| 研究課題/領域番号 |
19K07843
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| 研究機関 | 滋賀医科大学 |
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研究代表者 |
WALKER DOUGLAS 滋賀医科大学, 神経難病研究センター, 特任教授 (10813694)
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| 研究期間 (年度) |
2019-04-01 – 2022-03-31
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| キーワード | TFEB / alpha synuclein / Lewy body / autophagy / Parkinson's disease / polo-like kinase |
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| 研究実績の概要 |
A number of transfected cell lines have been prepared and characterized. These include HEK and SH-SY5Y neuroblastoma cells lines that are overexpressing TFEB or alpha-synuclein. We have not been able to use the LA-N-5 neuroblastoma cell line as originally proposed as they were not able to be transfected. The SH-SY5Y neuroblastoma was used instead. Cell lines have been used in experiments involving transient transfection of the other plasmid gene (TFEB or alpha synuclein). To address the central issue of how TFEB activation affects the metabolism of alpha-synuclein and in particular phosphorylated alpha synuclein, chemical and biological activators of TFEB have been tested. The TFEB expressed protein from the plasmid is labeled with green fluorescent protein and should be used to show nuclear localization upon TFEB activation. For unknown reasons at present, identification of TFEB activation has not been shown by GFP fluorescence and has needed direct TFEB immunocytochemistry. Recent experiments have utilized a plasmid for polo-like Kinase-2 (PLK2) to transfect alpha synuclein expressing cells. PLK3 transient expression increased levels of alpha synuclein and its phosphorylated forms. Activation of endogenous cellular TFEB in PLK3/alpha synuclein transfected cells showed both reduced alpha synuclein and phosphorylated alpha synuclein. A number of control experiments are needed to confirm this observation, including the construction of triple transfected cell lines.
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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
Overall, the project has achieved several milestones. We believe we have shown that TFEB activation can increase degradation of alpha synuclein to the same degreee as phosphorylated alpha synuclein. The only issue has been demonstrating TFEB activation using GFP; this did not work but have need to use immunocytochemistry. The project has made greater progress since acquiring and using plasmid to PLK2. This has given more easily interpretable results than the use of chemical agents. We now have to do a range of control and replicate experiments to confirm our findings.
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| 今後の研究の推進方策 |
The second year will involve replication of initial findings with PLK2 combined with alpha synuclein and TFEB activation to demonstrate alpha synuclein removal. Control experiments will require preparing different double transfected cell lines to compare to the triple transfection. One additional feature will be attempts to characterize and manipulate the forms of alpha synuclein prepared in our cells. At present, the alpha synuclein and phosphorylated alpha synuclein in our cell lines is soluble and has not aggregated to the extent observed in human diseased brains. We have done preliminary experiments to show that treatment with iron compounds can promote aggregation. To complete this project we need to show whether TFEB activation can promote the degradation of aggregated and phosphorylated alpha synuclein. This will require further manipulations of overexpressing cell lines.
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| 次年度使用額が生じた理由 |
Amount remaining was due to cost savings.
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