以果蠅模式探討TDP-43蛋白在神經退化性疾病所扮演的角色與治療對策

A Drosophila Model to Investigate the Role of TDP-43 in Neurodegenerative Diseases and Its Therapeutic Application

程靜暐(Ching-Wei Cheng)

指導教授 ： 沈哲鯤

國立臺灣大學/醫學院/分子醫學研究所/博士(2015年)

https://doi.org/10.6342/NTU.2015.00496

摘要

TDP-43是一種多功能的RNA / DNA結合蛋白，存在於多種物種中，如哺乳動物和果蠅；在某些類型的肌萎縮性脊髓側索硬化症(ALS-TDP)與額顳葉型失智症病人(FTLD-TDP)中，TDP-43會以異常的形式出現在堆積蛋白內，目前為止，並無有效的藥物可以治療這些神經退化疾病。在此論文中，我利用果蠅TDP-43的同源蛋白－dTDP，建立一套果蠅模型來探討TDP-43的功能，同時作為藥物測試的平台。藉由調控dTDP的表現，發現果蠅個體若喪失dTDP的表現，會嚴重影響其發育以及其運動能力；而在蘑菇體、運動神經元或是PDF（+）神經元中過量表現dTDP，則會出現神經退化的病變，而這些病變和dTDP的含量或是個體年齡成正相關；此外在蘑菇體過量表現dTDP，還會影響學習能力。由此可見dTDP對於個體的發育與神經功能的維持是很重要的。接著，利用‘TARGET’系統建立ALS-TDP果蠅模型，以溫度調控dTDP在運動神經元的表現，進而避免dTDP對發育早期的不良影響。如同ALS病人般，ALS-TDP果蠅的壽命會縮短且運動能力會下降；進一步觀察胸腔神經節的T1/T2區域，發現神經元較正常果蠅少，同時會有dTDP的堆積蛋白形成。因此利用這個ALS-TDP果蠅模型作為藥物測試的平台；我選用一個會活化自噬作用的藥物－雷帕黴素來測試。結果發現高劑量的雷帕黴素會些微改善ALS-TDP果蠅的運動缺陷和存活天數，以及減少含有dTDP堆積蛋白的神經元數目。另外利用此ALS-TDP果蠅模型，還發現S6K是dTDP的genetic modifier。綜合以上結果，意味著TDP-43在神經發育過程中扮演重要角色，若TDP-43失去調控，將導致神經退化疾病。而本論文所建立的ALS-TDP模型，適合用來快速篩選治療ALS新藥的平台；同時亦佐證近幾年的研究，即自噬作用的活化劑，包括雷帕黴素和其衍生物，在治療ALS與TDP-43 proteinopathies是有開發潛力的藥物。

關鍵字

肌萎縮性脊髓側索硬化症 ； 額顳葉型失智症 ； TDP-43蛋白 ； 果蠅模型 ； 果蠅蘑菇體 ； 運動能力 ； 生存曲線

並列摘要

TDP-43 is a multi-functional RNA/DNA-binding protein well-conserved among many species including mammals and Drosophila. However, it is also a major component of the pathological inclusions associated with degenerating motor neurons of amyotrophic lateral sclerosis (ALS-TDP). Further, TDP-43 is a signature protein in one subtype of frontotemporal degeneration, FTLD-TDP. Unfortunately, there are no effective drugs for these neurodegenerative diseases. In this thesis, fly models are generated to explore the function and dysfunction of TDP-43. By both down- and up-regulation of the levels of Drosophila ortholog of TDP-43, dTDP, ubiquitously or in specific cell types/ tissues, the important role of the dTDP protein and by implication the mammalian TDP-43 protein as well, in development and in neuronal functioning are examined. For the latter in particular, I show that dTDP regulates learning ability and locomotion of the fruit flies. As a model of the TDP-43 proteinopathies, flies with overexpression of dTDP, either in the mushroom body, in motor neurons, or in PDF (+) neurons, also exhibit dose-dependent and/or age-dependent pathogenesis behavior.
Next, I establish a fly model of ALS-TDP with transgenic expression of dTDP in adult flies under the control of a temperature sensitive motor neuron-specific GAL4, thus bypassing the deleterious effect of dTDP during development. These ALS-TDP flies also exhibit diminished lifespan as well as locomotor defects following induction of dTDP. Dissection of the T1/T2 region of the thoracic ganglia has revealed loss of the neurons and the formation of dTDP (+) aggregates. Since the ALS-TDP flies exhibit diseased phenotypes, I utilize this model to examine the therapeutic effect of rapamycin, a TOR-dependent autophagy activator. Although harmful to the control flies, administration of 400

並列關鍵字

TDP-43 ； amyotrophic lateral sclerosis ； frontotemporal lobar degeneration ； Drosophila ； lifespan ； locomotor activity ； mushroom body

參考文獻

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