2022 Fiscal Year Research-status Report
Synaptic vesicle transport revealed by electrophysiological and imaging studies
| Project/Area Number |
21K06445
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| Research Institution | Okinawa Institute of Science and Technology Graduate University |
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Principal Investigator |
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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| Co-Investigator(Kenkyū-buntansha) |
DUTTA Soumyajit 沖縄科学技術大学院大学, 細胞分子シナプス機能ユニット, ポストドクトラルスカラー (20899372) [Withdrawn]
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | Vesicle transport / The calyx of Held / Hippocampal CA1 synapse / Endocytosis / Release-site clearance / Synaptic depression / Recovery from STD / Physiological condition |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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| Causes of Carryover |
I had to cancel two international meetings (FENS, 2023 and GRC meeting) this year to present my work, due to medical reasons. The incurring amount would be used to attend SfN meeting later this year.
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