研究課題/領域番号 |
21K06445
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研究種目 |
基盤研究(C)
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配分区分 | 基金 |
応募区分 | 一般 |
審査区分 |
小区分46030:神経機能学関連
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研究機関 | 沖縄科学技術大学院大学 |
研究代表者 |
MAHAPATRA Satyajit 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, スタッフサイエンティスト (40832861)
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研究分担者 |
DUTTA Soumyajit 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, ポストドクトラルスカラー (20899372)
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研究期間 (年度) |
2021-04-01 – 2024-03-31
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研究課題ステータス |
交付 (2022年度)
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配分額 *注記 |
4,030千円 (直接経費: 3,100千円、間接経費: 930千円)
2023年度: 1,040千円 (直接経費: 800千円、間接経費: 240千円)
2022年度: 1,560千円 (直接経費: 1,200千円、間接経費: 360千円)
2021年度: 1,430千円 (直接経費: 1,100千円、間接経費: 330千円)
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キーワード | Vesicle transport / The calyx of Held / Hippocampal CA1 synapse / Endocytosis / Release-site clearance / Synaptic depression / Recovery from STD / Physiological condition / SV transport / STD / Synaptic vesicles (SVs) / Release site clearance / Calyx of Held synapse |
研究開始時の研究の概要 |
A. Target identification: Finding the molecules/proteins from three functionally different categories that should enhance the STD and delay the recovery from STD - by using brain slice patch-clamp electrophysiology (EP) at physiological conditions. B. Target validation and live-imaging with EP: Confirming the targets efficacy in the calyceal terminals in cultures by EP and tracking the SV trajectories. C. Live-imaging with EP: Completion of the SV tracking experiments and paper writing.
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研究実績の概要 |
Toward the aim of understanding synaptic vesicles (SVs) transport mechanisms, our first attempt of arresting the vesicle docking through the block of endocytic release site clearance didn’t show any delay in the recovery from short term depression (STD) upon use of endocytosis inhibitors (Dynasore and Pitstop 2) physiologically. These findings are in sharp contrast to the previous report (Hosoi et al., Neuron. 2009).
1) Since inhibition of endocytic proteins by Dynasore and Pitstop-2 resulted decrease of synaptic strength by nearly half, with an ultrafast onset of 10 milliseconds. This fast inhibition cannot be explained by the common belief that clathrin mediated slow endocytosis underlies the release site clearance effect, and some fast endocytic form likely is involved. To test this, at 37C and in 2.0 mM calcium, using endocytic inhibitors (Dynasore, Dynamin1 PRD peptide and Pitstop 2) we found all these blockers effectively block fast endocytosis in addition to slow endocytosis. 2) Next, we tested the physiological implication of blocking the active zone scaffold protein intersectin, inhibition of which has been shown to strongly delay the fast time course of recovery from STD at the calyx of Held (Sakaba et al., PNAS. 2013). 3) To inhibit the intersectin pathway, we targeted its downstream effectors, CDC42 and F actin, by use of ML141 and Latrunculin B, respectively. First, we tested the effect of these drugs on endocytosis.Neither of the blockers had any effect on endocytic recovery. Both drugs strongly enhanced the STD, without altering the recovery from STD.
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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 electrophysiological assessment of the SV transport mechanism is going smoothly. Out of three functionally distinct target groups, the first one (role of endocytic proteins) is completed and the second one (role of scaffold proteins) is nearing completion. For the first time, our data show that in physiological conditions how endocytic proteins (dynamin and clathrin) and scaffold protein intersectin affect the SV release pattern in milliseconds to seconds scale in two functionally distinct synapses owing to their release-site clearance property. The manuscript writing is in final phase.
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今後の研究の推進方策 |
The physiological role of endocytic proteins in promoting SV docking through their release-site clearance property at the auditory-brainstem calyx of Held and Hippocampal CA1 synapses is completed. In addition, the scaffold protein intersectin mediated (CDC42 and f-actin) vesicle replenishment at the calyx also completed. Currently its role on hippocampal CA1 synapses is being investigated, and manuscript of this finding would be submitted. Next, I would test the role of presynaptic G-proteins and GTPases (small and large) on regulating the SV transportation.
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