Comprehensive understanding of refilling mechanism of excitatory neurotransmitter glutamate into synaptic vesicles

• Project/Area Number: 19H03330
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 46010:Neuroscience-general-related
• Research Institution: Doshisha University
• Principal Investigator: Takamori Shigeo 同志社大学, 脳科学研究科, 教授 (10397002)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2021: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2020: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2019: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
• Keywords: シナプス / シナプス小胞 / グルタミン酸 / 神経伝達物質 / エンドサイトーシス / クラスリン / 海馬 / 神経伝達物質・受容体 / VGLUT / SNAREタンパク質 / シナプス伝達 / 蛍光イメージング

Outline of Research at the Start

哺乳類中枢神経系における主要な興奮性シナプス伝達には、シナプス小胞膜上に存在する小胞型グルタミン酸トランスポーター(VGLUT)によるグルタミン酸のシナプス小胞への濃縮過程が必須である。VGLUTの発現量や活性が興奮性シナプス伝達強度の制御に繋がると考えられるが、その詳細なメカニズムは不明である。本研究では、各種pH感受性蛍光タンパク質と蛍光グルタミン酸センサーを利用した蛍光イメージングを基盤技術とし、【１】 グルタミン酸再充填メカニズムの解明、【２】 VGLUT発現量とイソ型の違いとシナプス小胞放出特性の解明、【３】 VGLUTタンパク質のシナプス小胞への回収機構の解明、の３点に取り組む。

Outline of Final Research Achievements

The vast majority of excitatory synaptic transmission in our brain is mediated by the principal neurotransmitter glutamate, which is accumulated into presynaptic vesicles (SVs) by vesicular glutamate transporters (VGLUTs). We have discovered that the reduction of VGLUT expression slowed the rate of glutamate transport into SVs, consequently resulting in synaptic failure during high repetitive stimulation. In a separate study, we found that SVs are not functionally homogeneous, but a subpopulation of SVs defined by the endosomal SNARE, Syntaxin 7, undergoes exocytosis preferentially during high repetitive stimulation. Thus, our studies reveal that glutamate refilling into SVs can be a rate-limiting step for synaptic transmission and that the segregation of SV pools by endosomal SNAREs can be one of the key features for functional heterogeneity of individual synapses.

Academic Significance and Societal Importance of the Research Achievements

統合失調症、自閉症、認知症などの脳神経疾患の多くは、シナプス異常が原因で起こることがわかってきており、総称してシナプトパチーと呼ばれています。これらの病気の発症予防や治療法を確立するためには、シナプス伝達がどのように起こっているか分子レベルで解明することが必要です。今回の我々の研究成果では、VGLUT及びSyntaxin 7をはじめとした脳での発現が低いSNAREタンパク質の働きがシナプス伝達効率の維持に重要な役割を果たしていることがわかりました。これらの知見を蓄積することは、病態の理解、薬物候補のアセスメントの厳格化に寄与することが期待されます。

Research Products

• [Int'l Joint Research] FMP-Berlin 分子薬理学研究所/マックスプランク実験医学研究所(ドイツ)
• [Int'l Joint Research] マックスプランク実験医学研究所/ベルリン分子薬理学研究所/マックスプランク生物物理学研究所(ドイツ)
• [Int'l Joint Research] マックスプランク実験医学研究所(ドイツ)
• [Int'l Joint Research] ベルリン分子薬理学研究所(ドイツ)
• [Journal Article] Clathrin-independent endocytic retrieval of SV proteins mediated by the clathrin adaptor AP-2 at mammalian central synapses (2022)
  • Author(s): Lopez-Hernandez Tania、Takenaka Koh-ichiro、Mori Yasunori、Kongpracha Pornparn、Nagamori Shushi、Haucke Volker、Takamori Shigeo
  • Journal Title: eLife Volume: 11 Pages: 71198-71198
  • DOI: 10.7554/elife.71198
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Physiological Perspectives on Molecular Mechanisms and Regulation of Vesicular Glutamate Transport: Lessons From Calyx of Held Synapses (2022)
  • Author(s): Hori Tetsuya、Takamori Shigeo
  • Journal Title: Frontiers in Cellular Neuroscience Volume: 15 Pages: 811892-811892
  • DOI: 10.3389/fncel.2021.811892
  • Peer Reviewed / Open Access
• [Journal Article] Quantitative Analysis of Presynaptic Vesicle Luminal pH in Cultured Neurons (2022)
  • Author(s): Egashira Yoshihiro、Katsurabayashi Shutaro、Takamori Shigeo
  • Journal Title: Methods Mol Biol Volume: 2417 Pages: 45-58
  • DOI: 10.1007/978-1-0716-1916-2_4
  • ISBN: 9781071619155, 9781071619162
  • Peer Reviewed
• [Journal Article] The endosomal Q-SNARE, Syntaxin 7, defines a rapidly replenishing synaptic vesicle recycling pool in hippocampal neurons (2021)
  • Author(s): Mori Yasunori、Takenaka Koh-ichiro、Fukazawa Yugo、Takamori Shigeo
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 981-981
  • DOI: 10.1038/s42003-021-02512-4
  • Peer Reviewed / Open Access
• [Journal Article] Hidden proteome of synaptic vesicles in the mammalian brain (2020)
  • Author(s): Taoufiq Zacharie、Ninov Momchil、Villar-Briones Alejandro、Wang Han-Ying、Sasaki Toshio、Roy Michael C.、Beauchain Francois、Mori Yasunori、Yoshida Tomofumi、Takamori Shigeo、Jahn Reinhard、Takahashi Tomoyuki
  • Journal Title: Proceedings of the National Academy of Sciences Volume: 117 Issue: 52 Pages: 33586-33596
  • DOI: 10.1073/pnas.2011870117
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Vesicular Glutamate Transporter Expression Ensures High-Fidelity Synaptic Transmission at the Calyx of Held Synapses (2020)
  • Author(s): Nakakubo Yutaro、Abe Saeka、Yoshida Tomofumi、Takami Chihiro、Isa Masayuki、Wojcik Sonja M.、Brose Nils、Takamori Shigeo、Hori Tetsuya
  • Journal Title: Cell Reports Volume: 32 Issue: 7 Pages: 108040-108040
  • DOI: 10.1016/j.celrep.2020.108040
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Cargo-selective sorting function of clathrin adaptor protein AP-2 during clathrin-independent endocytosis at hippocampal synapses. (2020)
  • Author(s): Takenaka K, Lopez-Hernandez T, Mori Y, Nagamori S, Haucke V, Takamori S.
  • Organizer: 第72回日本細胞生物学会大会
• [Presentation] Intersectin 1が形成する液滴は、SNAP-25によって形質膜に係留されたエンドサイトーシス関連タンパク質の貯蔵庫として働く. (2020)
  • Author(s): 吉田知史、竹中康一郎、塙京子、森靖典、高森茂雄
  • Organizer: 第72回 日本細胞生物学会.
• [Presentation] Cargo-selective sorting function of clathrin adaptor protein AP-2 during clathrin-independent endocytosis at hippocampal synapses. (2020)
  • Author(s): Takenaka K, Lopez-Hernandez T, Mori Y, Nagamori S, Haucke V, Takamori S.
  • Organizer: 生理学研究所 シナプス研究会
• [Presentation] 形質膜に係留されている ITSN1 によって形成される相分離環境はエンドサイトーシス関連タンパク質の動的な貯蔵庫として機能する. (2020)
  • Author(s): 吉田知史
  • Organizer: 生理学研究所 シナプス研究会
• [Presentation] Cargo-selective sorting function of clathrin adaptor protein AP-2 during clathrin-independent endocytosis at hippocampal synapses. (2020)
  • Author(s): Takenaka K, Lopez-Hernandez T, Mori Y, Nagamori S, Haucke V, Takamori S.
  • Organizer: JSPS Core-to-core Program Symposium “Nanobiology of neural plasticity based on optical nanoscopy”
  • Int'l Joint Research
• [Presentation] Intersectin 1 condensates, the reservoir for various endocytic proteins, are anchored at plasma membrane by SNAP-25. (2020)
  • Author(s): Yoshida T.
  • Organizer: JSPS Core-to-core Program (A) Symposium “Nanobiology of neural plasticity based on optical nanoscopy”
  • Int'l Joint Research
• [Presentation] Intersectin-1 liquid phase serves as the cytoplasmic reservoir for endocytic proteins. (2019)
  • Author(s): Shigeo Takamori
  • Organizer: Core-to-Core Symposium: Understanding synapses: Form molecules to function
  • Int'l Joint Research / Invited
• [Presentation] Q-SNARE Syntaxin 7 defines a reluctant recycling pool of synaptic vesicles in hippocampal neurons (2019)
  • Author(s): 森靖典、竹中康一郎、深澤有吾、高森茂雄
  • Organizer: 生理学研究所シナプス研究会「ミクロからマクロに至る脳の構造と機能のダイナミクス
  • Invited
• [Presentation] 小胞型グルタミン酸輸送体タイプ1(VGLUT1)欠損が神経活動低下を引き起こす様々な要因の検証 (2019)
  • Author(s): 阿部冴華、中久保佑太郎、吉田知史、伊佐真幸、高森茂雄、堀哲也
  • Organizer: 第42回日本分子生物学学会
• [Presentation] 神経終末におけるSyntaxin 7を含むシナプス小胞の機能解析 (2019)
  • Author(s): 森靖典、竹中康一郎、深澤有吾、高森茂雄
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] Cargo- Selective Sorting Function of Clathrin Adaptor Protein Complex AP-2 During Clathrin- Independent Endocytosis at Hippocampal Synapses (2019)
  • Author(s): Koichiro Takenaka, Tania Lopez Hernandez, Yasunori Mori, Shushi Nagamori, Volker Haucke and Shigeo Takamori
  • Organizer: Core-to-Core Symposium: Understanding synapses: Form molecules to function
  • Int'l Joint Research
• [Presentation] Vesicular Glutamate Transporter Expression Ensures the High Fidelity Synaptic Transmission at the Calyx of Held Synapses (2019)
  • Author(s): Yutaro Nakakubo, Saeka Abe, Tomofumi Yoshida, Chihiro Takami Masayuki Isa, Sonja M. Wojcik, Nils Brose , Shigeo Takamori and Tetsuya Hori
  • Organizer: Core-to-Core Symposium: Understanding synapses: Form molecules to function
  • Int'l Joint Research
• [Presentation] A liquid phase of Intersectin1 serves as the cytoplasmic reservoir for endocytic proteins (2019)
  • Author(s): Tomofumi Yoshida, Koichiro Takenaka, Manabu Kitamata, Kyoko Hanawa-Suetsugu, Yasunori Mori, Shiro Suetsugu and Shigeo Takamori
  • Organizer: Core-to-Core Symposium: Understanding synapses: Form molecules to function
  • Int'l Joint Research
• [Remarks] 高森茂雄教授のVGLUTに関する総説がFront Cell Neurosciに掲載
  • URL: https://brainscience.doshisha.ac.jp/news/2022/0114/news-detail-255.html
• [Remarks] 脳科学研究科・神経膜分子機能部門の論文がElife誌に掲載
  • URL: https://brainscience.doshisha.ac.jp/news/2022/0112/news-detail-254.html
• [Remarks] 森靖典准教授の論文がCommunications Biology誌に掲載されました
  • URL: https://brainscience.doshisha.ac.jp/news/2021/0823/news-detail-242.html