆之²，田代朋子¹ 青山学院大学大学院理工学研究科理工学専攻¹，名城大学薬学部薬学科² Thyroid hormone（TH）is required for normal brain development. In the cerebellum, TH deficiency during the perinatal period results in delayed proliferation, migration, maturation and also apoptosis of cerebellar granule neurons（CGNs）. To understand the role of TH in the developing cerebellum, we examined the effect of TH on the survival and morphology of CGNs in culture. Cultured CGNs require chronic depolarization in a medium with high K⁺ concentration（25 mM, K25）. When cultured in physiological low K⁺ concentration（5 mM, K5）, CGNs undergo cell death. This low K+-induced death of CGNs is a well known model for studying apoptosis. Cell death detected in CGNs cultured in K5 for 2 days was inhibited by the addition of 50-200 nM thyroxine（T4）, but not 3,5,5'-triidothyronine（T3）or 3,3',5'-triiodothyronine（reverse T3；rT3）in a dose-dependant manner. Furthermore, T4 was effective in maintaining the neurites in K5 by stabilizing microtubules through reduction of tau phosphorylation at two developmentally regulated sites of Ser199 and Ser422. T4 in K5 down-regulated the activities of the two tau kinases, GSK-3β and c-jun N-terminal kinase（JNK）, by maintaining inhibitory phosphorylation of GSK-3β and suppressing phosphorylation of JNK necessary for its activation. Since these kinases are involved in low K⁺-induced death of CGNs, suppression of their activities is likely to be a key to the function of T4 in both cell survival and neurite maintenance in K5.

P2-24 γ-グルタミルシステイン合成酵素の産生を促進する新規オキシインドール化合物の創製井川貴礼¹，古田享史^2,3，大橋憲太郎^1,2，木内一壽^1,2，平田洋子^1,2 岐阜大学大学院工学研究科生命工学専攻¹，岐阜大学大学院連合創薬医療情報研究科創薬科学専攻²，岐阜大学大学院医学系研究科再生医科学専攻³ Glutathione（GSH）is especially important for antioxidant defense system, which is synthesized in two sequential enzymatic reactions catalyzed by γ-glutamylcysteine ligase（GCL）and GSH synthetase. GCL composed of a catalytic（GCLC）and modifier（GCLM）subunit catalyzes the rate-limiting step in de novo synthesis of GSH. Accumulating evidence suggests that substances for boosting the GSH synthesis could be a therapeutic tool for treating neurodegenerative disorders and schizophrenia in which a deficit in brain GSH has been observed. In the present study, we have attempted to develop small organic compounds which increase GSH levels. Rat GCLC promoter（from -1758 to+2）was cloned into the pGL3 Basic luciferase reporter vector, which was co-transfected with hygromycin resistance vector into mouse hippocampal HT22 cells. Using the cells stably expressing the GCLC（-1758/+2）-luciferase reporter, we confirmed that tert-butylhydroquinone, a synthetic phenolic antioxidant known as an inducer of GSH, up-regulated the GCLC promoter activity. We chose GIF-0726-r, a novel oxindole compound, as a lead compound because GIF-0726-r protects HT22 cells from glutamate-induced oxidative stress. From over 200 compounds newly synthesized, 5 compounds showed over 200％ increase in the GCLC promoter activity, 16 compounds showed approximately 150％ increase and 26 compounds showed approximately 120％ increase at 50 μM with statistical significance. The most effective 5 compounds including GIF-0726-r also increased GCLC mRNA levels in HT22 cells as well as rat pheochromocytoma PC12 cells. Therefore, oxindole derivatives may promote neuronal survival by augmenting resistance to oxidative stress.