Investigate the effects of natural anti-oxidative compounds and helicase on the zebrafish and cell models of trinucleotide repeat disease

• Main Authors: Guan-Yu Lin, 林冠宇
• Other Authors: Hui-Chin Pan
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/93355e

碩士 === 中山醫學大學 === 生物醫學科學學系碩士班 === 106 === Part 1: Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by an abnormal expansion of a coding trinucleotide (CAG) repeat, which is translated into an elongated glutamine (Q) tract in the respective mutant proteins. It leads to protein misfolding and aggregation, altering the function of neuronal cells and eventually resulting in neurodegeneration. Currently there is no treatment to prevent or cure the disease, only symptomatic treatments exist. We have established transgenic zebrafish and N2a cell lines expressing polyQ proteins. The polyQ zebrafish showed growth retardation, delayed hatching and increased mortality rate. The escape response and swimming pattern were also affected. In N2a cells, polyQ protein aggregation in the cytoplasm was observed and expanded polyQ interrupts branching of neuronal processes upon differentiation. Since ROS is increased in cells expressing polyQ proteins, we tested the effects of curcumin and resveratrol, both known as antioxidative compounds, in rescuing the phenotypes in the polyQ fish and cell models. After treating with curcumin and resveratrol, the mortality rate, hatching rate and escapes response were all improved significantly. In addition, both compounds decreased ROS production in the polyQ fish and cell models. The results demonstrated that curcumin and resveratrol are beneficial for the polyQ diseases and that the zebrafish model is a feasible platform for polyQ disease drug test. Part 2: Myotonic dystrophy type1 (DM1) is an autosomal dominant disorder caused by the expansion of a CTG trinucleotide repeat in the 3′-UTR of the dystrophia myotonica-protein kinase (DMPK) gene. The transcripts of CUG repeats accumulate in nucleus and disrupt a variety of cellular regulatory pathways including splicing and transcription by sequestration of MBNL proteins. Previous study using a DM1 C. elegans model and RNAi library screening has revealed several modifier genes that could mitigate the disease phenotype. One of the candidate genes is WRN (Werner syndrome helicase), a DNA helicase of the RecQ family that plays a critical role in repairing DNA and maintaining the structure of DNA. The endogenous expression of WRN in C2C12-CUG200 cells and DM1 cells are higher than in control. To investigate the role of WRN in DM1 disease, we used lentiviral shRNA of WRN to knock down its expression in C2C12-CUG200 cells. Decreased expression of WRN could rescue cell differentiation defect caused by the CUG repeats. In addition, knockdown of WRN reduced the expression of EGFP in CUG200 cells, suggesting that WRN could modify the CUG repeats and facilitate its expression at the RNA level. We also used siRNA to suppress WRN expression in human DM1 cells. Similarly, knockdown of WRN decreased the expression of DMPK gene and RNA foci in the nucleus. These results indicate that WRN is required for the toxic effect caused by expended CUG repeats and down-regulation of WRN may provide an avenue for treatment of DM1 disease.