亞歷山大氏症是一種罕見但致命的神經退化性疾病，好發於幼年期且尚未有治癒方法，超過95%的亞歷山大氏症是由第三類中間型蛋白絲GFAP基因顯性突變所造成。亞歷山大氏症患者的臨床症狀有很大的差異，發病年紀、病變腦區、疾病嚴重程度等皆因個體而有不同；最明顯的病理特徵為GFAP表現量上調以及羅森塔爾纖維堆積，這些含有突變GFAP的蛋白質團塊會傷害星狀細胞的正常功能，導致神經損傷。在本篇研究中，選擇11個位於中間型蛋白絲高度保留序列的GFAP點突變，探討這些突變如何影響絲狀結構的聚合。體外聚合實驗顯示，突變GFAP會形成非典型的結構，包含較短的絲狀、不一致的直徑、粗糙的表面以及絲狀團塊。在細胞實驗中，突變GFAP在初級星狀細胞內會形成明顯的團塊；透過生化特性分析，發現這些GFAP團塊對於溶劑萃取有較低的溶解性。這些實驗資料皆可證實在rod domain上高度保留的胺基酸對於GFAP絲狀結構的影響雖然不太相同，但同樣干擾劇烈；另外也證實GFAP突變導致絲狀聚合及生化特性的改變是亞歷山大氏症關鍵的誘發因素。

Alexander disease is a rare and often fatal neurodegenerative disease of childhood with no known treatment. More than 95% cases are caused by missense mutations in the gene coding for the type III intermediate filament protein GFAP. Patients with Alexander disease exhibit a wide range of clinical phenotypes, with ages of onset ranging from fetal through the eighth decade, differing distributions of lesions, and mild to severe courses. Pathologically, Alexander disease is characterized by reactive astrogliosis, upregulation of GFAP, and accumulation of Rosenthal fibers. These fibrous globs are abnormal protein aggregates containing mutated forms of GFAP that impair astrocyte functions and lead to progressive loss of neurons. In this study, I have analyzed 11 missense mutations located in the highly conserved ends of the GFAP rod domain to investigate how these mutations affect filament assembly. In vitro assembly studies showed that the GFAP rod end mutants assembled into one or more of several atypical structures, including short filament length, irregular diameter, roughness of filament surface, and filament aggregation. In the context of the cell, GFAP mutants with in vitro assembly defects usually formed cytoplasmic aggregates in transduced primary astrocytes. Using a fractionation protocol that solubilized non-aggregated forms of GFAP while retaining GFAP aggregates, protein complexes containing GFAP mutants are more resistant to solvent extraction. These data support the conclusion that amino acids throughout the rod domain are not equal with respect to their roles in the GFAP filament assembly and suggest that altered filament assembly and properties as a results of GFAP mutation as critical initiating factors for the pathogenesis of Alexander disease.

Abstract i
摘要 ii
誌謝 iii
Abbreviation iv
Table of Contents v
Chapter 1 Introduction 1
1.1 Intermediate filament and GFAP 1
1.2 Mutations in GFAP are associated with Alexander disease 4
1.3 Outline of this study 6
Chapter 2 Material & Methods 8
2.1 Plasmid construction and side direct mutagenesis 8
2.2 Cell culture 8
2.2.1 Cell line culture and transient transfection 8
2.2.2 Primary astrocyte culture 9
2.2.3 Lentiviral production and infection of astrocytes 9
2.3 Immunofluorescence microscopy 10
2.4 Subcellular fractionation and immunoblotting 11
2.5 Bacterial expression and purification of recombinant GFAP 13
2.6 In vitro assembly, transmission electron microscopy and sedimentation assay 14
Chapter 3 Results 17
3.1 Effects of mutant GFAP in SW13 (Vim-) cells 17
3.2 Effect of mutant GFAP in primary astrocytes 17
3.3 Dominant effect of mutant GFAP co-transduced with WT GFAP in primary astrocytes 21
3.4 Characterization of the filament-forming ability of GFAP mutants through in vitro assembly 22
3.5 Dominant effect of mutant GFAP revealed by co-assembly studies 25
Chapter 4 Discussion 26
4.1 Mutations in 1A and 2B domain of IFs highly conserved region cause a strong effect on filament formation 26
4.2 Further prospects 31
Conclusion 34
Tables 35
Table 1.1. The list of diseases associated with mutations in IFs 35
Table 2.1. Primers used for GFAP plasmid construction 36
Table 2.2. Primary antibody list 37
Table 4.1. Summary of mutations characterization 38
Figures 40
Figure 1.1. A schematic view of GFAP structural organization and localization of Alexander disease-causing mutations 40
Figure 1.2. Primary structure analysis of IFs rod domain 41
Figure 1.3. Flow diagram of this study 42
Figure 3.1. Transient expression of GFAP in SW13 (-) cells 43
Figure 3.2.1. Effects of GFAP mutations on IF network formation 44
Figure 3.2.2. Effects of GFAP mutations on IF network formation 45
Figure 3.3. The extent of mutant GFAP aggregation did not change appreciably with time (1A domain) 47
Figure 3.4. The extent of mutant GFAP aggregation did not change appreciably with time (2B domain) 49
Figure 3.5. Analysis of the solubility properties of the GFAP mutants 51
Figure 3.6.1. Three types of staining pattern observed in primary astrocytes co-transduced with WT and mutant GFAP 52
Figure 3.6.2. Solubility properties of GFAP co-transduction in primary astrocytes 54
Figure 3.7. Expression and purification of recombinant human GFAP 56
Figure 3.8. Effects of GFAP 1A mutations on the in vitro assembly of IFs 59
Figure 3.9. Effects of GFAP 2B mutations on the in vitro assembly of IFs 61
Figure 3.10. The effect of mutants on GFAP assembly is dominant over the WT 62
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