シナプス後膜における受容体の局在・動態へのアミロイドβの作用

• Project Area: Generation of synapse-neurocircuit pathology
• Project/Area Number: 25110717
• Research Category: Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Kyoto University
• Principal Investigator: 平野 丈夫 京都大学, 理学(系)研究科(研究院), 教授 (50181178)
• Project Period (FY): 2013-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥8,320,000 (Direct Cost: ¥6,400,000、Indirect Cost: ¥1,920,000); Fiscal Year 2014: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2013: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
• Keywords: シナプス / グルタミン酸受容体 / 蛍光イメージング / 全反射顕微鏡 / エンドサイトーシス / アミロイドβ

Outline of Annual Research Achievements

アルツハイマー病等ではシナプス可塑性に異常があると考えられている。シナプス可塑性に際しては、シナプス後膜上のAMPA型グルタミン酸受容体数がダイナミックに増減し、シナプス後膜上の受容体数の制御には、エキソサイトーシス・エンドサイトーシス・側方移動がかかわる。私たちはこれら受容体動態を可視化する実験手法を開発してきた。培養神経細胞のシナプス後膜構造をガラスに面する形で形成させ、その神経細胞にpH依存性蛍光タンパク質SEPを融合したAMPA受容体を発現させ、その蛍光を全反射顕微鏡で観察することにより、シナプス後膜内外でのAMPA受容体のエキソサイトーシスおよび側方移動をライブイメージングした。本研究では、新たな実験手法を加えて、AMPA受容体の個別のエンドサイトーシスをシナプス後膜内外で可視化した。AMPA受容体のエンドサイトーシスを細胞膜上のSEP標識受容体からの蛍光シグナルの減少として同定することは、SN比が悪いために困難であった。そこで、エンドサイトーシスされた直後のSEP標識AMPA受容体のみを検出することを試みた。ガラス管を用いて細胞外液のpHを数ミリ秒で交換できるシステムを構築し、pHを7.3から6に換える実験を行ったところ、細胞膜上のSEPからの蛍光シグナルは消光し、エンドサイトーシスされた直後のSEP標識AMPA受容体シグナルのみを検出することができた。こうしたエンドサイトーシス直後の蛍光シグナルは、シナプス後膜外の他にシナプス後部に隣接した部位でも認められ、シナプス後膜隣接領域でAMPA受容体のエンドサイトーシスが起こることが明らかになり、エンドサイトーシスの頻度は長期抑圧を引き起こす刺激によって増加することも判明した。また、Aβのシナプス可塑性への作用を検討し、長期増強時のAMPA受容体数の増加がAβによって抑えられることを見出した。

Research Progress Status

26年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

26年度が最終年度であるため、記入しない。

Research Products

• [Journal Article] Opposite regulation of sustained CaMKII activity by alpha and beta subunits impacts inhibitory synaptic plasticity. (2014)
  • Author(s): Nagasaki, N., Hirano, T. and Kawaguchi, S.
  • Journal Title: J. Physiol. Volume: 592 Issue: 22 Pages: 4891-4909
  • DOI: 10.1113/jphysiol.2014.280230
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Around LTD hypothesis in motor learning. (2014)
  • Author(s): Hirano, T.
  • Journal Title: Cerebellum Volume: 12 Issue: 5 Pages: 645-650
  • DOI: 10.1007/s12311-014-0581-4
  • NAID: 120005669284
  • Peer Reviewed
• [Journal Article] Control of spontaneous Ca2+ transients is critical for neuronal maturation in the developing neocortex. (2014)
  • Author(s): Y. Bando, K. Irie, T. Shimomura, H. Umeshima, Y. Kushida, M. Kengaku, Y. Fujiyoshi, T. Hirano and Y. Tagawa
  • Journal Title: Cerebral Cortex Volume: 1 Issue: 1 Pages: 1-12
  • DOI: 10.1093/cercor/bhu180
  • Peer Reviewed / Open Access
• [Journal Article] Live-cell imaging of receptors around postsynaptic membrane formed on adhesion-protein-coated glass (2014)
  • Author(s): Tanaka, H., Fujii, S. & Hirano, T.
  • Journal Title: Nature Protocols Volume: 9 Issue: 1 Pages: 76-89
  • DOI: 10.1038/nprot.2013.171
  • Peer Reviewed
• [Journal Article] Regulation and functional roles of rebound potentiation at cerebellar stellate cell - Purkinje cell synapse (2014)
  • Author(s): Hirano, T. & Kawaguchi, S.
  • Journal Title: Frontiers in Cellular Neuroscience Volume: 8 Pages: 1-8
  • DOI: 10.3389/fncel.2014.00042
  • NAID: 120005385884
  • Peer Reviewed
• [Journal Article] Contribution of postsynaptic GluD2 to presynaptic R-type Ca2+ channel function, glutamate release and long-term potentiation at parallel fiber to Purkinje cell synapses (2013)
  • Author(s): Yamashita, M., Kawaguchi, S. & Hirano, T.
  • Journal Title: Cerebellum Volume: 12 Issue: 5 Pages: 657-666
  • DOI: 10.1007/s12311-013-0474-y
  • NAID: 120005575262
  • Peer Reviewed
• [Journal Article] Long-term potentiation of inhibitory synaptic transmission onto cerebellar Purkinje neurons contributes to adaptation of vestibulo-ocular reflex (2013)
  • Author(s): Tanaka, S., Kawaguchi, S. Shioi, G. & Hirano, T.
  • Journal Title: J. Neurosci. Volume: 33 Issue: 43 Pages: 17209-17220
  • DOI: 10.1523/jneurosci.0793-13.2013
  • NAID: 120005342657
  • Peer Reviewed
• [Journal Article] Long-term depression and other synaptic plasticity in the cerebellum (2013)
  • Author(s): Hirano, T.
  • Journal Title: Proceedings of the Japan Academy, Series B Volume: 89 Issue: 5 Pages: 183-195
  • DOI: 10.2183/pjab.89.183
  • NAID: 130003365114
  • ISSN: 0386-2208, 1349-2896
  • Peer Reviewed
• [Journal Article] Dysfunction of KCNK potassium channels impairs neuronal migration in the developing mouse cerebral cortex. (2012)
  • Author(s): Yuki Bando
  • Journal Title: Cerebral Cortex Volume: in press Issue: 4 Pages: 1017-1029
  • DOI: 10.1093/cercor/bhs387
  • Peer Reviewed
• [Presentation] Imaging of individual endocytosis of AMPA receptor around postsynaptic membrane. (2015)
  • Author(s): Hirano, T.
  • Organizer: Symposium “Forefront of exo- and endocytosis research” in the 92nd Annual Meeting of the Physiological Society of Japan
  • Place of Presentation: Kobe
  • Year and Date: 2015-03-21
  • Invited
• [Presentation] Molecular mechanisms and roles of synaptic plasticity. (2015)
  • Author(s): Hirano, T.
  • Organizer: 1st Kyoto University and UC San Diego Joint Symposium, Section “Changing Brain”.
  • Place of Presentation: Kyoto
  • Year and Date: 2015-03-11
  • Invited
• [Presentation] Inhibitory synaptic plasticity in cerebellar Purkinje neurons and its differential contribution to motor learning paradigms. (2014)
  • Author(s): Hirano, T.
  • Organizer: Symposium “Synaptic regulation in the cerebellum and motor control” in the 37th annual meeting of the Japan Neuroscience Society
  • Place of Presentation: Yokohama
  • Year and Date: 2014-09-11
  • Invited
• [Presentation] Cerebellar inhibitory synaptic plasticity, rebound potentiation, contributes to adaptation of vestibulo-ocular reflex
  • Author(s): Tanaka, S., Kawaguchi, S. and Hirano, T.
  • Organizer: The 43rd Annual Meeting, Society for Neuroscience
  • Place of Presentation: San Diego, USA
• [Presentation] Regulation of neural activity is critical for the control of neural migration and dendrite formation of cortical layer 2/3 neurons in vivo
  • Author(s): Bando, Y., Irie, K., Kushida, Y., Shimomura, T., Fujiysohi, Y., Hirano, T. and Tagawa, Y.
  • Organizer: The 43rd Annual Meeting, Society for Neuroscience
  • Place of Presentation: San Diego, USA
• [Presentation] 「学習と記憶」のメカニズム
  • Author(s): 平野丈夫
  • Organizer: 千里ライフサイエンスフォーラム
  • Place of Presentation: 大阪
  • Invited
• [Presentation] Visualized dynamics of glutamate receptors around postsynaptic membrane
  • Author(s): Hirano, T., Tanaka, H. and Fujii, S.
  • Organizer: Workshop “Molecular architecture and regulation mechanism of synapses” in the 36th Annual Meeting of the Molecular Biology Society of Japan
  • Place of Presentation: Ｋｏｂｅ
  • Invited
• [Presentation] Visualization of glutamate receptors around postsynaptic membrane during synaptic plasticity
  • Author(s): Hirano, T.
  • Organizer: The 2nd Kyoto-Bristol Symposium 2014, Neuroscience Section
  • Place of Presentation: Kyoto
  • Invited
• [Presentation] Regulation mechanism and functional role of inhibitory synaptic plasticity in a cerebellar Purkinje neuron
  • Author(s): Hirano, T.
  • Organizer: Symposium “Mechanism and role of inhibitory synaptic plasticity” in the 91st Annual Meeting of the Physiological Society of Japan
  • Place of Presentation: Kagoshima
  • Invited
• [Presentation] Subtype-specific trafficking of AMPA receptors during hippocampal LTD
  • Author(s): Fujii, S., Tanaka, H. and Hirano, T.
  • Organizer: The 36th Annual Meeting of the Japan Neuroscience Society
  • Place of Presentation: Kyoto
• [Presentation] Regulation of excitability is critical for the control of migration and dendrite formation in the developing cerebral cortex
  • Author(s): Bando, Y., Irie, K., Kushida, Y., Shimomura, T., Fujiysohi, Y., Hirano, T. and Tagawa, Y.
  • Organizer: The 36th Annual Meeting of the Japan Neuroscience Society.
  • Place of Presentation: Kyoto
• [Remarks] シナプス形成と可塑性の分子機構
  • URL: http://www.neurosci.biophys.kyoto-u.ac.jp/physiology.html
• [Remarks] 平野研究室ホームページ
  • URL: http://www.neurosci.biophys.kyoto-u.ac.jp/main.html