細胞外ミトコンドリア放出による生体恒常性維持と加齢・疾患の関連について

2022 Fiscal Year Research-status Report

• Project/Area Number: 21K15627
• Research Institution: Osaka University
• 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)
• Project Period (FY): 2021-02-01 – 2024-03-31
• Keywords: Mitochondria / Extracellular release / Mitophagy

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 themachinery at molecular level and physiological relevance can pave the way for therapeutic implication.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

Currently, 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. Membrane trafficking proteins are involved in a number of processes, including neurotrasmitter and endocrine release, phagocytosis and lysosomal and proteasomal protein degradation. However, the screening protocol needs to be further optimized due to variations among wells.

Strategy for Future Research Activity

Future plan involves the optimization for lentivirus transduction condition, MOI determination, incubation period and the requirement of puromycin selection using the positive control lentivirus with EmGFP before performing screening.

Causes of Carryover

Usage plan as scheduled.