Lee In-Hwan, Kim Hui-Young, Kim Myunghye, Hahn Ji-Sook, Paik Seung R
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151-744, Republic of Korea.
Neurochem Res. 2008 Jul;33(7):1393-400. doi: 10.1007/s11064-008-9598-8. Epub 2008 Mar 7.
Intracellular toxic effects of the dequalinium-induced protofibrils of alpha-synuclein have been investigated with the yeast system expressing alpha-synuclein-GFP fusion protein in single copy, which appears in the green halo around the plasma membrane. Intracellular responses of the green fluorescent protein were analyzed as the cells were treated with dequalinium (DQ) and lactacystin. Yeast cells expressing alpha-synuclein-GFP were susceptible to both compounds in alpha-synuclein-dependent manner. Upon DQ treatment, the green halo became smeared throughout the cytoplasm while lactacystin induced a few discrete green dots, reflecting intracellular formation of the protofibrils and the protein inclusions, respectively. The DQ-treated yeast cells were intensely stained with the nucleic acid stains of cell-permeable Hoechst 33342 and cell-impermeable propidium imidione, indicating that nucleus has been disrupted in addition to plasma membrane destabilization. Those DQ-treated yeast cells, however, still contained active mitochondria identified with MitoTracker Red. Therefore, the DQ-induced protofibrillar state of alpha-synuclein-GFP has been suggested to cause the nuclear damage either independently or in combination with the membrane destabilization without affecting mitochondria.
利用在单拷贝中表达α-突触核蛋白-GFP融合蛋白的酵母系统，研究了地喹氯铵诱导的α-突触核蛋白原纤维的细胞内毒性作用，该融合蛋白出现在质膜周围的绿色光晕中。在用去甲喹啉（DQ）和乳胞素处理细胞时，分析了绿色荧光蛋白的细胞内反应。表达α-突触核蛋白-GFP的酵母细胞对这两种化合物均敏感，且呈α-突触核蛋白依赖性。用DQ处理后，绿色光晕在整个细胞质中变得模糊，而乳胞素诱导出一些离散的绿色斑点，分别反映了原纤维和蛋白质内含物的细胞内形成。用可透过细胞的Hoechst 33342和不可透过细胞的碘化丙啶进行核酸染色，结果显示DQ处理的酵母细胞被强烈染色，这表明除了质膜不稳定外，细胞核也受到了破坏。然而，那些用DQ处理的酵母细胞仍然含有用MitoTracker Red鉴定的活性线粒体。因此，有人认为，DQ诱导的α-突触核蛋白-GFP原纤维状态会独立或与膜不稳定共同导致核损伤，而不影响线粒体。
