Molecular and structural basis and modification mechanisms of the active zones regulating synaptic transmission efficiency

• Project/Area Number: 19H03324
• 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: University of Yamanashi
• Principal Investigator: Ohtsuka Toshihisa 山梨大学, 大学院総合研究部, 教授 (40401806)
• Co-Investigator(Kenkyū-buntansha): 萩原 明 東京理科大学, 理工学部応用生物科学科, 准教授 (70402849)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥17,290,000 (Direct Cost: ¥13,300,000、Indirect Cost: ¥3,990,000); Fiscal Year 2021: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2020: ¥5,850,000 (Direct Cost: ¥4,500,000、Indirect Cost: ¥1,350,000); Fiscal Year 2019: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
• Keywords: アクティブゾーン / CAST / ELKS / ビオチンリガーゼ / 液-液相分離 / 近位依存性ビオチン標識法 / Bassoon / シナプス

Outline of Research at the Start

神経伝達物質放出の調節が、いかにシナプス伝達の強度とその可塑性をコントロールし、最終的に個体レベルでどのように表現されるのか、その本質は未だ明らかになっていない。そこで本研究では、伝達物質放出の位置とタイミングを決定する構造体であるアクティブゾーンに着目した包括的研究を推進する。具体的には、CAST/ELKSの結合タンパク質の同定、修飾機構の解明およびマウス行動解析の３項目の研究を推進し、アクティブゾーンの分子構造基盤の全容解明とその異常がいかに病態・疾患につながるかを明らかにする。

Outline of Final Research Achievements

Using constructs with different linker lengths between the biotin ligase and CAST, we proceeded to analyze their expression and localization in cultured hippocampal neurons and quantified their co-localization with Bassoon, showing a statistically significant difference in the degree of co-localization between the biotin signal and the Bassoon signal depending on the linker length. The results showed a statistically significant difference in the degree, suggesting that different linker lengths can detect different binding molecule complexes depending on the distance from CAST at active zones.
In addition, no cytoplasmic droplet formation was observed when RIM1 was expressed alone, and significant droplet formation could only be observed when coexpressed with CAST/ELKS. These results suggest that liquid-liquid phase separation may play an important function in the formation of active zone complexes.

Academic Significance and Societal Importance of the Research Achievements

本研究で、アクティブゾーンにおける近位ビオチンラベル化法を確立した。アクティブゾーンタンパク質CAST/ELKSが形成するタンパク質複合体のさらなる解析から、複合体のタンパク質組成が脳内の異なる領域でどのように違っているか、分子レベルでの知見が期待される。分子・シナプスレベルでの違いが、最終的には個体レベルの機能（脳機能や行動）にどのような影響をおよぼすのか、その因果関係の解明につながる学術的な意義を有する。また、アルツハイマー病などの認知症においてもシナプスレベルの変化が知られており、本研究で開発した手法をモデルマウス等に用いることで、認知症研究の発展につながり、社会的な意義も高い。

Research Products

• [Journal Article] An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping (2021)
  • Author(s): Hamada Shun、Nagase Masashi、Yoshizawa Tomohiko、Hagiwara Akari、Isomura Yoshikazu、Watabe Ayako M.、Ohtsuka Toshihisa
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s42003-021-01977-7
  • Peer Reviewed / Open Access
• [Journal Article] Prickle2 and Igsf9b Coordinately Regulate the Cytoarchitecture of the Axon Initial Segment (2020)
  • Author(s): Chowdhury Md. Imrul Hasan、Nishioka Tomoki、Mishima Noriko、Ohtsuka Toshihisa、Kaibuchi Kozo、Tsuboi Daisuke
  • Journal Title: Cell Structure and Function Volume: 45 Issue: 2 Pages: 143-154
  • DOI: 10.1247/csf.20028
  • NAID: 130007894945
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Development of an L-type Ca 2+ channel-dependent Ca 2+ transient during the radial migration of cortical excitatory neurons (2020)
  • Author(s): Horigane S, Hamada S, Kamijo S, Yamada H, Yamasaki M, Watanabe M, Bito H, Ohtsuka T, Takemoto-Kimura S
  • Journal Title: Neurosci Res Volume: S0168-0102(20)30391-6 Pages: 30391-6
  • DOI: 10.1016/j.neures.2020.06.003
  • Peer Reviewed / Open Access
• [Journal Article] RFC1 repeat expansion in Japanese patients with late-onset cerebellar ataxia (2020)
  • Author(s): Tsuchiya Mai、Nan Haitian、Koh Kishin、Ichinose Yuta、Gao Lihua、Shimozono Keisuke、Hata Takanori、Kim Yeon-Jeong、Ohtsuka Toshihisa、Cortese Andrea、Takiyama Yoshihisa
  • Journal Title: Journal of Human Genetics Volume: 65 Issue: 12 Pages: 1143-1147
  • DOI: 10.1038/s10038-020-0807-x
  • Peer Reviewed / Open Access
• [Journal Article] Neuron-enriched phosphatase and actin regulator 3 (Phactr3)/ nuclear scaffold-associated PP1-inhibiting protein (Scapinin) regulates dendritic morphology via its protein phosphatase 1-binding domain (2020)
  • Author(s): Miyata Tomoaki、Kikuchi Keietsu、Ihara Daisuke、Kaito Maki、Ishibashi Yuta、Hakamata Tomoyuki、Yamada Tetsuya、Ishikawa Mitsuru、Mizukoshi Miho、Shoji Shizuku、Fukuchi Mamoru、Tsuda Masaaki、Hida Yamato、Ohtsuka Toshihisa、Kaneda Marisa、Tabuchi Akiko
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 528 Issue: 2 Pages: 322-329
  • DOI: 10.1016/j.bbrc.2020.05.006
  • Peer Reviewed / Open Access
• [Journal Article] Presynaptic development is controlled by the core active zone proteins CAST/ELKS. (2020)
  • Author(s): Radulovic, T., Dong, W., Goral, R., Thomas, C., Veeraraghavan, P., Montesinos, M., Guerrero-Given, D., Goff, K., Luebbert, M., Kamasawa, N., Ohtsuka, T., Young, S.M.*
  • Journal Title: J. Physiol. Volume: in press Issue: 12 Pages: 2431-2452
  • DOI: 10.1113/jp279736
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Impaired experience-dependent maternal care in presynaptic active zone protein CAST-deficient dams (2020)
  • Author(s): Hagiwara, A., Sugiyama, N., Ohtsuka, T.*
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 5238-5238
  • DOI: 10.1038/s41598-020-62072-1
  • Peer Reviewed / Open Access
• [Journal Article] CAST/ELKS in the mouse forebrain causes high mortality of newborn pups. (2020)
  • Author(s): Hagiwara, A., Hamada, S., Hida, Y., Ohtsuka, T.*
  • Journal Title: Molecular Brain Volume: 13 Issue: 1 Pages: 13-13
  • DOI: 10.1186/s13041-020-0557-x
  • Peer Reviewed / Open Access
• [Journal Article] VPS13D-related disorders presenting as a pure and complicated form of hereditary spastic paraplegia (2020)
  • Author(s): Koh K, Ishiura H, Shimazaki H, Tsutsumiuchi M, Ichinose Y, Nan H, Hamada S, Ohtsuka T, Tsuji S, Takiyama Y
  • Journal Title: Mol Genet Genomic Med Volume: 8 Issue: 3
  • DOI: 10.1002/mgg3.1108
  • Peer Reviewed / Open Access
• [Journal Article] Role of the active zone protein, ELKS, in insulin secretion from pancreatic β-cells (2019)
  • Author(s): Ohara-Imaizumi Mica、Aoyagi Kyota、Ohtsuka Toshihisa
  • Journal Title: Molecular Metabolism Volume: 27 Pages: 1213-1226
  • DOI: 10.1016/j.molmet.2019.06.017
  • Peer Reviewed / Open Access
• [Journal Article] Vangl2 interaction plays a role in the proteasomal degradation of Prickle2 (2019)
  • Author(s): Nagaoka Tadahiro、Furuse Mikio、Ohtsuka Toshihisa、Tsuchida Kunihiro、Kishi Masashi
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 2912-2912
  • DOI: 10.1038/s41598-019-39642-z
  • Peer Reviewed / Open Access
• [Journal Article] ELKS/Voltage-dependent Ca2+ channel-β Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic β-Cells. (2019)
  • Author(s): Ohara-Imaizumi, M., Aoyagi, K., Yamauchi, H., Yoshida, M., Mori, M.X., Hida, Y., Tran, H.N., Ohkura, M., Abe, M., Akimoto, Y., Nakamichi, Y., Nishiwaki, C., Kawakami, H., Hara, K., Sakimura, K., Nagamatsu, S., Mori, Y., Nakai, J., Kakei, M., and Ohtsuka, T.
  • Journal Title: Cell Reports Volume: 26 Issue: 5 Pages: 1213-1226
  • DOI: 10.1016/j.celrep.2018.12.106
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Neuroimaging, genetic, and enzymatic study in a Japanese family with a GBA gross deletion. (2019)
  • Author(s): Ichinose Y, Ishiura H, Tanaka M, Yoshimura J, Doi K, Umeda T, Yamauchi H, Tsuchiya M, Koh K, Yamashiro N, Mitsui J, Goto J, Onishi H, Ohtsuka T, Shindo K, Morishita S, Tsuji S, Takiyama Y.
  • Journal Title: Parkinsonism Relat Disord. Volume: - Pages: 57-63
  • DOI: 10.1016/j.parkreldis.2018.11.028
  • Peer Reviewed / Open Access
• [Journal Article] UBAP1 mutations cause juvenile-onset hereditary spastic paraplegias (SPG80) and impair UBAP1 targeting to endosomes (2019)
  • Author(s): Nan H, Ichinose Y, Tanaka M, Koh K, Ishiura H, Mitsui J, Mizukami H, Morimoto M, Hamada S, Ohtsuka T, Tsuji S, Takiyama Y
  • Journal Title: J Hum Genet Volume: 64 Issue: 11 Pages: 1055-1065
  • DOI: 10.1038/s10038-019-0670-9
  • Peer Reviewed / Open Access
• [Presentation] Role of CAST/ELKS in the active zone function via liquid-liquid phase separation (2021)
  • Author(s): Toshihisa Ohtsuka
  • Organizer: 第44回日本神経科学大会（CJK第１回国際会議）
  • Int'l Joint Research
• [Presentation] An engineered channelrhodopsin optimized for axon terminal activation and circuit mapping (2021)
  • Author(s): Shun Hamada, Masashi Nagase, Tomohiko Yoshizawa, Akari Hagiwara, Yoshikazu Isomura, Ayako M. Watabe, and Toshihisa Ohtsuka
  • Organizer: 第44回日本神経科学大会（CJK第１回国際会議）
  • Int'l Joint Research
• [Presentation] 光操作ツールの紹介 (2020)
  • Author(s): 大塚稔久
  • Organizer: CREST「オプトバイオ」領域の第5回領域会議
  • Invited
• [Presentation] 前シナプスアクティブゾーン因子群のプロテオーム解析と液相分離 (2020)
  • Author(s): 大塚稔久
  • Organizer: 第93回日本生化学会シンポジウム「生体膜ダイナミクス:細胞レベルの制御機構から個体レベルの生理機能まで」
  • Invited
• [Presentation] Neuropsychopharmacology of relaxin-3 (2019)
  • Author(s): Toshihisa Ohtsuka
  • Organizer: 第６回アジア神経精神薬理学会大会（AsCNP2019)
  • Int'l Joint Research
• [Presentation] シナプス分子間相互作用から捉える可塑性機構とその破綻 (2019)
  • Author(s): 大塚稔久
  • Organizer: 日本蛋白質科学会年会・日本細胞生物学会合同年次大会
  • Invited
• [Remarks] 山梨大学医学部生化学講座第一教室ホームページ
  • URL: http://www.med.yamanashi.ac.jp/basic/bioche01/
• [Remarks] 山梨大学医学部生化学講座第一教室ホームページ