| Summary: | 碩士 === 國立臺灣師範大學 === 生命科學研究所 === 102 === Parkinson’s disease (PD), the second most common neurodegenerative disorder, is pathologically characterized by loss of dopaminergic neurons in the substantia nigra of the midbrain. Mutations in the F-box only protein 7 gene (FBXO7), the substrate-specifying subunit of Skp1-Cullin-F-Box (SCF) E3 ubiquitin ligase complex, cause PD-15 (PARK15). Previously we identified an amino acid changed variant Y52C in association with decreased risk of developing PD. Upon expression in cells, Y52C variant displayed significantly reduced rate of decay in cycloheximide chase experiment. The human FBXO7 gene promoter has not been analyzed. To investigate cis elements controlling FBXO7 expression, we cloned FBXO7 promoter fragments from -1240, -694, -538, -438, -202 and -56 to +261 by PCR amplification and placed in front of GFP reporter for transfection assay in HEK-293T cells. The expression of GFP was monitored by both high content analysis and flow cytometry. When the expressed GFP level of the -56~+261 promoter fragment was set as 100%, significantly increased FBXO7 promoter activity was observed with the -202~+261 and -694~+261 fragments by both methods. As the protective role of increased FBXO7 level in PD was implicated, Flp-In 293 cell line expressing GFP reporter driven by FBXO7 -694~+261 promoter fragment was constructed and used as a platform to screen herbal extracts provided by Industrial Technology Research Institute for enhancing FBXO7 expression. Herbal extracts NTNU-319, 379, 395 and 439 were found to increase endogenous FBXO7 protein expression in both Flp-In 293 and SH-SY5Y cells. Treatment of the above herbal extracts protected SH-SY5Y cells against MPP+-induced cell death. In addition, herbal extracts NTNU-319, 395 and 439 significantly alleviated MPP+-induced loss of mitochondrial membrane potential. As lack of treatment to prevent or slow PD progression, the proposed study may provide new insights into the therapeutic approach to PD.
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