2017 Fiscal Year Annual Research Report
Deciphering molecular mechanisms underlying the unconventional extracellular mitochondria release
| Project/Area Number |
17F17413
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| Research Institution | Osaka University |
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Principal Investigator |
望月 秀樹 大阪大学, 医学系研究科, 教授 (90230044)
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| Co-Investigator(Kenkyū-buntansha) |
CHOONG CHI JING 大阪大学, 医学(系)研究科(研究院), 外国人特別研究員
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| Project Period (FY) |
2017-11-10 – 2020-03-31
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| Keywords | mitochondria / rotenone / parkin |
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| Outline of Annual Research Achievements |
Initial work involves establishment of in vitro models, both stable overexpression and knockout cell lines (PC12-DsRed2mito, PC12-empty, PC12-Parkin, PC12-Parkin -/-). PC12DsRed2mito facilitates the observation of movement of mitochondria labeled with red fluorescent protein. Overexpression and knockout cells permit the study of related mechanisms. Creation of stable cells starts with plasmid preparation by molecular cloning followed by cell transfection, selection and confirmation of protein levels.
For assessment of mitochondrial quality impairment on mitochondria release from PC12 cells, we treated cell with DMSO (control) or mitochondrial toxin rotenone. Conditioned media were collected, centrifuged at 300 x g, filtered through a 1.22um pore size syringe filter, centrifuged at 2000 x g and pellet obtained was resuspended in phosphate-buffered saline. DNA was extracted from pellet using DNA SP extractor kit and quantification of mitochondrial DNA (rat cytochrome C oxidase subunit III) by quantitative real time PCR was performed.Time lapse confocal microscopy imaging of PC12DsRed2mito was performed to capture release of red fluorescent labeled mitochondria. Data was validated by shotgun analysis with detection of mitochondrial proteins in pellet sample and also by electron microscopy.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
In vitro models, both stable overexpression and knockout cell lines, have been established. Protein expression and mRNA level were examined using western blotting and qPCR. By performing confocal time-lapse imaging, we observed the phenomenon involving direct release of vesicle-entrapped-mitochondrial related particles from PC12 cell line upon disruption of mitochondrial quality by rotenone treatment. This finding was validated by electron microscopy imaging showing blebs containing mitochondria on the cell membrane surface of rotenone treated PC12 cells. Quantitative PCR and western blotting also showed increased mitochondrial DNA and protein levels in pellet obtained from rotenone treated PC12 cells.
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| Strategy for Future Research Activity |
As it is widely recognized that mitochondrial dysfunction occurs in many neurodegenerative diseases, genetic approach manipulating the human Parkinson’s disease related Parkin gene, implicated in mitochondrial maintenance, may provide novel information on previously unrecognized role of mitochondria release in the process of neurodegeneration. Future plan will be focusing on assessment of effect of parkin overexpression and ablation on mitochondria release from PC12 cells. Measurement of mitochondrial DNA level in the pellet obtained from conditioned media of PC12empty, PC12Parkin, PC12 and PC12parkin-/- will be performed.
To assess if the released mitochondria have beneficial or adverse role, collected pellet will be added to 6-3 microglia cells and pro-inflammatory cytokines level in the conditioned media will be determined.
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