Development of new fusion proteins for visualizing amyloid-β oligomers in vivo.
Development of new fusion proteins for visualizing amyloid-β oligomers in vivo.
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DOI：
10.1038/srep22712
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发表时间：
2016-03-16
影响因子：
4.6
通讯作者：
Shimura H
中科院分区：
文献类型：
--
作者：
Ochiishi T;Doi M;Yamasaki K;Hirose K;Kitamura A;Urabe T;Hattori N;Kinjo M;Ebihara T;Shimura H
β-淀粉样蛋白（A β）寡聚体在细胞内的积聚是阿尔茨海默病（AD）疾病进展的关键因素，可能成为AD治疗的潜在靶点。在体内直接观察A β寡聚体的分子动力学是药物发现研究的关键，然而，由于A β聚集抑制融合蛋白的荧光，因此具有挑战性。在这里，我们开发了A β 1 - 42-GFP融合蛋白，这些融合蛋白是寡聚化的，即使在聚集时也可以观察到它们在细胞内的动力学。我们使用几种方法检查了A β-GFP融合蛋白的聚集状态，并证实它们没有组装成原纤维，而是在体外和活细胞中形成寡聚体。通过排列A β和GFP之间连接体的长度，我们得到了两种具有"长连接体"和"短连接体"的融合蛋白，并利用A β-GFP质粒转染的原代培养大鼠神经元和A β-GFP转基因C.优美的我们发现A β-GFP融合蛋白诱导COS 7细胞死亡。这些结果表明，新型A β-GFP融合蛋白可用于研究A β寡聚体在活细胞和动物体内的生理功能，以及通过分析A β毒性进行药物筛选。
The intracellular accumulation of amyloid-β (Aβ) oligomers critically contributes to disease progression in Alzheimer’s disease (AD) and can be the potential target of AD therapy. Direct observation of molecular dynamics of Aβ oligomers in vivo is key for drug discovery research, however, it has been challenging because Aβ aggregation inhibits the fluorescence from fusion proteins. Here, we developed Aβ1-42-GFP fusion proteins that are oligomerized and visualize their dynamics inside cells even when aggregated. We examined the aggregation states of Aβ-GFP fusion proteins using several methods and confirmed that they did not assemble into fibrils, but instead formed oligomers in vitro and in live cells. By arranging the length of the liker between Aβ and GFP, we generated two fusion proteins with “a long-linker” and “a short-linker”, and revealed that the aggregation property of fusion proteins can be evaluated by measuring fluorescence intensities using rat primary culture neurons transfected with Aβ-GFP plasmids and Aβ-GFP transgenic C. elegans. We found that Aβ-GFP fusion proteins induced cell death in COS7 cells. These results suggested that novel Aβ-GFP fusion proteins could be utilized for studying the physiological functions of Aβ oligomers in living cells and animals, and for drug screening by analyzing Aβ toxicity.