臺灣博碩士論文加值系統

漢丁頓舞蹈症是一種遺傳性神經退化疾病，造成此疾病的原因是因為huntingtin基因在外顯子1上的CAG核酸重複片段有異常的擴增。而此疾病最主要影響的區域為大腦皮質與基底核中的紋狀體。近年來有許多文獻指出，huntingtin基因突變導致polyQ蛋白聚集體的產生是造成疾病的主要原因。實驗室先前利用酵母菌篩選系統，發現了Spt4基因缺失時，具有選擇性減少過度增長的CAG表現量，進而降低polyQ蛋白聚集體的產生。在哺乳類細胞中，Spt4的同源基因Supt4h會和Supt5h結合在一起形成異質二聚體，此二聚體具有促進轉錄以及增加RNA polymerase II持續合成的能力。在此，以老鼠為動物模式，探討Supt4h表現量對突變Htt基因的影響及對疾病鼠病徵之改善。

Huntington’s disease (HD), an inherited neurodegenerative disorder, is caused by expanded CAG repeats in the exon 1 of huntingtin gene, and primarily affects the striatum and cortex in the brain. Several studies have indicated that polyQ aggregation of mutant huntingtin triggers HD pathogenesis, and polyQ aggregate is toxic to neuron. Recently, SPT4 was identified through genome-wide genetic screening in yeast and showed regulation in expression and aggregation of extended polyQ selectively. Supt4h, the mammalian ortholog of Spt4, associates with Supt5h that promote transcription elongation and RNA polymerase II processivity, and modulates the expression of mutant Htt in neuronal cells. In this study, a Supt4h genetic modified mouse was generated and crossed to R6/2 HD mice. The mutant Htt gene expression and phenotypes were analyzed.

Contents

Chinese abstract --------------------------------------------------------------------------------i
Abstract ------------------------------------------------------------------------------------------ii
Contents -----------------------------------------------------------------------------------------iii
List of Figures -----------------------------------------------------------------------------------v
Introduction -------------------------------------------------------------------------------------1
Huntington’s disease -------------------------------------------------------------------------1
Huntington’s disease and PolyQ aggregation ---------------------------------------------1
HD mouse model------------------------------------------------------------------------------2
Therapeutical targets in HD -----------------------------------------------------------------3
Biochemical function of Supt4h ------------------------------------------------------------4
Regulatory role of Supt4h in polyQ aggregation and in Huntington’s Disease ------4
Material and Methods ------------------------------------------------------------------------6
Animal ---------------------------------------------------------------------------------------6
Rotarod performance -----------------------------------------------------------------------6
Beam walk -----------------------------------------------------------------------------------7
Tissue collection and protein isolation --------------------------------------------------7
Western blot analysis -----------------------------------------------------------------------7
Filter-trap assay -----------------------------------------------------------------------------8
RNA extraction -----------------------------------------------------------------------------9
Quantitative real time RT-PCR amplification ------------------------------------------9
Immunohistochemistry -------------------------------------------------------------------10
Transfection --------------------------------------------------------------------------------10
Constructs ----------------------------------------------------------------------------------11
Supt4h Protein induction in E. coli -----------------------------------------------------11
Protein purification ------------------------------------------------------------------------12
Southern blot -------------------------------------------------------------------------------13
Statistical analysis -------------------------------------------------------------------------13
Results ------------------------------------------------------------------------------------------15
1. Generation of Supt4h knockout mice ---------------------------------------------------15
2. Generation of Supt4h antibody ----------------------------------------------------------15
3. Supt4h is ubiquitously expressed in different tissues of mice -----------------------16
4. Supt4h+/- mice show an decrease of Supt4h expression ------------------------------16
5. Supt4h heterozygous knockout attenuates HD symptoms in R6/2 mice with 220 ~ 240 CAG repeats --------------------------------------------------------------------------17
6. The effects of Supt4h heterozygous knockout in R6/2 mice with 240～260 CAG repeats ---------------------------------------------------------------------------------------18
i. Supt4h expression was reduced in HD；Supt4h+/- and WT；Supt4h+/- mice---------------------------------------------------------------------------------18
ii. Supt4h heterozygote reduces mutant, but not normal allele of Htt gene expression in R6/2 mice----------------------------------------------------------18
iii. Decrease of Supt4h attenuates polyQ aggregation in the brain of R6/2 mice---------------------------------------------------------------------------------19
iv. Supt4h heterozygote showed beneficial effects in R6/2 mice---------------19
Discussion---------------------------------------------------------------------------------------21
Reference ---------------------------------------------------------------------------------------25
Tables and Figures ---------------------------------------------------------------------------30








List of Figures

Figure 1. Conventional Supt4h knockout mice were validated by Southern blot
analysis-------------------------------------------------------------------------------32
Figure 2. Genotyping to identify the wild-type and knockout alleles of Supt4h by
PCR-----------------------------------------------------------------------------------33
Figure 3. Manufactured of Supt4h antibody------------------------------------------------34
Figure 4. Supt4h is ubiquitously expressed in mice tissues-------------------------------35
Figure 5. Supt4h expression levels in the brain of Supt4h+/- mice ---------------------37
Figure 6. Immunohistochemistry staining showed decreased protein level of Supt4h
in the cortex and striatum of Supt4h+/- mice ----------------------------------38
Figure 7. Decrease of Supt4h was beneficial to R6/2 mice with 220 to 240 CAG
Repeats ------------------------------------------------------------------------------40
Figure 8. Genotyping to examine the number of CAG repeats in R6/2 mice ----------42
Figure 9. Reduced Supt4h mRNA expression in the brain of Supt4h heterozygous
mice ----------------------------------------------------------------------------------43
Figure 10. Wild-type Htt expression is not affected by decrease of Supt4h in R6/2
mice --------------------------------------------------------------------------------44
Figure 11. Mutant Htt mRNA expression is down-regulated in the brain of
Supt4h+/-；R6/2 mice -----------------------------------------------------------45
Figure 12. Supt4h reduction attenuates polyQ aggregation in the brain of R6/2
mice --------------------------------------------------------------------------------46
Figure 13. Supt4h reduction had beneficial effects on R6/2 mice with 240 to 260 CAG
Repeats -----------------------------------------------------------------------------48

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