亞歷山大氏症（Alexander disease）是一種因為中樞神經內星狀細胞官能障礙(Astrocyte dysfunction)所引起的的中樞神經退化性疾病。此疾病主要是由於星狀細胞的中間型蛋白絲(Intermediate filaments; Ifs)，即神經膠質纖維酸性蛋白質(Glial fibrillary acidic protein; GFAP)基因突變引起。亞歷山大氏症的主要病理特徵是星狀細胞內有大量沉積的包含體(Inclusion body)，即Rosenthal fibers，其主要由GFAP、小分子量熱休克蛋白質(Small heat shock protein) Hsp27及alphaB-crystallin組成。雖然目前已知基因突變造成GFAP異常而引起亞歷山大氏症，但GFAP突變引起疾病的機制尚不清楚。大部分GFAP突變都是錯義突變(missense mutation)改變單一個或少數幾個胺基酸導致GFAP結構及功能異常而引起疾病。本研究目的為探討三種新型GFAP突變(∆4 GFAP, IDF GFAP, E312X GFAP)如何影響GFAP形成絲狀結構的能力及其穩定性。結果顯示，此三種GFAP突變皆會引起GFAP堆積(Aggregation)，破壞其絲狀結構，其中又以E312X GFAP影響最為嚴重。有表現E312X GFAP的細胞中有活化Caspases及細胞存活率下降的現象。活化的Caspases 會進一步降解GFAP產生片段(GFAP fragment)。藉由本次研究結果發現經Caspase降解的不同的GFAP突變會產生不同的GFAP fragments，可進一步探討GFAP 蛋白質水解(proteolysis)與亞歷山大氏症致病機轉的關係。

Alexander disease (AxD) is a primary genetic disorder of astrocytes caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein (GFAP). The mechanism of GFAP mutation causing the AxD remains unclear. The aim of this study is to investigate the effect of the novel AxD-causing mutation on GFAP filament formation and stability by using filament assembly in vitro and transient transfection in cultured cells. The results showed that all the GFAP mutations perturbed the filament assembly in vitro and in transiently transfected cells. The E312X GFAP caused the most dramatic effects on filament assembly both in vitro and in transiently transfected cells. This truncated mutant caused extensive filament aggregation coinciding with the activation of caspases, cleavage of GFAP, and a significant decrease in cell viability. These data provide a direct link of GFAP mutation on filament aggregation and loss of cell viability through the activation of caspases and cleavage of GFAP, suggesting that these could be contributing factors in the development of Alexander disease.

Abstract I
摘要 II
致謝 III
Abbreviation IV
Chapter 1 Introduction 1
1.1 Alexander disease 1
1.2 GFAP 2
1.3 Mutations in GFAP are associated with Alexander disease 4
1.4 Outline of this study 6
Chapter 2 Materials & Methods 8
2.1 Plasmid construction and site directed mutagenesis 8
2.2 Bacterial expression and purification of recombinant GFAPs 8
2.3 In vitro assembly assay and sedimentation assay 10
2.4 Electron microscopy 12
2.5 Cell cultures and transient transfection 12
2.6 Immunofluorescence microscopy 13
2.7 Subcellular fractionation 14
2.8 Immunoblotting 15
2.9 Immunoprecipitation 15
Chapter 3 Results 17
3.1 Effects of mutant GFAP upon the in vitro filament assembly 17
3.2 Epitope mapping of anti-GFAP antibodies 21
3.3 Effects of GFAP mutations upon IF network formation in SW13 (Vim+) cells 21
3.4 Assembly properties of KGFAP and mutant GFAP in transfected human astrocytoma U343MG cells 23
3.5 GFAP is cleaved by both caspase 3 and 6 in transfected SW13 cells. 25
3.6 The degradation product p20 was produced by caspase 3 cleavage. 27
Chapter 4 Discussion 29
4.1 Impact of GFAP Mutations on Filament Assembly and Network Organization 29
4.2 Paracrystal structure 31
4.3 Caspases-mediated proteolysis of GFAP 33
4.4 Further prospects 35
References 39
Tables 49
Table 1 The primers used for construction of variant GFAP constructs. 49
Table 2 Primary antibody list 50
Figures 51
Figure 1 Expression and purification of KGFAP , and electrophoretic analysis of GFAP variants. 53
Figure 2 In virto assembly of purified GFAP variants observed by TEM. 54
Figure 3 Paracrystals of the E312X GFAP. 55
Figure 4 Characterization of mutant GFAP by the in vitro assembly assay. 56
Figure 5 Effects of E312X on WT GFAP in vitro assembly assay 58
Figure 6 Immunoblotting analysis of purified recombinant GFAP variants. 61
Figure 7 The different staining patterns of the cells transfected with GFAP variants 63
Figure 8 Mutant GFAPs and KGFAP aggregate also disrupted endogenous vimentin networks 65
Figure 9 A dominant effect of E312X GFAP over WT GFAP 67
Figure 10 Mutant and KGFAP causes aggregation of GFAP in U343MG astrocytoma cells. 69
Figure 11 GFAP is cleaved by caspase 6 and 3. 71
Figure 12 The degradation product p20 was produced by caspase 3 cleavage. 72
Figure 13 Schematic representation of the molecular arrangement in the fundamental pattern observed in paracrystals of E312X GFAP. 73
Fig.14. Model summarizes the role of caspase-generated GFAP fragments. 74
Appendix 75
Appendix 1 Identification of a novel nonsense mutation in the rod domain of GFAP that is associated with Alexander disease 75

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Alexander disease :
http://www.waisman.wisc.edu/alexander-disease/index.html
Human intermediate filament database:
http://www.interfil.org