Cross-talk analysis between heterotypic cell-cell communication via siRNA/TLR7 and new therapeutic strategy

• Project/Area Number: 19K09899
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 56050:Otorhinolaryngology-related
• Research Institution: Keio University
• Principal Investigator: HAYASHI Ken 慶應義塾大学, 医学部(信濃町), 共同研究員 (80534528)
• Co-Investigator(Kenkyū-buntansha): 神崎 晶 慶應義塾大学, 医学部(信濃町), 講師 (50286556) ; 高橋 宏知 東京大学, 大学院情報理工学系研究科, 教授 (90361518)
• Project Period (FY): 2019-04-01 – 2023-03-31
• Project Status: Completed (Fiscal Year 2022)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: エクソソームmiRNA let-7b / 異種細胞間コミュニケーション / ミスマッチネガティビティ / 間歇的自発バースト / 初代神経培養細胞 / TLR-7 / 初代培養神経細胞 / CMOS-MEA / 内耳細胞過興奮性細胞死 / ミスマッチ陰性電位 / CMOS-アレイ / miRNA let-7b / オートファジー / Toll-Like-Receptor 7 / エクソソーム / 耳鳴

Outline of Research at the Start

昨年、脳内炎症を伴う内耳性片頭痛によるトップダウンシグナルが感音難聴や耳鳴を発症させると報告された。しかし、脳内からのトップダウンシグナルが内耳障害を起こすメカニズムは、明らかではない。本研究は、脳内炎症によって分泌されたエクソソーム内miRNAlet-7bがTLR7シグナルを刺激した場合、神経細胞ネットワーク機能を低下させ、内耳感覚細胞死を誘導するか否かを実験的に証明することを目的としている。本研究成果は、 miRNAlet-7bは脳内炎症による感音難聴・耳鳴のバイオマーカーとして有用であり、miRNAlet-7b inhibitorは新規治療薬になり得る可能性を持っている。

Outline of Final Research Achievements

Our objective is to clarify the impact of exosomal (exo-) miRNA let-7b inducing hyper excitability-induced auditory cell death to the mismatch negativity (MMN) reflecting an auditory memory traces in neural cells. Primary neuronal cells are prepared from Wister rat brains. The stimulation selectivity of the deviance detection was evaluated by stimulus-specific adaptation index (SSAI), and the spontaneous burst-firing was estimated by total spike rate (TSR). SSAI was significantly decreased in the secondary response in exo-miRNA let-7b-treated cells. The intermittent spontaneous burst-firing was confirmed by the increase of TSR in neural circuitry after exposure of exo-miRNA let-7b. These results indicate that exo-miRNA let-7b holds the potential to decease auditory MMN leading to the generation of the spontaneous burst-firing in neural cells. Our results suggest that exo-miRNA let-7b might disrupt the sensory memory network in the auditory-brain, decreasing the special perception.

Academic Significance and Societal Importance of the Research Achievements

本研究結果は、 内耳細胞の過興奮性細胞死を引き起こすmiRNA let-7bが認知症等の神経障害における耳鳴あるいは脳過敏反応の原因となる可能性を示唆している。即ち、miRNAlet-7bは、臨床的に聴覚中枢制御機能低下による耳鳴バイオマーカーとして有用であることを意味している。また、エクソソームは細胞への親和性も高く高率に細胞移入可能であり、miRNAlet-7b 阻害剤は耳鳴中枢制御機能をターゲットとした新規治療薬 になり得る。本研究は、客観的な評価法・的確な治療法のない耳鳴で苦しむ多くの患者にとって大きな意義がある。

Research Products

• [Journal Article] Fine-tuned cholesterol solubilizer, mono-6-O-α-D-maltosyl-γ-cyclodextrin, ameliorates experimental Niemann–Pick disease type C without hearing loss (2022)
  • Author(s): Yamada Y, Miwa T, Nakashima M, Shirakawa A, Ishii A, Namba N, Kondo Y, Takeo T, Nakagata N, Motoyama K, Higashi T, Arima H, Kurauchi Y, Seki T, Katsuki H, Okada Y, Ichikawa A, Higaki K, Hayashi K, Minami K, Yoshikawa N, Ikeda R, Ishikawa Y, Kajii T, Tachii K, Takeda H, Orita Y, Matsuo M, Irie T, Ishitsuka Y
  • Journal Title: Biomedicine & Pharmacotherapy Volume: 155 Pages: 113698-113698
  • DOI: 10.1016/j.biopha.2022.113698
  • Peer Reviewed / Open Access
• [Journal Article] Molecular Mechanisms and Biological Functions of Autophagy for Genetics of Hearing Impairment. (2020)
  • Author(s): Ken Hayashi, Yuna Suzuki Y, Chisato Fujimoto, Sho Kanzaki
  • Journal Title: Genes Volume: 11 Issue: 11 Pages: 1331-1331
  • DOI: 10.3390/genes11111331
  • Peer Reviewed / Open Access
• [Journal Article] Casper-8 Regulates Endoplasmic Reticulum Stress-Induced Necroptosis Independent of the Apotosis Pathway in Auditor Cells (2019)
  • Author(s): Kishino A, Hayashi K, Maeda M, Jike T, Hidai C, Nomura Y, Oshima T.
  • Journal Title: Int J. Mol. Sci. Volume: 20(23) Issue: 23 Pages: 5896-5896
  • DOI: 10.3390/ijms20235896
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Exosomal miRNA let-7b modulates the interaction between TLR7 and TRPA1, inducing hyperexcitability-induced cell death in auditory cell (2022)
  • Author(s): Ken Hayashi, Hideaki Sakata
  • Organizer: 49th Congress of the Neurootological and Equilibriometric Society
  • Int'l Joint Research
• [Presentation] The functions of exosomal miRNA let-7b as a top-down signal disrupting the neural network in the auditory-brain (2022)
  • Author(s): Ken Hayashi, Hideaki Sakata
  • Organizer: 49th Congress of the Neurootological and Equilibriometric Society
  • Int'l Joint Research
• [Presentation] TFEBによるオートファジー・リソソーム経路活性化を介した内耳細胞老化制御メカニズムの解明 (2022)
  • Author(s): 林 賢
  • Organizer: 第32回 日本耳科学会 総会・学術講演会
  • Invited
• [Presentation] The effect of exosomal miRNA let-7b to the mismatch negativity as an auditory memory traces in cellular neural network. (2021)
  • Author(s): Ken Hayashi, Yuna Suzuki, Fumiyuki Goto, Yasuyuki Nomura, Sho Kanzaki, Chisato Fujimoto.
  • Organizer: The 125th American Academy Otolaryngology-Head and Neck Surgery Foundation Annual Meeting & OTO EXPO
  • Int'l Joint Research
• [Presentation] Exosomal miRNA let-7b inducing hyperexcitability-induced auditory cell death decrease the auditory sensorial memory in cellular neural network (2021)
  • Author(s): Ken Hayashi Hideaki Sakata1
  • Organizer: The 48th NES Congress online
  • Int'l Joint Research
• [Presentation] The impact of extracellular miRNA let-7b as a top-down signal in the auditory-brain. (2020)
  • Author(s): Ken Hayashi, Yuna Suzuki, Fumiyuki Goto, Akihiro Kishino, and Kaoru Ogawa
  • Organizer: American Academy Otolaryngology-Head and Neck Surgery Foundation Annual Meeting & OTO EXPO
  • Int'l Joint Research
• [Presentation] The interaction of TRL7 with TRPA1 drives hyperexcitability cell death modulated by miRNA let-7b in auditory cells. (2020)
  • Author(s): Ken Hayashi, Yuna Suzuki, Akihiro Kishino, Fumiyuki Goto, Kaoru Ogawa
  • Organizer: 43RD Association of research in Otolaryngology, Midwinter Meeting
  • Int'l Joint Research
• [Presentation] TLR7-TRPA1 interaction drives hyperexcitability cell death modulated by miRNA let-7b in auditory cells (2019)
  • Author(s): Ken Hayashi, Fumiyuki Goto, Akihiro Kishino, Yuna Suzuki, Yasuyuki Nomura, Sho Kanzaki, Kaoru Ogawa
  • Organizer: ICORL 2019 Symposium, Basic research in hearing loss
  • Int'l Joint Research
• [Presentation] The cross-talk between exosomal miRNA, apoptosis and autophagy in auditory cell death (2019)
  • Author(s): Ken Hayashi, Fumiyuki Goto, Akihiro Kishino, Yuna Suzuki, Yasuyuki Nomura, Sho Kanzaki, Kaoru Ogawa
  • Organizer: 123thAAO-HNSF
  • Int'l Joint Research
• [Presentation] The effect of hyperexcitability-induced cell death modulated by miRNA let-7b in the auditory-brain (2019)
  • Author(s): Ken Hayashi, Fumiyuki Goto, Yuna Suzuki, Akihiro Kishino, Sho Kanzaki, Kaoru Ogawa
  • Organizer: 12thAPSCI
  • Int'l Joint Research
• [Presentation] Autophagy Signaling Mechanisms Controlling Cell Fate in Auditory Cells -Cell death and Cellular Senescence- (2019)
  • Author(s): Ken Hayashi
  • Organizer: The 25th Ajou Otology Symposium
  • Invited