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研究生:吳思瑢
研究生(外文):Sih-Rong Wu
論文名稱:探討抑制Supt4h表現對於小腦萎縮症疾病基因表現之影響
論文名稱(外文):Analysis of Supt4h knockdown effect on the expression of spinocerebellar ataxia associated genes
指導教授:鄭子豪
指導教授(外文):Tzu-Hao Cheng
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:48
中文關鍵詞:Supt4hAntisense oligonucleotide (ASO)小腦萎縮症
外文關鍵詞:Supt4hAntisense oligonucleotide (ASO)Spinocerebellar ataxia
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第一型和第三型小腦萎縮症是一種遺傳的神經退化性疾病,造成這個疾病的原因分別是ATXN1基因第八個外顯子、ATXN3基因第十個外顯子發生CAG三核苷酸重複序列的異常擴增,並在腦部特定區域造成多麩醯胺酸蛋白質的異常堆積,這個特性也發生在其他神經退化性疾病中,例如亨氏舞蹈症,因此這些疾病又統稱為多麩醯胺酸疾病。實驗室先前研究發現,降低轉錄因子Supt4h的表現可以選擇性抑制亨氏舞蹈症突變基因(Htt)的表現,並抑制突變蛋白質的聚集和毒性。由於CAG核苷酸重複序列在造成多麩醯胺酸疾病基因的相對位置不盡相同,因此目前並不清楚Supt4h對於小腦萎縮症疾病基因是否也有類似的調控機制。在本論文中,我們利用小鼠、大鼠神經細胞株以及取自第三型小腦萎縮症患者的血液單核球細胞株,探討降低Supt4h對於突變ATXN3基因表現的影響。此外,利用第一型小腦萎縮症動物模式,我們分析Supt4h antisense oligonucleotide (ASO)對於疾病病理及運動功能改善的狀況。
Spinocerebellar ataxia type 1 (SCA1) and type 3 (SCA3) are inherited neurodegenerative diseases caused by expansion of CAG repeats in eoxn8 of ataxin-1 (ATXN1) and exon10 of ataxin-3 (ATXN3) gene respectively. Deposition and accumulation of polyglutamine protein aggregates in the brain is a feature commonly associated with SCA1, SCA3, and other neurological disorders such as Huntington’s disease (HD), which are collectively designated as polyQ disorders. Our early study demonstrated that down-regulation of Supt4h, a transcription elongation factor, can selectively decrease mutant Htt (disease gene of HD) expression and reduce its protein aggregation and toxicity. However, it is unclear whether Supt4h also have a similar effect on ATXN1 and ATXN3 genes, in which the location of CAG repeats in these gene is distinct from that of Htt. In this study, we examined whether, under Supt4h deficiency, mutant ATXN3 gene expression is preferentially decreased and further checked the change of motor coordination and neuropathology of SCA1 animals by Supt4h ASO intervention. Our findings suggest that Supt4h might be a potential therapeutic target against SCA1 and SCA3.
Acknowledgement…………………………………………………………………….i
Chinese abstract …………………………………………………………………......ii
Abstract ……………………………………………………………………………iii
Contents ...…………………………………………………………………………...iv
List of Tables ………………………………………………………………………vii
List of Figures ………………………………………………………………………viii
Introduction ………………………………………………………………………….1
Spinocerebellar ataxia type 1 and type 3 and other polyQ diseases ……………….1
Therapeutic strategies for SCA3 and SCA1 ……………………………………….2
Spt4/Supt4h and its role in Huntington’s disease ………………………………….3
Antisense oligonucleotides (ASO) ………………………………………………...4
SCA1 disease mouse models ………………………………………………………5
The aims of this study ……………………………………………………………...6
Materials and Methods ………………………………………………………………7
PART I: for in vitro experiments ………………………………………………...7
DNA construction ………………………………………………………………….7
Cell lines and culture conditions …………………………………………………..7
Transfection and transduction ……………………………………………………...7
RNA extraction and semi-quantitative RT-PCR …………………………………...8
Western blot analysis ………………………………………………………………8
Filter trap assay ……………………………………………………………………9
Trypan blue assay ………………………………………………………………….9
PART II: for in vivo experiments ……………………………………………….10
Animals …………………………………………………………………………...10
Stereotaxic surgery ……………………………………………………………….10
Tissue collection and Western blot analysis ……………………………………...10
Rotarod performance ……………………………………......................................11
Open-field test …………………………………….................................................11
Beam walk ……………………………………......................................................11
Footprint analysis ………………………………………………………………..12
Nissl staining ……………………………………..................................................12
Immunohistochemistry ……………………………………...................................12
Quantification of the thickness of molecular layer and Purkinje cells density…13
Statistical analysis ………………………………………………………………...13
Results ………………………………………………………………………………14
1. Supt4h knockdown preferentially suppresses the expression of mutant
ATXN3 in murine neuronal cells ……………………………………………..14
2. Supt4h down-regulation reduces protein aggregation and toxicity of mutant ATXN3 in murine neuronal cells ……………………………………………14
3. Supt4h deficiency results in a decrease of mutant ATXN3 expression in lymphoblastoid cell lines derived from SCA3 patients ……………………...15
4. Intracerebral ventricle injection of antisense oligonucleotides against Supt4h reduces mutant ATXN1 expression in SCA1 mice model …………………...15
5. Supt4h ASO treatment moderately improves motor function of SCA1 mice ………………………………………………………………………….16
6. Supt4h ASO intervention attenuates the loss of Purkinje cells in SCA1 mice ………………………………………………………………………….18
Discussion …………………………………………………………………………...19
Reference ……………………………………………………………………………22
Tables and Figures ………………………………………………………………….30
List of Tables
Table 1. Primers used for RT-PCR and semi-quantitative PCR ……………………30
Table 2. Nucleotide sequence of siRNA, shRNA, and antisense oligonucleotides …31

List of Figures
Figure 1. Supt4h knockdown preferentially decreases mutant ATXN3 expression, aggregation and toxicity in murine neuronal cells ………………………..32
Figure 2. Supt4h knockdown suppresses mutant ATXN3 expression in lymphobla-stoid cells derived from SCA3 patients …………………………………..35
Figure 3. Supt4h down-regulation by antisense oligonucleotide (ASO) decreases mutant ATXN1 protein level in SCA1 mice ……………………………..36
Figure 4. Supt4h ASO treatment moderately improves rotarod performance of
SCA1 mice ………………………………………………………………..38
Figure 5. Autonomous activity of SCA1 mice treated with Supt4h ASO did not significantly differs from that of mice treated with control ASO ………...40
Figure 6. Supt4h ASO treatment moderately ameliorates motor deficits of SCA1
mice in beam walk test ……………………………………………………42
Figure 7. Supt4h ASO treatment moderately attenuates ataxia of hindlimbs in SCA1 mice ……………………………………………………………………….44
Figure 8. SCA1 mice treated with control or Supt4h ASO show no difference in the thickness of molecular layer ……………………………………………...45
Figure 9. Supt4h ASO intervention rescues the loss of Purkinje cells in SCA1
mice ……………………………………………………………………….47

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