建立斑馬魚成為Tauopathy研究與藥物篩選之模式動物

Establishment of zebrafish as an animal model for Tauopathy research and drug screening

吳百凱(Bo-Kai Wu)

指導教授 ： 張俊哲

共同指導教授 ： 黃銓珍(Chang-Jen Huang)

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2016年)

https://doi.org/10.6342/NTU201600979

摘要

Tau蛋白是一個微管結合蛋白，其主要的工作在於維持微管的形成與穩定。當tau基因產生突變時，會伴隨產生家族性的第17號染色體與額顳葉性癡呆合併巴金森氏症 (frontotemporal dementia with Parkinsonism linked to chromosome-17, FTDP-17)。而在阿茲海默症 (Alzheimer’s disease, AD) 的患者腦部中，通常發現在細胞內會有由磷酸化Tau蛋白所組成的神經纖維糾結以及在細胞間會有β-類澱粉蛋白沈積。為了瞭解Tau蛋白在個體層次之功能，在轉基因的動物模式是非常重要的。而在這之中，包含斑馬魚等模式生物，也已被用於研究由Tau蛋白所導致的神經退化機制。在此篇的研究，我們利用專一表現在神經系統的HuC啟動子，來表現人類跟斑馬魚Tau螢光融合蛋白，建立一套Tau螢光蛋白瞬時表現系統。相較於果蠅及老鼠等模式生物，此系統可藉由活體影像來追蹤Tau螢光蛋白所造成的高度神經元細胞死亡。而為了想要降低Tau螢光蛋白所造成的神經細胞死亡，我們發現在表現Bcl2-L1，Nrf2以及GDNF等訊息因子時，能有效地保護神經細胞免於Tau螢光蛋白所引起的死亡。在使用抗氧化反應或促進神經營養作用等化合物之下，同樣也能產生類似的神經保護作用，並且使人類的Tau螢光蛋白維持在磷酸化的狀態下，同時可以被pT212以及AT8等抗體偵測到。於是，我們接著便利用此系統去篩選了400種的中草藥萃取物，並在其中找到了45種萃取物能有效降低Tau螢光蛋白所造成的神經元細胞死亡。雷公藤 (Tripterygium wilfordii) 便是其中一個有效的草本萃取物。在透過高效能液相層析法 (HPLC) 分析以及功能測試上，證明了表兒茶素 (epicatechin, EC) 是雷公藤莖部萃取物中能降低Tau螢光蛋白所造成神經毒性的主要化合物。ARE (antioxidant response elements)-luciferase reporter gene assay主要用於偵測Nrf2的活性，而利用此實驗，我們可以在斑馬魚活體之中，確認表兒茶素可以增加Nrf2的活性。以上這些研究結果顯示，雷公藤莖部萃取物中表兒茶素具由降低Tau螢光蛋白所造成的神經細胞死亡的功用，是藉由透過活化Nrf2來達成。

關鍵字

tauopathy ； Bcl-2 ； Nrf2 ； GDNF ； 神經毒性 ； 斑馬魚 ； 表兒茶素 ； 雷公藤

並列摘要

Tau protein is a tubulin-binding protein, which plays important roles in the formation and stability of the microtubule. Mutations in the tau gene are associated with familial forms of frontotemporal dementia with Parkinsonism linked to chromosome-17 (FTDP-17). Neurofibrillary tangles (NFTs) of Tau and extracellular plaques containing amyloid-β (Aβ) are found in the brain of Alzheimer’s disease (AD) patients. Transgenic models, including those of zebrafish, have been employed to elucidate the mechanisms by which Tau protein causes neurodegeneration. In this study, a transient expression system was established to express GFP fusion proteins of zebrafish and human Tau under the control of a neuron-specific HuC promoter. The expression of Tau-GFP was observed to cause high levels of neuronal death which could be directly traced in vivo. Multiple signaling factors, such as Bcl2-L1, Nrf2, and GDNF, were found to effectively protect neuronal cells expressing Tau-GFP from death. Treatment with compounds that induce anti-oxidative or neurotrophic effects also resulted in a similar neuronal protective effect and maintained human Tau-GFP protein in a phosphorylated state, as detected by antibodies pT212 and AT8. Therefore, we used this model to screen 400 herbal extracts and found 45 of them to be effective on reducing Tau-GFP-induced neuronal death. One of the effective herbal extracts is the Tripterygium wilfordii. HPLC analysis and functional assay demonstrated that epicatechin (EC) is the major compound of Tripterygium wilfordii stem extract to decrease the neurotoxicity induced by Tau-GFP. ARE (antioxidant response elements)-luciferase reporter gene assay is usually used to detect the activity of Nrf2. We used the assay to demonstrate that EC could increase the activity of Nrf2 in zebrafish. These data suggest that EC from the Tripterygium wilfordii stem extract could diminish Tau-GFP-induced neuronal death through the activation of Nrf2.

並列關鍵字

tauopathy ； Bcl-2 ； Nrf2 ； GDNF ； neurotoxicity ； zebrafish ； epicatechin ； Tripterygium wilfordii

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