- 學位論文
以生化及細胞分析來探討亞歷山大疾病之GFAP突變蛋白對星狀細胞的影響
Biochemical and cellular analyses to study the effects of Alexander disease-associated GFAP mutations in astrocytes
摘要
膠質纖維酸性蛋白（GFAP）是第III型的中間絲蛋白，主要是存在在中樞神經系統的星狀膠質細胞中。GFAP的功能是細胞骨架，從細胞核延伸到細胞膜，使細胞成形。過去已經報導突變的GFAP與某些疾病有很多關係;最著名的是亞歷山大疾病（AxD），一種罕見的神經退行性疾病，多發於兒童。AxD的特徵是星形膠質細胞內存在大量的膠質聚集，稱Rosenthal fibers，由GFAP、αB-crystallin 和HSP27、及其他未鑑定的蛋白質所組成。有許多報導指出62種不同的GFAP突變，其中一些是missense或frame-shift mutations。然而，GFAP突變的Transient expression和動物模型對於表現出phenotype（在AxD中的Rosenthal fibers）有一些挑戰和困難。為此，我們在小鼠DBT細胞建立了tet -on系統來表現GFAP，接著又建立了pLEX -MCS病毒來感染大鼠星狀細胞進而表現GFAP。 在tet -on系統中，我們發現GFAP突變R416W的細胞質聚集增加了細胞活力，並通過Autophagy和UPS來分解蛋白。在 PLEX -MCS病毒感染系統，我們發現，某些不同類型的突變GFAP（Δ4，κGFAP，IDF，ΔT 和E312 *）的聚集會導致的大鼠星形細胞細胞中的線粒體也聚集。我們另外發展出特定的小鼠GFAP專一性抗體以檢測來自轉基因動物，人類患者或是AxD動物模型中的GFAP。
並列摘要
Glial Fibrillary Acidic Protein (GFAP) is type III intermediate filament and major astrocytic protein in central nerve system. The functions of GFAP are cytoskeleton that extend from nucleus to cell membrane to give cells their shape. Mutant GFAPs have been described to have lots of relation with some disorders; most famous is the Alexander disease (AxD), a rare neurodegenerative disease mostly in children. AxD is the presence of inclusion bodies within astrocytes known as Rosenthal fibers, consisting of ubiquitinated GFAP, the small stress proteins αB-crystallin and HSP27, and likely other unidentified proteins. There are many reports point out 62 different GFAP mutation, some of them are missense or frame-shift mutations. However, transient expression system and animal model for GFAP mutation have some challenge to show the phenotype of inclusion (Rosenthal fiber). For this reason, we have stablished the doxycycline inducible tet-on system in mouse DBT stable cell lines and pLEX-MCS lentivirus infection system in rat primary astrocyte cells. In tet-on system, we found the cytoplasmic aggregation of GFAP mutant R416W (the amino acid residue 416 Arginine was changed to Tryptophan) increased the cell viability and degraded by autophagy and UPS synchronously. In pLEX-MCS lentivirus infection system, we demonstrated that different types of mutants GFAP (∆4, κGFAP, IDF, ∆T and E312*) aggregations would cause mitochondrial co-localization in rat primary astrocyte cells. Moreover, we characterized the GFAP antibody epitope mapping and generated the mouse GFAP-specific antibody to detect GFAP from the samples of the transgenic animal, human patients, and cell-based models of AxD.