亞歷山大氏症 (Alexander disease, AxD) 是一罕見中樞神經退化性疾病，其致病原因為主要表現於星狀細胞 (astrocyte) 內的中間型蛋白絲 (intermediate filament, IF)-神經膠質纖維酸性蛋白質 (glial- specific intermediate filament protein, GFAP) 基因發生突變，導致其在結構與功能上發生異常，並且在星狀細胞中產生大量且不正常聚集，進而形成羅森塔爾纖維 (Rosenthal fibers)。研究發現，此不正常聚集纖維也能在罕見疾病-巨軸突神經病變 (giant axon neuropathy, GAN)患者中看到，而引起此疾病的主因為 gigaxonin 基因產生突變，由患者的病理學上可以看到大量神經絲 (neurofilament, NF) 聚集於軸突當中，又經由結構預測 gigaxonin 為一E3泛素接合酶 (E3 ligase adaptor)，而E3泛素接合酶也參與了蛋白質降解之重要路徑-泛素-蛋白酶體系統 (Ubiquitin-proteasome system, UPS)，所以基於 gigaxonin 之潛在功能，前人研究 gigaxonin 功能正常是否能將大量且不正常聚集之神經絲降解，結果顯示 gigaxonin 確實會藉由泛素-蛋白酶體系統將神經絲表現量下降之外，也會降解其他中間型蛋白絲-波形蛋白 (vimentin)、外周蛋白 (peripherin)；目前 gigaxonin 對於降解同樣屬於中間型蛋白絲之 GFAP 仍未知，因此，本研究利用慢性病毒轉導技術將正常 gigaxonin 大量表現於初代星狀細胞 (primary astrocyte) 以及將未具有內生性中間型蛋白絲之 SW13 細胞株表現與亞歷山大氏症相關之 GFAP突變，利用免疫染色法 (immunofluorescence microscopy) 和免疫轉漬法 (immunoblotting) 分析 gigaxonin 是否會降解 GFAP 表現量，本研究結果發現，gigaxonin 會降解正常 GFAP，但是卻只能降低部分之突變 GFAP 表現量；研究結果顯示 gigaxonin 對於亞歷山大氏症及因異常中間型蛋白絲所導致的相關疾病，在未來可能提供一個新的治療契機。

Abnormal aggregates resulting from mutations in the intermediate filament (IF) proteins are pathological hallmarks of a wide range of neurodegenerative diseases. This is exemplified by Alexander disease (AxD), a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding the glial filament protein GFAP. This disease is characterized by excessive accumulation of GFAP in an aggregated form known as Rosenthal fibers within astrocyte cell bodies and processes. Abnormal GFAP aggregation also occurs in other pathological conditions, such as giant axon neuropathy (GAN). GAN is caused by recessive mutations in the gene encoding gigaxonin, which is predicted to be an E3 ligase adaptor. Although GAN represents the only disease that causes a generalized disorganization of IFs in a range of cell types, the molecular mechanisms by which mutations in gigaxonin affect the IF cytoskeletal system are unknown. Recent studies have demonstrated that the main function of gigaxonin is to target IF proteins for degradation through the proteasomal pathway. We therefore sought to determine whether gigaxonin is involved in degradation of GFAP. Using a lentiviral transduction system, we demonstrated that gigaxonin levels influence the degradation of GFAP IFs in primary rat astrocytes and in SW13 cell lines stably expressing this IF protein. Gigaxonin was similarly involved in the degradation of some but not all AxD-associated GFAP mutants. Gigaxonin directly binds to GFAP, and proteasomal inhibition by MG-132 reversed the clearance of GFAP in cells achieved by overexpressing gigaxonin. Together, these findings identified gigaxonin as an important factor that targets the glial-specific IF protein GFAP for degradation through the proteasome pathway. Our studies provide a critical foundation for future studies with a goal towards reducing or reversing pathological accumulation of GFAP as a potential therapeutic strategy for AxD and other related diseases characterized by IF aggregation.

Abstract-1
摘要-2
Chapter 1 : Introduction-5
1.1 Intermediate filaments and GFAP-5
Table 1.1-1. Intermediate filaments: expression pattern and involvement in human diseases.-6
Figure 1.1-1. A schematic view of GFAP structural organization and localization of disease-causing mutations.-8
Figure 1.1-2. A hypothetical model of GFAP assembly pathway.-10
1.2 Functions of GFAP-11
Figure 1.2-1. Schematic representation of gigaxonin.-14
Figure 1.2-2. A model of gigaxonin-mediated protein degradation through ubiquitin proteasome system (UPS).-15
1.3 Outline of the study-16
Chapter 2 : Materials and methods-17
2.1 Plasmid construction and site-directed mutagenesis.-17
2.2 Cell culture-17
Figure 2.2-1. Establishment of stable cell lines.-18
Figure 2.2-2. Representative images of SW13 stable lines expressing either wild type or mutant GFAPs.-19
2.3 Primary cortical astrocyte cultures-20
2.4 Lentiviral production and transduction-20
Figure 2.4-1. Expression vectors used for production of lentiviruses. 21
Figure 2.4-2. Quantification of the concentration of lentivirus.-23
Figure 2.4-3. Visualization of lentivirus by transmission electron microscopy.-24
Table 2.4-1. Summary of conditions for lentiviral transduction.-25
2.5 Immunofluorescence microscopy-26
Table 2.5-1. Summary of antibodies used for immunoblotting, immunostaining and immunoprecipitation.-27
2.6 Cell fractionation-28
2.7 Immunoprecipitation-28
2.8 Immunoblotting-28
Chapter 3 : Results-30
3.1 Gigaxonin overexpression specifically cleared GFAP IFs in primary astrocytes-30
Figure 3.1-1. Gigaxonin cleared GFAP IFs in primary rat astrocyte.-31
Figure 3.1-2. Expression of gigaxonin did not affect organization of actin-containing microfilaments and microtubules in primary astrocytes.-33
Figure 3.1-3. Clearance of GFAP IFs by gigaxonin did not affect other cytoskeletal structures.-34
3.2 Ubiquitin proteasomal pathway involves in gigaxonin-mediated clearance of GFAP-35
Figure 3.2-1. Interaction of GFAP with gigaxonin determined by co-immunoprecipitation.-36
3.3 Proteasome is involved in the clearance of GFAP-37
Figure 3.3-1. GFAP was cleared by gigaxonin through ubiquitin proteasome system.-38
3.4 Gigaxonin degraded human wild type GFAP IFs.-39
Figure 3.4-1. Gigaxonin cleared human wild type GFAP IFs in SW13 cells.-40
Figure 3.4-2. Microtubules (MTs) appeared normal in gigaxonin-transfected cells.-41
3.5 Gigaxonin cleared some but not all GFAP mutants.-42
Figure 3.5-1. A disease-causing GFAP mutant was similarly cleared by gigaxonin. SW13 cells stably expressing R416W GFAP were infected with Flag-GAN lentiviruses.-43
Figure 3.5-2. Gigaxonin cleared a disease-linked form of GFAP mutant.-44
Figure 3.5-3. Gigaxonin cleared a mutant form of GFAP. SW13 cells stably expressing R88C GFAP were infected with lentiviruses containing Flag-gigaxonin for 72 hours.-46
Figure 3.5-4. Gigaxonin cleared some but R239H GFAP mutants.-47
Figure 3.5-5. Gigaxonin was unable to clear Δ4 GFAP mutant.-48
3.6 Disease-linked mutations in GFAP reduced its interaction with gigaxonin-49
Figure 3.6-1. Disease-causing mutations in GFAP reduces its interaction with gigaxonin.-51
Chapter 4 : Discussion-52
4.1 How gigaxonin exerts its effect on GFAP-52
Figure 4.1-1. A schematic diagram shows that gigaxonin functions as an E3 ubiquitin ligase adaptor that facilitates proteasomal degradation of GFAP.-54
4.2 Why gigaxonin degrades some but not all GFAP mutants-55
4.3 Therapeutic implication for AxD and related disorders-57
References-60
Appendix-68

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