Development of screening methods to find novel therapeutic drugs for neurodegenerative disease using fluorescence-based probe and high-content microscope.

2013 Fiscal Year Final Research Report

• Project/Area Number: 24659117
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: General pharmacology
• Research Institution: Hiroshima University
• Principal Investigator: SAKAI NORIO 広島大学, 医歯薬保健学研究院, 教授 (70263407)
• Co-Investigator(Kenkyū-buntansha): SEKI Takahiro 熊本大学, 生命科学研究部, 准教授 (50335650) ; HIDE Izumi 広島大学, 大学院医歯薬保健学研究院, 助教 (20253073) ; TANAKA Shigeru 広島大学, 大学院医歯薬保健学研究院, 助教 (20512651)
• Project Period (FY): 2012-04-01 – 2014-03-31
• Keywords: 神経変性疾患 / タンパク質分解 / イメージング / タンパク質リン酸化酵素

Research Abstract

1. We developed novel fluorescence-based probe that can access the activity of chaperone-mediated autophagy (CMA) in a single cell level. Using this probe, we identified the decreased level of CMA in a cellular model of spinocerebellar ataxia type 14 (SCA14), indicating that CMA is a novel therapeutic target for neurodegenerative disease.
2.Imaging analysis using SCA14 mutant gamma PKC,-GFP elucidated that HSP70, a member of molecular chaperone, is involved in the compensation of cytotoxicity induced by SCA14 mutant gamma PKC.

Research Products

• [Journal Article] The role of Pak-Interacting Exchange Factor-13 phosphorylation at Serine 340 and 583 by PKCy in the dopamine release (2014)
  • Author(s): Shirafuji, T., Ueyama, T., Yoshino, K., Takahashi, H., Adachi, N., Ago, Y., Koda, K., Nashida, T., Hiramatsu, N., Matsuda, T., Toda, T., Sakai, N. and Saito, N.
  • Journal Title: J. Neuroscience. Volume: (In press)
  • Peer Reviewed
• [Journal Article] Selective blockade of N-methyl-D-aspartate channels in combination with dopamine receptor antagonism induces loss of the righting reflex in mice, but not immobility (2014)
  • Author(s): Kikuchi, N., Irifune, M., Shimizu, Y., Yoshida, K., Morita, K., Kanematsu, T., Morioka, N., Nakata, Y., Sakai, N.
  • Journal Title: Psychopharmacology Volume: (In press)
  • Peer Reviewed
• [Journal Article] Principles for the use of in vivo transgene techniques : overview and an introductory practical guide for the selection of tetracycline-controlled transgenic mice (2014)
  • Author(s): Sakai, N.
  • Journal Title: Methods Mol Biol. Volume: 1142 Pages: 33-40
  • DOI: 10.1007/978-1-4939-0404-44
  • Peer Reviewed
• [Journal Article] Developmental expression of GPR3 in rodent cerebellar granule neurons is associated with cell survival and protects neurons from various apoptotic stimuli (2014)
  • Author(s): Tanaka, S., Miyagi, T., Dohi, E., Seki, T., Hide, I., Sotomaru, Y., Saeki, Y., Chiocca,A. Matsumoto, M. and Sakai, N.
  • Journal Title: Neurobiol. Dis. Volume: 68 Pages: 215-227
  • DOI: 10.1016/j.nbd.2014.04.007
  • Peer Reviewed
• [Journal Article] Deregulation of the actin cytoskeleton and macropinocytosis in response to phorbol ester by the mutant protein kinase C gamma that causes spinocerebellar ataxia type 14 (2014)
  • Author(s): Yamamoto, K., Seki, T., Adachi, N., Tanaka, S., Hide, I., Saito, N. and Sakai, N.
  • Journal Title: Frontiers in Physiology Volume: 5 Pages: 126
  • DOI: 10.3389/fphys.2014.00126
  • Peer Reviewed
• [Journal Article] A brain-Specific Grb2-associated regulator of Erk/MAPK(GAREM) subtype, GAREM2, contributes to neurite outgrowth of neuroblastoma cell by regulating Erk signaling (2013)
  • Author(s): Taniguchi, T., Tanaka, S., Ishii, A., Watanabe, M., Fujitani, N., Sugeo, A., Gotoh, S. Ohta, T., Hiyoshi, M., Matsuzaki, H., Sakai, N. and Konishi, H.
  • Journal Title: J. Biol. Chem. Volume: 288 Pages: 29934-29942
  • DOI: 10.1074/jbc.M113.492520
  • Peer Reviewed
• [Journal Article] Mutant yPKC that causes spinocerebellar ataxia type 14 upregulates Hsp70, which protects cells from the mutant's cytotoxicity (2013)
  • Author(s): Ogawa, K., Seki, T., Onji, T., Adachi, N., Tanaka, S., Hide, I., Saito, N. and Sakai, N.
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 440 Pages: 25-30
  • DOI: 10.1016/j.bbrc.2013.09.013
  • Peer Reviewed
• [Journal Article] a membrane-targeted deubiquitinating enzyme, is activated by ubiquitination and regulates membrane dynamics, cell motility and endocytosis (2013)
  • Author(s): Seki, T., Gong, J., Williams, A. J., Sakai, N., Todi, S. V. and Paulson, H. L. JosD1.
  • Journal Title: J. Biol. Chem. Volume: 288 Pages: 17145-17155
  • DOI: 10.1074/jbc.M113.463406
  • Peer Reviewed
• [Journal Article] Effects of the chemical chaperone 4-phenylbutylate on the function of the serotonin transporter (SERT) expressed in COS-7 cells (2013)
  • Author(s): Fujiwara, M., Yamamoto, H., Miyagi, T., Seki, T., Tanaka, S., Hide, I. and Sakai, N.
  • Journal Title: J. Pharmacol. Sci. Volume: 122 Pages: 71-83
  • DOI: 10.1254/jphs.12194FP
  • Peer Reviewed
• [Journal Article] Long-term exposure of RN46A cells expressing serotonin transporter (SERT) to a cAMP analog up-regulates SERT activity and is accompanied by neural differentiation of the cells (2013)
  • Author(s): Yamamoto, H., Tanaka S., Tanaka, A., Hide, I. Seki, T. and Sakai, N.
  • Journal Title: J. Pharmacol. Sci. Volume: 21 Pages: 25-38
  • DOI: 10.1254/jphs.12229FP
  • Peer Reviewed
• [Journal Article] Hypoxic stress activates chaperone-mediated autophagy and modulates neuronal cell survival (2012)
  • Author(s): Dohi, E., Tanaka, S., Seki, T., Miyagi, T., Hide, I., Takahashi, T., Matsumoto, M. and Sakai, N.
  • Journal Title: Neurochem. Int. Volume: 60 Pages: 431-442
  • DOI: 10.1016/j.neuint.2012.01.020
  • Peer Reviewed
• [Journal Article] Establishment of a novel fluorescence-based method to evaluate chaperone-mediated autophagy in a single neuron (2012)
  • Author(s): Seki, T., Yoshino, K., Tanaka, S., Dohi, E., Onji, T., Yamamoto, K., Hide, I., Paulson, H.L., Saito, N. and Sakai. N.
  • Journal Title: PLoS ONE Volume: 7 Pages: e31232
  • DOI: 10.1371/journal.pone.0031232
  • Peer Reviewed
• [Presentation] PKCγノックアウトパーキンソン症候群モデル : Pak-interacting Exchange Factorβの役割 (2014)
  • Author(s): 白藤俊彦、上山健彦、吉野健一、足立直子、高橋英之、平松直樹、吾郷由希夫、松田敏夫、酒井規雄、齋藤尚亮
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-21
• [Presentation] 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GM-CSF受容体の上方制御が関与する (2014)
  • Author(s): 神垣真由美、秀和泉、柳瀬雄輝、関貴弘、白藤俊彦、秀道広、酒井規雄
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-21
• [Presentation] TLR4を介したアデノシン2a受容体活性化によるミクログリア機能の調節 (2014)
  • Author(s): 秀和泉、神垣真由美、柳瀬雄輝、関貴弘、白藤俊彦、秀道広、酒井規雄
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-21
• [Presentation] GPR3は様々なアポトーシス誘導刺激に対し神経保護的に作用する (2014)
  • Author(s): 田中茂、宮城達博、秀和泉、白藤俊彦、E.Antonio Chiocca、酒井規雄
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-20
• [Presentation] 神経細胞における恒常的Gs活性化受容体GPR3の細胞内局在と機能 (2014)
  • Author(s): 宮城達博、田中茂、秀和泉、白藤俊彦、酒井規雄
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-19
• [Presentation] 脱ユビキチン化酵素JosD1は細胞形態、細胞移動及びエンドサイトーシスを調節する (2014)
  • Author(s): 関貴弘、Sokol V.Todi、酒井規雄、Henry L.Paulson、香月博志
  • Organizer: 第87回日本薬理学会年会
  • Place of Presentation: 仙台
  • Year and Date: 2014-03-19
• [Presentation] 齧歯類脳虚血モデルにおけるCMA関連蛋白LAMP-2Aの経時的発現変化 (2013)
  • Author(s): 土肥栄祐、田中茂、関貴弘、宮城達博、秀和泉、高橋哲也、松本昌泰、酒井規雄
  • Organizer: 第54回神経学会総会
  • Place of Presentation: 東京
  • Year and Date: 20130529-0601
• [Presentation] Possible relationship between decreased expression of lysosomal-associated membrane protein type 2A and delayed neuronal death after brain ischemia (2013)
  • Author(s): Dohi E., Tanaka S., Seki T., Miyagi T.,, Hide I.,, Sakai, N
  • Organizer: Neuroscience meeting 2013
  • Place of Presentation: San Diego, USA
  • Year and Date: 2013-11-12
• [Presentation] GPR3 protects neurons from apoptosis via Phosphatidylinositol 3-Kinase and mitogen-activated protein-kinase signaling pathway (2013)
  • Author(s): Tanaka S. Miyagi T, Hide I Seki, T., Sakai, N
  • Organizer: Neuroscience meeting 2013
  • Place of Presentation: San Diego, USA
  • Year and Date: 2013-11-11
• [Presentation] 黒質線条体におけるPKCγ基質の解析 : βPIXリン酸化のドパミン遊離での役割 (2013)
  • Author(s): 白藤俊彦、上山健彦、吉野健一、足立直子、高橋英之、香田健、平松直樹、吾郷由希夫、松田敏夫、酒井規雄、齋藤尚亮
  • Organizer: 第124回日本薬理学会近畿部会
  • Place of Presentation: 京都
  • Year and Date: 2013-11-01
• [Presentation] セロトニントランスポーター機能調節に対するケミカルシャペロン4-phenylbutylate(4-PBA)の効果 (2013)
  • Author(s): 酒井規雄、藤原雅幸、山本光、田中茂、関貴弘、秀和泉
  • Organizer: 第43回神経精神薬理学会総会
  • Place of Presentation: 沖縄
  • Year and Date: 2013-10-26
• [Presentation] 中枢神経系における恒常的Gs活性型受容体GPR3の局在と機能 (2013)
  • Author(s): 宮城達博、田中茂、秀和泉、関貴弘、酒井規雄
  • Organizer: 第43回神経精神薬理学会総会
  • Place of Presentation: 沖縄
  • Year and Date: 2013-10-25
• [Presentation] 蛍光イメージングを用いた神経細胞でのシャペロン介在性オートファジー活性の解析 (2013)
  • Author(s): 関貴弘、吉野健一、田中茂 土肥栄祐、隠地智也、山本和央、秀和泉、PAULSON Henly L、齋藤尚亮、酒井規雄
  • Organizer: 第43回神経精神薬理学会総会
  • Place of Presentation: 沖縄
  • Year and Date: 2013-10-25
• [Presentation] 膜輸送によるセロトニントランスポーター調節機構 (2013)
  • Author(s): 酒井規雄、藤原雅幸、山本光、浅野昌也、田中茂、関貴弘、秀和泉
  • Organizer: 第17回活性アミンに関するワークショップ
  • Place of Presentation: 福井
  • Year and Date: 2013-08-24
• [Presentation] 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 (2013)
  • Author(s): 神垣真由美、秀和泉、柳瀬雄輝、田中芳樹、原田佳奈、関貴弘、田中茂、秀道広、酒井規雄
  • Organizer: Neuro2013
  • Place of Presentation: 京都
  • Year and Date: 2013-06-21
• [Presentation] 神経細胞における恒常的Gs活性化受容体GPR3の局在と機能 (2013)
  • Author(s): 宮城達博、田中茂、秀和泉、関貴弘、酒井規雄
  • Organizer: Neuro 2013
  • Place of Presentation: 京都
  • Year and Date: 2013-06-21
• [Presentation] イメージングを活用した新たなシャペロン介在性オートファジーの解析方法の確立 (2013)
  • Author(s): 酒井規雄、吉野健一、田中茂、土肥栄祐、隠地智也、山本和央、秀和泉、PAULSON Henly L、齋藤尚亮、関貴弘
  • Organizer: 第54回神経学会総会
  • Place of Presentation: 東京
  • Year and Date: 2013-05-31
• [Presentation] Toll様受容体4活性化により生存するミクログリアは穎粒球マクロファージコロニー刺激因子を自己誘導する (2013)
  • Author(s): 神垣真由美 秀和泉 酒井規雄, 他
  • Organizer: 第122回薬理学会近畿部会
  • Place of Presentation: 福岡
  • Year and Date: 2013-03-23
• [Presentation] GPR3を介した突起伸長にGbetagannmaを介する経路が部分的に関与する (2013)
  • Author(s): 田中茂 宮城達博 酒井規雄, 他
  • Organizer: 第86回日本薬理学会年会
  • Place of Presentation: 福岡
  • Year and Date: 2013-03-22
• [Presentation] ミクログリアの死細胞食食におけるP2Y2受容体の役割 (2012)
  • Author(s): 秀和泉 神垣真由美 酒井規雄, 他
  • Organizer: 第122回薬理学会近畿部会
  • Place of Presentation: 大阪
  • Year and Date: 2012-11-16
• [Presentation] Phosphatidylinositol 3-Kinase and mitogen-activated protein-kinase signaling pathway play a role in the GPR3-mediated neurite outgrowth (2012)
  • Author(s): Tanaka, S. Miyagi, T. Sakai, N. et al.
  • Organizer: Neuroscience 2012 (Annual meeting of society for neuroscience)
  • Place of Presentation: New Orleans USA
  • Year and Date: 2012-10-15
• [Presentation] Long-term exposure of cAMP analogue up-regulates the function of serotonin transporter (SERT) in RN46A cells (2012)
  • Author(s): Yamamoto, H. Tanaka, S. Sakai, N et al.
  • Organizer: Neuroscience 2012 (Annual meeting of society for neuroscience)
  • Place of Presentation: New Orleans USA
  • Year and Date: 2012-10-14
• [Presentation] Enhanced survival and phagocyticactivity by Toll-like receptor 4 activation in ratmicroglia (2012)
  • Author(s): Hide I.Yanase, Y Sakai N.et al.
  • Organizer: 第55回日本神経化学会
  • Place of Presentation: 神戸
  • Year and Date: 2012-10-01
• [Presentation] GPR3依存的な神経突起伸長にPI3キナーゼ、MAPキナーゼの活性化が寄与する (2012)
  • Author(s): 田中茂 宮城達博 酒井規雄, 他
  • Organizer: 第35回日本神経科学大会
  • Place of Presentation: 名古屋
  • Year and Date: 2012-09-18
• [Presentation] GPR3 protects neurons from apoptosis under the hypoxic condition (2012)
  • Author(s): Tanaka S., Miyagi,M Sakai, N.et al.
  • Organizer: 第55回日本神経化学会
  • Place of Presentation: 神戸
  • Year and Date: 2012-09-13
• [Presentation] 神経細胞株SH-SY 5Y細胞におけるニコチン誘発性PKCトランスロケーションの観察 (2012)
  • Author(s): 酒井規雄 宮原岳史 田中茂, 他
  • Organizer: 第16回活性アミンに関するワークショップ
  • Place of Presentation: 札幌
  • Year and Date: 2012-08-24
• [Presentation] 低酸素ストレスにより活性化されるシャペロン介在性オートファジーの細胞保護効果 (2012)
  • Author(s): 土肥栄佑 田中茂 酒井規雄, 他
  • Organizer: 第121回薬理学会近畿部会
  • Place of Presentation: 徳島
  • Year and Date: 2012-06-29