オートファジー性神経細胞死の分子細胞生物学的研究

2008 Fiscal Year Annual Research Report

• Project/Area Number: 16GS0315
• Research Institution: Juntendo University
• Principal Investigator: 内山 安男 Juntendo University, 医学部, 教授 (10049091)
• Co-Investigator(Kenkyū-buntansha): 柴田 昌宏 新潟大学, 医歯学系, 准教授 (10343253) ; 小池 正人 順天堂大学, 医学部, 准教授 (80347210) ; 佐々木 光穂 順天堂大学, 医学部, 助教 (20432536) ; 和栗 聡 福島県立医科大学, 医学部, 教授 (30244908) ; 上野 隆 順天堂大学, 医学部, 准教授 (10053373)
• Keywords: オートファジー性神経細胞死 / リソソーム / 低酸素-脳虚血 / Atg7 / LC3 / ミュータントマウス / リソソームプロテアーゼ / 脂肪代謝

Research Abstract

本年度は、中枢神経系で特異的にAtg7を欠損する(オートファジーができない)7日齢のマウスを用いて低酸素-脳虚血(H/I)負荷実験を実施して、海馬錐体細胞がアポトーシスでもネクローシスでもないオートファジー性神経細胞死でその多くが死に至ることを初めて報告した(AJP, Koike et al.)。さらに、成獣でもH/I負荷で死ぬ神経細胞はオートファジー性であることを示すため、CaMKIIトランスジェニックマウスとAtg7F/Fマウスを掛け合わせて、成獣になってAtg7を海馬で欠損するマウスを作製している。オートファジー性細胞死の存在は、H/I負荷後の神経細胞以外でも認められることを移植肝の実験で示した。長期保存同所移植実験で、24時間冷保存液/移植-再潅流すると2時間以内に移植肝細胞は変性に陥り、個体も肝不全で死に至る。私達は、この変化にオートファジーが関与することを突き止め、PI3Kの阻害剤でオートファジーを抑制することが知られているWortmanninやLY294002で抑制すると,移植肝の細胞死や個体死が抑制されることを明らかにした。さらに、オートファジーに続いて、リソソームプロテアーゼも活性化され、このプロテアーゼ活性をleupeptinとpepstatinAを投与することで抑制すると、個体死も完全に抑制されることも明らかにした(Autophagy, Gotoh et al.)。また、オートファゴソーム形成に必要な膜の供給源を探るためLC3の局在を検討した所、オートファゴソームの隔離膜のみならず、脂肪滴に局在することが分かった。Atg7を肝臓で特異的に欠損したマウスの肝細胞では(LC3の膜型への変換ができない)、絶食によっても脂肪滴の形成が抑制されることも明らかにした。LC3-IIが脂肪代謝に必須であることが分かった(BBRC, Shibata et al.)。
学術創成研究費の補助の基にオートファジー性神経細胞死の解析をどのように実施して、その成果が何かを公表すると共に、私達と共同研究をして下さった人達の研究を紹介するために、国際シンポジウムを施行した。即ち、平成21年7月4日に"International Symposium on autophagy and cell death"を順天堂大学医学研究科老人性疾患病態・治療研究センターで行なった。プログラムは3つのバートに別けて組まれた。
オートファジーとリソソーム輸送について、1)谷田以誠(国立感染症研究所)、2)和栗聡(福島県立医科大学)、3)Bernald Hoflack(BIOTEC, Technical University of Dresden BioInnovations Zentrum)に、細胞死について4)Ralph Nixon(New York University Langone Medical Center)、5)Kevin Roth(University of Alabama at Birmingham)、6)三浦正幸(東京大学)、さらに、オートファジーと脳虚血について、7)Julien Puyal(DBCM, University of Lausanne)、8)内山安男(順天堂大学)。
以上についての講演を行い、十分な討論を行なった。当日は、100人を超す盛況で、ポスター発表も15あった。

Research Products

• [Journal Article] LC3, a microtubule-associated proteinlA/B light chain3, is involved in cytoplasmic lipid droplet formation (2010)
  • Author(s): Shibata M, Yoshimura K, Tamura H, Ueno T, Nishimura T, Inoue T, Sasaki M, Koike M, Arai H, Kominami E, Uchiyama Y
  • Journal Title: Biophys Biochem Res Comm 393 Pages: 274-279
  • Peer Reviewed
• [Journal Article] Chondrogenesis during the ES cell differentiation in vitro (2010)
  • Author(s): Yagita Y, Horie K, Yamanaka I, Koinuma S, Shigeyoshi Y, Kawakami K, Takeda J, Uchiyama Y
  • Journal Title: PNAS 107 Pages: 3846-3851
  • Peer Reviewed
• [Journal Article] Atg9A protein, an autophagy-related membrane protein, is localized in the neurons of mouse brains (2010)
  • Author(s): Tamura H, Shibata M, Koike M, Sasaki M, Uchiyama Y
  • Journal Title: J Histochem Cytochem Pages: DOI 10.1369 2010.955690
  • Peer Reviewed
• [Journal Article] The MAP1-LC3 conjugation system is involved in lipid droplet formation (2009)
  • Author(s): Shibata M^*, Yoshimura K^*, Furuya M, Koike M, Ueno T, Komatsu M, Arai H, Tanaka K, Kominami E, Uchiyama Y(^* equally contributed)
  • Journal Title: Biophys Biochem Res Comm 382 Pages: 419-423
  • Peer Reviewed
• [Journal Article] The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK-ERK pathway to late endosomes (2009)
  • Author(s): Nada S^*, Hondo A^*, Kasai A^*, Koike M^*, Saito K, Uchiyama Y, Okada M(^*equally contri buted)
  • Journal Title: EMBO J 28 Pages: 477-489
  • Peer Reviewed
• [Journal Article] Participation of autophagy in the initiation of graft dysfunction after rat liver transplantation (2009)
  • Author(s): Gotoh K, Lu Z, Morita M, Shibata M, Koike M, Waguri S, DonoK, DokiY, Kominami E, Sugioka A, Monden M, Uchiyama Y
  • Journal Title: Autophagy 5 Pages: 351-360
  • Peer Reviewed
• [Journal Article] Phosphorylation of highly conserved NF-M KSP repeats is not required for myelin-dependent radial axonal growth (2009)
  • Author(s): Garcia M, Rao M, Fujimoto J, Garcia V, Shah S, Crum J, Gotow T, Uchiyama Y, Ellisman MH, Calcutt N, Cleveland D
  • Journal Title: J Nurosci 29 Pages: 1277-1284
  • Peer Reviewed
• [Journal Article] Nesfatin-1-regulated paraventricular oxytocinergic signaling causes anorexia through a leptin-independent melanocortin pathway (2009)
  • Author(s): Maejima Y, Sedbazar U, Suyama S, Kohno D, Onaka T, Takano E, Yoshida N, Koike M, Uchiyama Y, Fujiwara K, Yashiro T, Horvath TL, Dietrich MO, Tanaka S, Dezaki K, Oh-I S, Hashimoto K, Shimizu H, Nakata M, Mori M, Yada T
  • Journal Title: Cell Metabolism 10 Pages: 355-365
  • Peer Reviewed
• [Journal Article] Ablation of C/EBP beta alleviates ER stress and pancreatic beta cell failure through induction of GRP78 (2009)
  • Author(s): Matsuda, T., Kido, Y., Nakano-Hashimoto, N., Kaisho, T., Tanaka, T., Asahara, S., Shigeyama, Y., Takeda, A., Inoue, T., Shibutani, Y., Koyanagi, M., Hosooka, T., Matsumoto, M., Inoue, H., Uchida, T., Koike, M., Uchiyama, Y., Akira, S., Kasuga, M.
  • Journal Title: J Clin Invest 120 Pages: 115-126
  • Peer Reviewed
• [Journal Article] Mini Screening of Kinase Inhibitors Affecting Period-length of Mammalian Cellular Circadian Clock (2009)
  • Author(s): Yagita K, Yamanaka I, Koinuma S, Shigeyoshi Y, Uchiyama Y
  • Journal Title: Acta Histochem Cytochem 42 Pages: 89-93
  • Peer Reviewed
• [Journal Article] Acquisition of T regulatory function in cathepsin L-inhibited T cells by eye-derived CTLA-2alpha during inflammatory conditions (2009)
  • Author(s): Sugita S, Horie S, Nakamura O, Maruyama K, Takase H, Usui Y, Takeuchi M, Ishidoh K, Koike M, Uchiyama Y, Peters C, Yamamoto Y, Mochizuki M.
  • Journal Title: J Immunol 183 Pages: 5013-5022
  • Peer Reviewed
• [Journal Article] Caspase-9 activation revealed by semaphorin 7A cleavage is independent of apoptosis in the aged olfactory bulb (2009)
  • Author(s): Ohsawa S, Hamada S, Asou H, Kuida K, Uchiyama Y, Yoshida H, Miura M.
  • Journal Title: J Neurosci 29 Pages: 11385-11292
  • Peer Reviewed
• [Journal Article] Very-high-dose alpha-tocopherol supplementation increases blood pressure and causes possible adverse central nervous system effects in stroke-prone spontaneously hypertensive rats (2009)
  • Author(s): Miyamoto K, Shiozaki M, Shibaita M, Koike M, Uchiyama Y, Gotow T
  • Journal Title: J Neurosci Res 87 Pages: 556-566
  • Peer Reviewed
• [Journal Article] (2009)
  • Author(s): Uchiyama Y, et al.
  • Journal Title: Chapter 3 Autophagic neuron death. In : Autophagy in Disease and Clinical Applications, Part C (Edited by : Daniel J.Rlionsky Vol.453)(Elsevier Inc.) Pages: 33-51
• [Journal Article] (2009)
  • Author(s): Uchiyama Y, et al.
  • Journal Title: Autophagy and cell death. In : Molecular imaging for integrated medical therapy and drug development.( Ed.YujiKuge)(Springer Japan) Pages: 176-188
• [Journal Article] Autophagy-Physiology and Pathophysiology (2008)
  • Author(s): Uchiyama, Y., Shibata, M., Koike, M., Yoshimura, K., Sasaki, M.
  • Journal Title: Histochem Cell Biol 129 Pages: 407-420
  • Peer Reviewed
• [Journal Article] Autophagic neuron death in neonatal brain ischemia/hypoxia (2008)
  • Author(s): Uchiyama, Y., Koike, M., Shibata, M.
  • Journal Title: Autophagy 4 Pages: 404-408
  • Peer Reviewed
• [Journal Article] Interleukin-6 blockade suppresses autoimmune arthritis in mice by the inhibition of inflammatory Th17 responses (2008)
  • Author(s): Fujimoto M, Serada S, Mihara M, Uchiyama Y, Yoshida H, Koike N, Ohsugi Y, Nishikawa T, Ripley B, Kimura A, Kishimoto T, Naka T.
  • Journal Title: Arthritis Rheum 58 Pages: 3710-3719
  • Peer Reviewed
• [Journal Article] Lysosomal enzyme cathepsin D protects against alpha-synuclein aggregation and toxicity (2008)
  • Author(s): Qiao L, Hamamichi S, Caldwell KA, Caldwell GA, Yacoubian TA, Wilson S, Xie Z-L, Speake LD, Parks R, Crabtree D, Liang Q, Crimmins S, Schneider L, Uchiyama Y, Iwatsubo T, Zhou Y, Peng L, Lu YM, Standaert DG, Walls KC, Shacka JJ, Roth KA, Zhang J
  • Journal Title: Mol Brain 1 Pages: 17
  • Peer Reviewed
• [Journal Article] The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice (2008)
  • Author(s): Sou YS, Waguri S, Iwata JI, Ueno T, Fujimura T, Hara T, Sawada N, Yamada A, Mizushima N, Uchiyama Y, Kominami E, Tanaka K, Komatsu M
  • Journal Title: Mol Biol Cell 19 Pages: 4762-4775
  • Peer Reviewed
• [Journal Article] GPHR is a novel anion channel critical for acidification and functions of the Golgi apparatus (2008)
  • Author(s): Maeda Y, Ide T, Koike M, Uchiyama Y, Kinoshita T
  • Journal Title: Nat Cell Biol 10 Pages: 1135-1145
  • Peer Reviewed
• [Journal Article] Human herpesvirus-6 induces MVB formation and virus egress occurs via an exosomal release pathway (2008)
  • Author(s): Mori Y^*, Koike M^*, Moriishi E, Kawabata A, Tang H, Oyaizu H, Uchiyama Y, Yamanishi K^*equal contributors
  • Journal Title: Traffic 9 Pages: 1728-1742
  • Peer Reviewed
• [Journal Article] Inefficient phagosome maturation in infant macrophages (2008)
  • Author(s): Saito F, Kuwata H, Oiki E, Koike M, Uchiyama Y, Honda K, Takeda K
  • Journal Title: Biochem Biophys Res Commun 375 Pages: 113-118
  • Peer Reviewed
• [Journal Article] LC3, an autophagosome marker, is highly expressed in gastrointestinal cancers (2008)
  • Author(s): Yoshida A, Miyata H, Doki Y, Yamasaki M, Sohma I, Gotoh K, Takiguchi S, Fujiwara Y, Uchiyama Y, Monden M
  • Journal Title: Int J Oncol 33 Pages: 461-468
  • Peer Reviewed
• [Journal Article] Developing postmitotic mammalian neurons in vivo lacking. Apaf-1 and ergo programmed cell death by a caspase-independent, nonapoptotic pathway involving autophagy (2008)
  • Author(s): Oppenheim RW, Blomgren K, Ethell DW, Koike M, Komatsu M, Prevette D, Roth KA, Uchiyama Y, Vinsant S, Zhu C
  • Journal Title: J Neurosci 28 Pages: 1490-1497
  • Peer Reviewed
• [Journal Article] Morphological and biochemical signs of age-related neurodegenerative changes in Klotho mutant mice (2008)
  • Author(s): Shiozaki M, Yoshimura K, Shibata M, Koike M, Uchiyama Y, Gotow T
  • Journal Title: Neurcience 152 Pages: 924-941
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• [Journal Article] Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector based strategy (2008)
  • Author(s): Kiyohara Y, Nishii K, Ukai-Tadenuma M, Ueda H, Uchiyama Y, Yagita K
  • Journal Title: Nucl Acids Res 36(4) Pages: e23
  • Peer Reviewed
• [Journal Article] Participation of autophagy in renal ischemia/reperfusion injury (2008)
  • Author(s): Suzuki C, Takabatake Y, Tanaka H, Koike M, Shibata M, Uchiyama Y, Takahara S, Imai E, Isaka Y
  • Journal Title: Biochem Biophys Res Comm 368 Pages: 100-106
  • Peer Reviewed
• [Journal Article] Human RME-8 is involved in membrane trafficking through early endosomes (2008)
  • Author(s): Fujibayashi A, Taguchi T, Misaki R, Ohtani M, Dohmae N, Takio K, Yamada M, Gu J, Yamakami M, Fukuda M, Waguri S, Uchiyama Y, Yoshimori T, Sekiguchi K
  • Journal Title: Cell Struct Funct 33 Pages: 35-50
  • Peer Reviewed
• [Journal Article] Ablation of Csk in neural crest lineages causes corneal anomaly by deregulating collagen fibril organization and cell motility (2008)
  • Author(s): Takatsuka A, Yagi R, Koike M, Oneyama C, Nada S, Schmedt C, Uchiyama Y, Okada M
  • Journal Title: Dev Biol 315 Pages: 474-488
  • Peer Reviewed
• [Journal Article] The protocadherin-alpha family is involved in axonal coalescence of olfactory sensory neurons into glomeruli of the olfactory bulb in mouse (2008)
  • Author(s): Hasegawa S, Hamada S, Kumode Y, Esumi S, Katori S, Fukuda E, Uchiyama Y, Hirabayashi T, Mombaerts P, Yagi T
  • Journal Title: Mol Cell Neurosci 38 Pages: 66-79
  • Peer Reviewed
• [Journal Article] Inhibition of autophagy prevents hippocampal pyramidal neuron death after hypoxic-ischemic injury (2008)
  • Author(s): Koike M, Shibata S, Tadakoshi T, Gotoh K, Komatsu M, Waguri S, Kawahara N, Kuida K, Nagata S, Kominami E, Tanaka K, Uchiyama Y
  • Journal Title: Am J Pathol 172 Pages: 454-469
  • Peer Reviewed
• [Journal Article] Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes (2008)
  • Author(s): Klionsky DJ, et al.
  • Journal Title: Autophagy 4 Pages: 151-175
  • Peer Reviewed
• [Journal Article] JE Loss of ALS2/Alsin Exacerbates Motor Dysfunction in a SOD1^<H46R_> Expressing Mouse ALS Model by Disturbing Endolysosomal Trafficking
  • Author(s): Hadano S, Otomo A, Kunita R, Suzuki-Utsunomiya A, Akatsuka A, Koike M, Aoki M, Uchiyama Y, Itoyama Y, Ikeda
  • Journal Title: PLoSONE (in press)
  • Peer Reviewed
• [Journal Article] Cathepsin L in bone marrow-derived cells is required for retinal and choroidal neovascularization
  • Author(s): Shimada N, Ohno-Matsui K, Iseki S, Koike M, Uchiyama Y, Wang J, Yoshida T, Sato T, Peters C, Mochizuki M, Morita I
  • Journal Title: Am J Pathol (in press)
  • Peer Reviewed
• [Presentation] Autophagy and cell death (2009)
  • Author(s): 内山安男
  • Organizer: 5^<th> International Symposium on Autophagy
  • Place of Presentation: 大津プリンスホテルコンベンションホール
  • Year and Date: 20090924-20090928
• [Presentation] Morphological aspects of autophagy : Identification and possible mechanism of autophagosome formation in neuronal cells (2009)
  • Author(s): 内山安男
  • Organizer: Microscopy Conference 2009
  • Place of Presentation: オーストリア グラーツ
  • Year and Date: 20090901-20090904
• [Presentation] Morphological features of programmed cell death across the eukaryotic kingdom (2009)
  • Author(s): 内山安男
  • Organizer: 6^<th> ISEM09 Committee
  • Place of Presentation: 神戸大学
  • Year and Date: 2009-09-17
• [Presentation] Cell Death and Autophagy (2009)
  • Author(s): 内山安男
  • Organizer: International Symposium on Autophagy and Cell Death
  • Place of Presentation: 順天堂大学医学研究科老人性疾患病態・治療研究センター
  • Year and Date: 2009-07-04
• [Presentation] Autophagy and other pathomechanisms (2009)
  • Author(s): 内山安男
  • Organizer: 12^<th> International Congress on Neuronal Ceroid Lipofuscinoses(NCL)
  • Place of Presentation: ドイツ ハンブルグ
  • Year and Date: 2009-06-05
• [Presentation] p62/A170/SQSTM1, a binding partner of ubiquitin and LC3, and lysosomal storage Disorder (2008)
  • Author(s): 内山安男
  • Organizer: 第31回日本神経科学会シンポジウム(SY3AE1)
  • Place of Presentation: 東京国際フォーラム
  • Year and Date: 20080709-20080711
• [Presentation] p62/A170/SQSTM1, a binding partner of ubiquitin and LC3, and lysosomal storage Disorder (2008)
  • Author(s): 内山安男
  • Organizer: 第5回アジア太平洋解剖学会(APICA)
  • Place of Presentation: イラン テヘラン
  • Year and Date: 20080516-20080519
• [Presentation] オートファジー/リソソーム制裁防止の分子細胞生物学的研究 (2008)
  • Author(s): 内山安男
  • Organizer: 平成20年度顕微鏡学会瀬藤賞受賞講演
  • Place of Presentation: 千葉大学
  • Year and Date: 2008-10-17