2019 Fiscal Year Research-status Report
Studies on activation of TFEB to promote autophagic degradation of alpha synuclein
| Project/Area Number |
19K07843
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
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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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | TFEB / alpha synuclein / Lewy body / autophagy / Parkinson's disease / polo-like kinase |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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| Causes of Carryover |
Amount remaining was due to cost savings.
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