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標題: | 突變果蠅支鏈甲型酮酸脫氫複合物導致肌肉以及神經性損傷 Mutations of the Drosophila branched-chain α-keto acid dehydrogenase complex (BCKDH) result in muscular and neuronal dysfunctions |
作者: | Hui-Ying Tsai 蔡惠穎 |
指導教授: | 陳俊宏(Chun-Hong Chen) |
關鍵字: | 楓糖尿症,支鏈胺基酸,支鏈甲型酮酸脫氫複合物缺乏,神經性退化,果蠅,二甲雙胍, Maple Syrup Urine Disease,BCKDH deficiency,Drosophila melanogaster,BCAA degradation pathway,Neurodegeneration,Metformin, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 楓糖尿症為罕見的代謝性胺基酸異常疾病,由於患者缺乏支鏈胺基酸代謝酵素-支鏈甲型酮酸脫氫複合物(BCKDH),導致過量的支鏈胺基酸(BCAAs)累積在血液中,進而對神經系統造成傷害。此病在台灣的發病率為十萬分之一,在嬰兒時期就會發病,如果未立即處理將會有死亡的風險。目前治療方法有兩種,一為飲食控制,避免攝入過多的支鏈胺基酸以減少體內過多的累積,二為嚴重者需要進行肝臟移植手術。本篇研究中,我們以果蠅為模式生物,將參與支鏈胺基酸代謝路徑中的五個酵素基因分別進行剔除。支鏈甲型酮酸脫氫複合物(BCKDH)包含四個亞基E1α、E1β、E2和E3分別對應到果蠅基因的CG8199、CG17691、CG5599和CG7430,此外路徑上游的支鏈胺基酸轉移酶(BCAT)針對基因則是CG1673,也將進行基因剔除。實驗結果顯示,支鏈胺基酸的代謝地點在肌肉,不同於其他胺基酸在肝臟代謝。表現量在肌肉較高的是E1α、E2、E3,表示著這三個是主要代謝支鏈胺基酸的亞基,而其中又以E3表現量最高,被認為是整個代謝路徑中最主要的基因。另外,確認了在五株基因剔除果蠅中支鏈胺基酸會上升之後,發現脂肪細胞的脂滴大小也會改變,顯示支鏈胺基酸的累積也會改變著脂肪的代謝。此外,我們利用視網膜電圖(ERG),紀錄在果蠅視神經在光刺激下電位的變化,E2和E3的變異株在神經傳導的功能上有明顯的受損,若解開導致神經性退化的機制,將對解決楓糖尿症嬰兒大腦受到支鏈胺基酸堆積是一大里程碑。此外,我們利用一種降血糖藥物-二甲雙胍進行測試,並且看到幼蟲體內支鏈胺基酸含量以及爬行能力有一定程度的恢復,在未來能進行更多的測試,為楓糖尿症帶來藥物治療的契機。 Maple Syrup Urine Disease (MSUD) is a rare autosomal recessive inherited disease caused by dysfunctions of the branched-chain α-keto-acid dehydrogenase (BCKDH) complex; this complex is involved in the branched-chain amino acid (BCAA) degradation pathway. Although it has been reported that patients with MSUD suffer from neuronal injuries, the causes are currently unknown. In order to investigate the underlying mechanisms of MSUD, we generated Drosophila melanogaster BCKDH enzyme and BCAA degradation pathway mutants using CRISPR-Cas9 techniques. The BCKDH enzyme consists of three components, each of which was targeted for mutation; alpha-keto acid decarboxylase (E1/ composed of the sub-units α (CG8199) and β (CG17691)), dihydrolipoyl transacylase (E2/CG5599) and dihydrolipoamide dehydrogenase (E3/CG7430). Branched-chain amino acid aminotransferase (BCAT), which is upstream of the BCKDH complex, was also targeted (CG1673). We found that BCAAs accumulated and lipid droplet (LD) sizes changed in mutant lines as compared to controls. Furthermore, we used GMR Gal4 to drive RNAi for each of the five genes and found that changes to rhabdomere morphology were only observed in CG5599 and CG7430 RNAi lines, indicating these two genes may be the most important for neuronal development. Using a Drosophila electroretinogram (ERG) to examine mutant physiology, we found that CG5599 and CG7430 RNAi lines lacked on- and off- transients and displayed defective depolarization, indicating that they had a neurodegenerative phenotype. Our results suggest that dihydrolipoyl transacylase and dihydrolipoamide dehydrogenase may be the most promising targets for further research into MSUD associated chronic neuropsychiatric symptoms. Moreover, we treated Metformin in this Drosophila model by detecting BCAAs level and larval locomotor, finding that an extent of rescuing. These results indicated that Metformin probably could be a potential drug for curing MSUD. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78663 |
DOI: | 10.6342/NTU201902658 |
全文授權: | 有償授權 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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