2018 Fiscal Year Research-status Report
ATP-dependent coupling of pre- and post-synaptic organelles movements at central synapses and its implication in synaptic plasticity and transmission
| Project/Area Number |
18K06494
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
Guillaud Laurent 沖縄科学技術大学院大学, 細胞分子シナプス機能ユニット, 研究員 (90596222)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Synapse / Mitochondria / ATP / Liquid phase separation / synaptic vesicle / active zone / cytosol / solubility |
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| Outline of Annual Research Achievements |
1) I have quantified the movements of synaptic vesicles toward new release sites during repetitive stimulations and showed that repetitive stimulations induced gradual decrease of mitochondria activity at specific release sites, concomitantly with an increase of mitochondrial activity localized in the proximity of newly established release sites. This data demonstrated a direct coordination between the transport of synaptic vesicles and the translocation of the release machinery during sustained synaptic activity.
2) I have also demonstrated that the activity of mitochondria is highly heterogenous in giant terminals in resting condition as well as during stimulations.
3) In addition, I have established that the liquid phase transition of peri-active zone cytoplasm is highly dependent on the local activity of mitochondria. Pre-synaptic swellings containing active mitochondria show significantly higher fluidity than swellings without active mitochondria.
4) I have successfully shown that blocking mitochondria activity in giant terminals with bath incubation of FCCP or rotenone drastically reduced peri-active zone cytosol fluidity as observed by a significant reduction in cytosolic GFP fluorescence recovery after bleaching (FRAP) experiments and indicating a clear reduction in the cytosol liquid phase compared to control terminals.
5) In addition, blocking mitochondria activity also induced liquid to solid phase transition of active zone component and synaptic vesicles pool as observed by a significant reduction in the mobile fraction of CFP-RIM1 and Venus-VGLUT1 measured by FRAP.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
Collectively, the data I obtained so far provide conclusive evidences that heterogenous mitochondria activity regulate liquid phase transition of peri-active zone proteins, active zone components and synaptic vesicles pool in cultured giant terminals. These changes in liquid phase transition result most likely from the modification in local ATP concentration due to differential mitochondria activity.
All experiments went as planned and were highly reproducible for both peri-active zone, active zone and vesicles components.
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| Strategy for Future Research Activity |
I am now starting to measure local ATP concentration using ATP/ADP fluorescent sensor in cultured giant terminals and in vitro luminescence assay. Preliminary results showed that the concentration of ATP measured with the fluorescent sensor PercevalHR varies locally and is correlated with the activity of mitochondria in the region of interest.
I will then measure the concentration of ATP in the terminal after FCCP or rotenone treatment and try to rescue the liquid phase transition by delivering various concentration of ATP to the cells.
In addition, I will perform in vitro assay of liquid phase separation of various proteins found in the pre-synaptic cytosol and known to be involved in neurodegenerative diseases such as Parkinson and Alzheimer.
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| Causes of Carryover |
Some cost of reagents were partially covered by OIST internal budget and as experiments went more smoothly than expected, the quantity of reagents and materials needed to complete the first phase of my research plan was reduced.
The remaining budget of FY2018 will be used to purchased new reagents and goods, and travel expenses to complete the second phase of my research project (ATP measurements and in vitro assays)
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Research Products
(3 results)
