- 學位論文
探討UQCRC1於神經退化性疾病模式之調控機制
Molecular investigation of UQCRC1 in a disease model of neurodegeneration
摘要
UQCRC1 (Ubiquinol-Cytochrome C Reductase Core Protein 1)是粒線體中電子傳遞鍊上的complex III其中一個次單元蛋白，先前多將其視為粒線體標記，偶有研究提及UQCRC1和神經退化性疾病間的關聯，但多為DNA/RNA階段。近年來開始亦有研究探討UQCRC1蛋白於神經細胞中的功能性。與本實驗室合作之林靜嫻醫師在先前針對遺傳性帕金森氏症患者及其家族進行全外顯子定序(whole exome sequence，WES)檢測，試圖發現與帕金森氏症相關之未知基因，而從其中發現UQCRC1單位點變異可能與帕金森氏症有關。先前對於UQCRC1於細胞之功能了解甚微，因此本研究希望以果蠅與人類神經細胞為模式，探討UQCRC1於粒線體或於神經細胞中所扮演之角色。從實驗中發現，於果蠅神經系統減少UQCRC1同源異構物uqcrc1，會導致果蠅爬行能力降低和多巴胺分泌神經數量減少，與人類帕金森氏症之病之類似；且發現完全剔除uqcrc1會導致果蠅於發育階段即死亡，可能是由於細胞凋亡(apoptosis)的發生。這些因缺少uqcrc1而導致的現象，都可因重新表現UQCRC1而減緩，表示人類與果蠅於該蛋白有些許功能方面之保守性。此外，我們亦發現減少uqcrc1會導致cyt-c移動至細胞質中，在先前研究也認為cyt-c從粒線體移至細胞質會與細胞凋亡的活化起始相關。後續實驗中，減少cyt-c表現量亦或表現細胞凋亡抑制因子p35都可減緩由於失去uqcrc1造成的神經退化現象。由此可知，UQCRC1或有調控cyt-c影響細胞凋亡發生之作用。此外，我們發現失去uqcrc1也會導致autophagy相關蛋白表現增加，且可能會與PINK1共同調控autophagy的進行。最後，我們還看到糖解作用酵素表現會受uqcrc1或cyt-c影響，可能是以此維持細胞中能量的生成。
並列摘要
Ubiquinol-cytochrome c reductase core protein 1 (UQCRC1) is an evolutionarily conserved subunit of complex III in the mitochondrial respiratory chain. We have recently identified from patients using whole-exon sequencing (WES) that the mutations in UQCRC1 are associated with familial Parkinsonism. However, its cellular role remains unclear. To reveal its endogenous function, we investigated through the Drosophila nervous system and human neuron cell line. Neuronal knockdown of fly uqcrc1 causes age-dependent Parkinsonism-assembling deficits including locomotor decline and dopaminergic neuron reduction and could be rescued by UQCRC1 expression. Knockout of uqcrc1 causes lethality during Drosophila development and also induces morphologic and functional mitochondria defects in larval brain and muscle. The lethality is also rescued by neuronal expression of UQCRC1, but not the disease-associated mutation. Finally, we found that knockdown of uqcrc1 causes cyt-c release to cytoplasmic fraction and activates the caspase cascade for apoptosis. Regulation of cyt-c and apoptosis inhibitor p35 ameliorates uqcrc1-mediated neurodegeneration. Our data identify the endogenous function of UQCRC1 in the regulation of cyt-c for apoptosis.