臺灣博碩士論文加值系統

亨丁頓氏舞蹈症 (Huntington’s disease, HD) 是一種顯性遺傳神經退化性疾病，致病原因為 Huntington 基因 exon1 的位置出現三個核苷酸 (CAG) 重複序列異常擴增的現象，此突變基因產生的蛋白質 (mutant-HTT, mHTT) 具有細胞毒性，最終導致神經細胞的死亡。在臨床診斷中發現，HD病患中基底核的紋狀體區域神經細胞萎縮的情形最為嚴重。Spt4為轉錄延伸因子，可以提升RNA polymerase II在 DNA 模板上進行轉錄作用的穩定性。為了進一步探討SUPT4H在人類神經細胞中對於HD致病基因表現的影響，我們利用HD病患的誘導型多功能幹細胞 (induced pluripotent stem cell, iPSC) 及其經基因工程改造的穩定細胞株，將其分化成病理相關的神經細胞型態，並藉由三種不同的分子機制 (shRNA, antisense-oligonucleotide 和小分子藥物) 使SUPT4H失去活性，探討其對於HD致病基因的影響；並進一步探討致病基因表現的改變，是否有助於改善HD神經細胞的存活能力。結果發現，在三種不同讓SUPT4H降低活性的情況下，皆可抑制SUPT4H的活性並降低mHTT的表現量，而其減少的幅度與處理的劑量呈現正相關性。同時，我們也觀察到帶有致病基因的神經細胞相較於健康個體，其細胞存活能力較差，並且在有氧化壓力情況下，其差異性顯著的拉大，但藉由抑制SUPT4H活性能夠改善HD神經細胞的存活能力。這個研究顯示，在HD病患誘導型多功能幹細胞所分化成的神經細胞中，透過抑制SUPT4H的活性可以降低mHTT的表現，並且能夠改善HD神經細胞存活能力的缺失。

Huntington’s disease (HD), a dominantly inherited neurodegenerative disease, is caused by abnormal expansion of CAG trinucleotide repeats in the exon 1 of the huntingtin (HTT) gene. The most striking pathological feature is the degeneration of striatum, which is comprised of more than 90% GABAergic medium spiny neurons (MSNs). Spt4 is a transcription elongation factor that associates with RNA polymerase II to regulate gene transcription by enhancing of RNA polymerase II processivity. In order to investigate the effect of SUPT4H on mHTT expression in clinical relevant samples, we use three different approaches (shRNA, antisense-oligonucleotide and small compound) to inactive SUPT4H function in human GABAergic neurons derived from induced pluripotent stem cells of HD patients (HD-iPSCs). By using HD-iPSCs derived GABAergic neurons (HD-GABAergic neuron), we demonstrated that inactivation of SUPT4H decreases the expression level of mutant HTT (mHTT) transcripts. Similar level of mHTT and wild-type HTT (wt-HTT) reduction by SUPT4H shRNA knockdown is noticeable using Taqman qPCR assay in which mHTT and wt-HTT is measured by allele-specific probes. Furthermore, the degree of mHTT reduction is shown in a dose dependent manner by various SUPT4H inactivation approaches. More importantly, SUPT4H downregulation can partially reverse the cell viability of HD-GABAergic neurons in the presence of H2O2. These results suggest that inactivation of SUPT4H can modulate mHTT expression and improve cell viability in human GABAergic neurons with HD pathology.

致謝 1
中文摘要 2
Abstracts 3
Introduction 4
3.1 Huntington’s disease (HD) 4
3.2 Induced pluripotent stem cells (iPSCs) 4
3.3 iPSCs of Huntington’s disease (HD-iPSCs) 5
3.4 Differentiation of iPSCs into disease-relevant neuronal type 5
3.5 The role of SUPT4H in transcription regulation 6
3.6 Tet-on system 7
3.7 Antisense-oligonucleotide (ASO) 7
3.8 Small compound (NU101) 7
3.9 The aim of this study 8
Material and methods 9
4-1 iPSCs maintenance 9
4-2 Neuronal differentiation from iPSCs 9
4-3 SUPT4H inactivation 10
4-4 Immunocytochemistry (ICC) 11
4-5 RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) 11
4-6 Western blot 12
4-7 Cell viability test 12
4-8 Taqman qPCR 13
4-9 Statistical Analysis 14
Results 15
5.1 Strategies of SUPT4H inactivation 15
5.2 Differentiation of iPSC into GABAergic neurons 15
5.3 SUPT4H inactivation by shRNA 16
5.3.1 SUPT4H knockdown in GABAergic neurons derived from 45Q-iPSC stable clones (221 and L36) 16
5.4 SUPT4H inactivation by Antisense oligonucleotide 17
5.4.1 SUPT4H knockdown in GABAergic neurons derived from 45Q-iPSC (iHDA1) and healthy individual (iCFB50) 17
5.5 SUPT4H inactivation by small compound (NU101) 18
5.5.1 Pharmacological inhibition of SUPT4H in GABAergic neurons derived from 45Q-iPSCs (iHDA1) and healthy iPSCs (iCFB50) 18
5.6 SUPT4H downregulation improves the cellular viability of HD-iPSCs derived GABAergic neurons 19
5.6.1 Viability of GABAergic neurons derived from HD-iPSCs (iHDA1) and healthy-iPSCs 19
5.6.2 iHDA1-derived GABAergic neurons are more susceptible to H2O2-induced oxidative stress 20
5.6.3 SUPT4H ASO knockdown rescue the cellular viability of iHDA1-derived GABAergic neurons with H2O2 insults 20
5.7 Implementation of taqman assay for detection of mHTT and wt-HTT in HD GABAergic neurons 21
Discussion 22
References 24
Figures 26
Tables 39

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