臺灣博碩士論文加值系統

亨氏舞蹈症是一種顯性遺傳的神經退化性疾病，造成此疾病的主因是在huntingtin 基因的1號外顯子上的CAG核苷酸重複序列有異常的擴增情形，並且導致大腦皮質以及基底核中的紋狀體發生神經細胞衰亡的現象。目前已有文獻指出，huntingtin基因突變所產生的多麩胺酸蛋白質聚集體具有細胞毒性，是導致腦部細胞退化或死亡而出現病徵的主要原因。實驗室先前研究發現，酵母菌中的SPT4 基因產生缺失時，會選擇性抑制過度增常的CAG基因表現量，進而降低多麩胺酸蛋白質聚集體的產生。在哺乳類細胞中，SPT4的同源基因Supt4h 會和Supt5h結合形成一個異質二聚體，此結構會促進基因轉錄功能，並且增加RNA polymerase II持續合成的能力。當在亨氏舞蹈症小鼠模式中降低Supt4h的表現時，已證明可以減少多麩胺酸蛋白質聚集體的產生。在此論文研究，我利用多種不同型式的試劑想要達到在疾病小鼠的大腦皮質以及紋狀體中降低Supt4h的表現，並分析其對運動功能受損恢復的狀態。實驗結果發現腦部Supt4h的減少，具有減緩突變huntingtin基因所造成的毒性，進而延緩R6/2亨氏舞蹈症小鼠發病的情況。

Huntington’s disease (HD) is a dominant inherited neurodegenerative disorder resulting from degeneration of neurons mainly in the striatum and cerebral cortex and caused by expansion of CAG repeats in the exon 1 of huntingtin (Htt) gene. Accumulated evidences suggest that polyglutamine (polyQ)-mediated protein aggregation of mutant Htt is toxic to neurons and triggers HD pathogenesis. In our recent studies, we have demonstrated that SPT4 is able to selectively regulate the expression and aggregation of proteins containing extended polyQ in yeast cells. Supt4h, the mammalian ortholog of yeast SPT4, is associated with Supt5h to promote transcription elongation and RNA polymerase II processivity, and modulates the expression of mutant Htt and polyQ aggregation both in neuronal cells and mouse brain. In this study, multiple approaches that aim to suppress Supt4h expression was applied, and the effect of Supt4h down-regulation on mutant Htt gene expression and corresponding changes in motor function of R6/2 HD mice was assessed. Our initial results suggest that Supt4h down-regulation in the brain of R6/2 HD mice might have an ameliorated effect to delay the onset of HD.

Contents
Chinese abstract ............................................................................................................ i
Abstract ......................................................................................................................... ii
Contents ...................................................................................................................... iii
List of tables.................................................................................................................. v
List of figures ............................................................................................................... vi
Introduction .................................................................................................................. 1
Huntington’s disease ................................................................................................ 1
HD is a polyglutamine disorder ............................................................................... 1
HD and PolyQ aggregation ...................................................................................... 2
HD mouse model ....................................................................................................... 3
Therapeutic targets of HD .......................................................................................... 4
Function of Supt4h ..................................................................................................... 5
Regulation between supt4h and polyQ aggregation .................................................. 5
Antipsychotic treatment ............................................................................................. 6
Adeno-associated virus, AAV ................................................................................... 6
Antisense oligonucleotides ........................................................................................ 7
Research rationale ...................................................................................................... 8
Material and Methods ................................................................................................. 9
Animal ..................................................................................................................... 9
Thioridazine hcl administration ............................................................................... 9
Tissue collection and protein isolation .................................................................. 10
Western blot analysis ............................................................................................. 10
a. Mouse brain protein ............................................................................ 10
b. Cell lysate protein ............................................................................... 11
RNA extraction ...................................................................................................... 12
Quantitative real time RT-PCR amplification ....................................................... 12
Cell lines and culture conditions ............................................................................ 13
Plasmid construction and preparations .................................................................. 13
Transfection of viral vectors and asos ................................................................... 14
Stereotaxic injection of AAV8 ............................................................................... 14
a. wild-type mice test .............................................................................. 14
b. R6/2 mice injection ............................................................................. 15
Immunohistochemistry .......................................................................................... 15
Rotarod performance ............................................................................................. 16
Beam walk ............................................................................................................. 16
Statistical analysis .................................................................................................. 17
Results ......................................................................................................................... 18
PART I: Reducing supt4h expression by pharmacological administration ..... 18
1. Effect of thioridazine HCl treatment in wild-type mice ..................... 18
PART II: Using aav8-mediated shrna on aso to study the effect of supt4h
down-regulation in HD mice ................................................................. 19
1. Down-regulation of Supth4 with AAV8 viral vector in Neuro2A cell
line ...................................................................................................... 19
2. Suppression of Supt4h by AAV8-shRNA delivery in the brain of
WT mice ............................................................................................. 19
3. Supt4h down-regulation attenuates HD symptoms in female R6/2
mice .................................................................................................... 20
4. Reduction of Supt4h with Antisense oligonucleotides in Neuro2A
cell line ............................................................................................... 21
Discussion ................................................................................................................... 22
Reference .................................................................................................................... 27
Tables and Figures ..................................................................................................... 33

List of Tables
Table 1. Primers sequence used for qPCR and genotyping ....................................... 33
Table 2. Supt4h shRNA sequences ............................................................................ 34
Table 3. Supt4h antisense oligonucleotide sequences ................................................ 35

List of Figures
Figure 1. Supt4h mRNA and protein levels in the brain of WT mice after thioridazine
HCl treatment for 7 days .............................................................................. 36
Figure 2. Supt4h expression levels in the brain of WT mice after Thioridazine HCl
Treatment for 28 days ................................................................................ 37
Figure 3. Supt4h expression levels in the brain of WT mice after Thioridazine HCl
treatment for 56 days ................................................................................. 38
Figure 4. Thioridazine HCl treatment cause sever physiological dysfunction .......... 39
Figure 5. Supt4h protein expression is down-regulated by AAV8 viral vector in
Neuro2A cell line ....................................................................................... 40
Figure 6. Supt4h mRNA levels after AAV8 injection in the striatum of WT mice ... 42
Figure 7. Immunohistochemistry staining showed decreased Supt4h protein level in
the striatum after AAV-Supt4h-S2 treatment ............................................ 44
Figure 8. Decreasing Supt4h was beneficial to female R6/2 mice motor function ... 46
Figure 9. Supt4h protein levels with Antisense oligonucleotides treatment in Neuro2A
cells ............................................................................................................ 48

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