| Project/Area Number |
21K15627
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 51030:Pathophysiologic neuroscience-related
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| Research Institution | Osaka University |
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Principal Investigator |
鐘 其静 大阪大学, 大学院医学系研究科, 特任研究員(常勤) (50796076)
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| Project Period (FY) |
2021-02-01 – 2025-03-31
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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000)
Fiscal Year 2023: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2022: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2021: ¥2,600,000 (Direct Cost: ¥2,000,000、Indirect Cost: ¥600,000)
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| Keywords | Mitochondria / Extracellular release / Mitophagy / ミトコンドリア / 細胞外放出 / マイトファジー |
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| Outline of Research at the Start |
最近我々は、マイトファジーと両輪で働く制御機構として、ミトコンドリアの細胞外放出機構を見出した。今回我々は、ミトコンドリア放出機構の分子機序の解明と、ミトコンドリア放出が与える細胞自律的な、または非自律的な影響について研究を進める。簡潔に、機序の解明においては、CRISPR crRNAライブラリーを用いて放出ミトコンドリアを制御する因子のスクリーニングを行い、ミトコンドリア放出機構に関わる因子の同定を行う。ミトコンドリア放出の意義については、放出に関わる因子の発現を変化させた際の細胞自律的変化と、または放出ミトコンドリアを培養細胞やマウス個体に投与した際の周辺細胞の変化を解析する。
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| Outline of Annual Research Achievements |
Mitochondrial quality control, which is crucial for maintaining mitochondrial function and cell fate determination, is mainly achieved through mitophagy. We recently discovered an alternative quality control pathway mediated by extracellular mitochondria release. Using mitochondrial toxin treatment and parkin gene overexpression and knockdown approaches, we found that mitochondrial stress and perturbation of mitophagy pathway induce greater extracellular mitochondria release, suggesting that impairment of conventional mitophagy prompts alternative pathway.
However, the detailed mechanisms and the resulting biological consequences remain indefinite. Here I propose to study the mechanisms of mitochondria secretion and its cell-autonomous and non-cell autonomous biological consequences. Briefly, the study of mechanisms involves CRISPR library screening assay to identify intrinsic factors that regulate mitovesicle release. Elucidation of the biological significance of mitochondria release includes determination on how mitochondria release affects the donor and recipient cells. A better understanding of the machinery at molecular level and physiological relevance can pave the way for therapeutic implication.
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| Current Status of Research Progress |
Current Status of Research Progress
4: Progress in research has been delayed.
Reason
The project progression is delayed due to the maternity and childcare leave of the principle investigator. However, we have established Hela Cas9 stably overexpressing cells with DsRed2-labelled mitochondria and purchased LentiArray Human Membrane Trafficking CRISPR library for screening. This library targets 141 genes with up to 4 gRNA per gene target.
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| Strategy for Future Research Activity |
Future plan involves the optimization for lentivirus transduction condition, MOI determination, incubation period and the requirement of polybrene using the positive control HPRT1-targeting lentivirus with EmGFP before performing screening. Gene editing efficiency has to be verified using genomic cleavage detection kit. Detection of extracellular mitochondria will be performed using CQ1 confocal quantitative image cytometer,
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