| 研究課題/領域番号 |
18K06494
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| 研究機関 | 沖縄科学技術大学院大学 |
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研究代表者 |
Guillaud Laurent 沖縄科学技術大学院大学, 分子神経科学ユニット, 研究員 (90596222)
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| 研究期間 (年度) |
2018-04-01 – 2021-03-31
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| キーワード | Liquid phase separation / Synapse / Mitochondria / ATP / Neurodegeneration |
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| 研究実績の概要 |
For the second year my research project has expanded and progressed according to plan as described below:
1) I have analyzed the variations in intracellular and extracellular ATP concentration using ATP fluorescent sensor in live imaging experiments and using in vitro luminescence assays. The results show that the concentration of ATP in giant pre-synaptic terminals is heterogeneous and correlates with mitochondria activity locally.
2) In addition, blocking mitochondria activity with FCCP significantly reduced the intracellular concentration of ATP measured by live fluorescence quantification or bioluminescence assays. These results suggest that the perturbation of liquid phase separation observed at the synapse after FCCP treatment is induced by the reduction of ATP in the terminal.
3) I have also successfully rescued the effect of FCCP on liquid phase separation by delivering ATP directly to the synapses or by pharmacologically promoting ATP production in cultured giant terminals.
4) Lastly, I performed in vitro assays to observe and quantify liquid phase separation of several proteins implicated in Parkinson and Alzheimer diseases. The results demonstrate that the majority of these proteins undergo liquid phase separation and form condensates at 37C. I also determined that the formation of these bio-condensates can be prevented or reverted by increasing ATP concentration.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The characterization and quantification of liquid phase transitions of peri-active zone cytosol, active zone components and synaptic vesicles in cultured giant synapses are now completed. The data clearly established strong correlation between the decrease in mitochondrial activity (activity blocked by FCCP or rotenone) and the perturbation of liquid phase separation in the synapse.
The measurement of ATP concentration and its correlation with mitochondrial activity are also completed and the data were highly consistent and reproducible.
The rescue experiments were more challenging and needed more time for optimization.
In vitro assays went rather smoothly, methods and analysis were straight forward with reproducible results as well.
I am now preparing the manuscript and expect to be ready for submission by the end of the second quarter of 2020.
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| 今後の研究の推進方策 |
My next research plan is to increase the number of protein candidates for in vitro assay and to determine the threshold concentration of ATP that triggers liquid phase separation for each candidate proteins.
Lastly, I am planning to study liquid phase separation using FRAP and live imaging in neurons derived from human IPSCs, obtained from patients with Parkinson or Alzheimer disease, in order to determine whether my observations in physiological condition from mouse cultured giant synapses can also be correlated with neurodegeneration in human neurons.
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| 次年度使用額が生じた理由 |
The remaining budget will be used to cover the cost of publication in 2020, as well as the expenses for the attendance of an international conference to present the outcome of my research.
In addition, I will purchase several human IPSCs lines and the necessary reagents for their culture and differentiation into neurons to complete the analysis of ATP-dependent liquid phase separation in neurons derived from patients with Parkinson, Alzheimer and ALS diseases.
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