亞歷山大氏症 (Alexander disease, AxD) 是一種罕見的中樞神經系統 (Central nervous system, CNS) 退化性疾病，已知主要致病原因為表現在星狀細胞 (astrocyte) 內的中間型蛋白絲 (intermediate filament, IF) ，也就是膠質纖維酸性蛋白質 (glial fibrillary acidic protein, GFAP) 發生基因突變，影響細胞骨架支持星狀細胞基本型態的功能，進而失去維持中樞神經系統恆定的能力。先前的研究發現，當GFAP在細胞中大量表現時，會導致在星狀細胞中產生大量且不正常的蛋白質聚集，稱為羅森塔爾纖維 (Rosenthal fiber, RF) ，是亞歷山大氏症主要的病理特徵，由GFAP、泛素 (ubiquitin) 、αB-crystallin以及其他蛋白質組成並堆積於組織中。本研究利用體外聚合實驗和以及慢病毒轉導實驗，探討與AxD相關的突變如何影響GFAP在體外的聚合和星狀細胞中間型蛋白絲網絡的形成，以及轉譯後修飾，例如泛素-蛋白酶體系統，是如何促進蛋白質病理性聚集的相關致病機轉。透過生化數據表明，從病患檢體分離出的羅森塔爾纖維中，可以偵測到泛素化的GFAP、透過雙硫鍵結合的高分子量GFAP，以及經由蛋白酶降解的蛋白質片段。根據此研究，我們提出了一個潛在的病理模型，以突變後的GFAP為引起疾病的首要條件，由於突變改變了GFAP聚合的特性，誘導GFAP被泛素修飾，進而導致蛋白質異常聚集。另一個可能的致病途徑為，突變後的異常GFAP聚集，會抑制蛋白酶體的功能，導致進一步被泛素化，最終引發疾病。
本篇碩士論文中，已有部分發表於我最近刊登於期刊之文章(Lin et al., 2024)，並對內容進行修改和更新，而該文章部分結果來自於就讀國立清華大學（NTHU）碩士在學期間，並且由國家科學及技術委員會(NSTC)提供計畫資助。

Alexander disease (AxD) is a rare degenerative disorder of the central nervous system (CNS), which mainly caused by mutations in the gene encoding for glial fibrillary acidic protein (GFAP), an intermediate filament (IF) protein expressed in astrocytes. These mutations disrupt the cytoskeletal system of astrocytes, affecting their ability to maintain CNS homeostasis. Previous studies have shown that overexpression of GFAP leads to the formation of abnormal protein aggregates in astrocytes, known as Rosenthal fibers (RFs), which are a hallmark of AxD. RF is primarily composed of GFAP, ubiquitin, αB-crystallin and other proteins. This study uses in vitro assembly assay and lentivirus transduction experiment to investigate how AxD-associated mutations affect the assembly of GFAP in vitro and the formation of IF networks in astrocytes, as well as the role of ubiquitination of GFAP in promoting pathological protein aggregation. Biochemical data indicate that RFs isolated from AxD patient brains contain ubiquitinated GFAP, high molecular weight (HMW) GFAP modified by crosslinking through disulfide bonds, and proteolytic fragments. Based on this study, we propose a potential pathogenic model in which mutated GFAP is a primary trigger of the disease. The mutations alter assembly properties of GFAP, leading to the induction of GFAP ubiquitination, which causes further aggregation. Another circuit of abnormal GFAP aggregation inhibits the function of the proteasome, causing additional ubiquitination and ultimately contributing to disease progression.
Some parts of this thesis are modified and updated from my recent publication (Lin et al., 2024), which was supported by grants from National Science and Technology Council (NSTC) and is in partial fulfillment for the requirement of the Master degree in NTHU.

Abstract-----------Ι
摘要---------------Ⅱ
致謝---------------Ⅲ
Abbreviation-------Ⅳ
Chapter 1 Introduction---------1
1.1 Intermediate filaments and the function of GFAP in Astrocyte-1
1.2 Genetics of Ax-------------4
1.3 Mechanisms of disease------5
1.4 Gain or loss of function---7
Chapter 2 Material and Methods-9
2.1 Plasmid construction and site direct mutagenesis---------------9
2.2 Bacterial expression and purification of recombinant GFAPs-----9
2.3 In vitro assembly assay and sedimentation assay----------------11
2.4 Negative staining and transmission electron microscopy---------12
2.5 Rodent models and genotyping-----------------------------------12
2.6 Primary astrocyte culture--------------------------------------13
2.7 Cell line culture----------------------------------------------14
2.8 Lentiviral production and transduction-------------------------14
2.9 Subcellular fractionation--------------------------------------15
2.10 Immunoblotting------------------------------------------------16
2.11 Immunofluorescence microscopy---------------------------------17
2.12 Preparation of Rosenthal fiber fraction from brain tissue-----18
Chapter 3 Results--------------------------------------------------19
3.1 Scope and outline of this study--------------------------------19
Figure 1. Outline of experiments in this study---------------------20
3.2 Expression and purification of recombinant human GFAP----------21
Figure 2. A schematic view summarized the structural organization of GFAP and the localization of mutations causing AxD used in this study--22
Figure 3. Inclusion body preparation of GFAP------------------------------23
Figure 4. Purification of recombinant human GFAP by column chromatography-24
3.3 In vitro assembly studies---------------------------------------------25
Figure 5. A Schematic diagram illustrates the in vitro assembly process and subsequent analysis----------------27
Figure 6. Effects of GFAP mutation upon the in vitro filament assembly-----------------------------------------28
3.4 Effects of GFAP mutants on IF network formation in Primary astrocytes--------------------------------------30
Figure 7. A flowchart for primary cell preparation and lentiviral transduction---------------------------------31
Figure 8. Characterization of primary astrocytes derived from GFAP KO rats-------------------------------------32
Figure 9. GFAP mutants formed aggregates when transduced into GFAP KO astrocytes-------------------------------36
Figure 10. Effect of GFAP mutations on IF network formation in GFAP KO astrocytes------------------------------37
Figure 11. Solubility properties of GFAP mutants in GFAP KO astrocytes-----------------------------------------39
Figure 12. Ubiquitin was colocalized with aggregates of mutant GFAP--------------------------------------------41
3.5 Mutant GFAPs alter anti-GFAP antibody binding sites on GFAP------------------------------------------------43
Figure 13. Epitope mapping of anti-GFAP antibody---------------------------------------------------------------44
3.6 Effects of mutant GFAP on the endogenous GFAP networks in primary WT astrocyte-----------------------------46
Figure 14. Effect of GFAP mutation of the endogenous GFAP on the aggregation process in primary rat astrocytes-48
Figure 15. Effect of GFAP mutation on the aggregation process in GFAP heterozygous KO astrocytes---------------50
Figure 16. Solubility properties of GFAP mutation in GFAP WT astrocytes----------------------------------------52
3.7 Ubiquitination of GFAP in SW13 (Vim-) cells----------------------------------------------------------------54
Figure 17. Solubility properties and ubiquitination of mutant GFAP in SW13(Vim-)-------------------------------56
Figure 18. Aggregates formed by mutant GFAP was co-stained with GFAP and ubiquitin-----------------------------57
3.8 GFAP is ubiquitinated in mouse AxD model and human AxD patients--------------------------------------------59
Figure 19. GFAP was modified by ubiquitination in the enriched RF fraction in Tg mice brain--------------------61
Figure 20. GFAP was modified by ubiquitination in the enriched RF fraction in human patient of AxD-------------63
Figure 21. Oxidative modification of GFAP in human AxD brains--------------------------------------------------65
3.9 Sequences required for GFAP assembly in vitro--------------------------------------------------------------66
Figure 22. A schematic view of GFAP structure and localization the truncated GFAP------------------------------68
Figure 23. N-terminal truncated form of GFAP disrupted the filament assembly in vitro--------------------------69
Figure 24. Analysis of the solubility properties of N- or C-terminal deleted GFAP------------------------------70
Figure 25. C-terminal deletion affected GFAP assembly in vitro-------------------------------------------------73
Figure 26. Structures of C-terminal deletion variants before assembly in low ionic strength buffer-------------76
Chapter 4 Discussion-------------------------------------------------------------------------------------------77
4.1 Purification of recombinant human GFAP---------------------------------------------------------------------77
4.2 Ubiquitination of GFAP in AxD------------------------------------------------------------------------------78
4.3 A pathogenic cycle initiated by GFAP mutations-------------------------------------------------------------82
Figure 27. Pathogenic cycle of disease progression in AxD------------------------------------------------------84
4.4 GFAP segments required for filament assembly---------------------------------------------------------------85
4.5 Concluding Remarks-----------------------------------------------------------------------------------------88
Tables---------------------------------------------------------------------------------------------------------91
Table 1. Major types of intermediate filaments-----------------------------------------------------------------91
Table 2. GFAP isoforms and their characteristic----------------------------------------------------------------91
Table 3. Classification of AxD and its clinical features-------------------------------------------------------92
Table 4. List of Rosenthal fiber components--------------------------------------------------------------------92
Table 5. Different types of genetic mutation in AxD and their location-----------------------------------------93
Table 6. Primers used in this study----------------------------------------------------------------------------94
Table 7. Primary antibodies used in this study-----------------------------------------------------------------94
Table 8. Details of human post-mortem tissue samples analyzed by immnoblotting---------------------------------95
Table 9. List of GFAP mutation used for in this study----------------------------------------------------------95
Table 10. GFAP purification of column chromatography-----------------------------------------------------------96
References-----------------------------------------------------------------------------------------------------97

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