Liu Xiao-Ling, Hu Ji-Ying, Hu Meng-Yun, Zhang Yi, Hong Zheng-Yuan, Cheng Xiao-Qing, Chen Jie, Pang Dai-Wen, Liang Yi
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China.
Biochim Biophys Acta. 2015 Aug;1852(8):1561-73. doi: 10.1016/j.bbadis.2015.04.015. Epub 2015 Apr 22.
A pathological hallmark of Alzheimer disease (AD) is the accumulation of misfolded hyperphosphorylated microtubule-associated protein Tau within neurons, forming neurofibrillary tangles and leading to synaptic dysfunction and neuronal death. Here we study sequence-dependent abnormal aggregation of human fragment Tau244-372 in an inducible cell model. As evidenced by confocal laser scanning microscopy, Western blot, and immunogold electron microscopy, fibril-forming motifs are essential and sufficient for abnormal aggregation of Tau244-372 in SH-SY5Y neuroblastoma cells induced by Congo red: when its two fibril-forming segments PHF6 and PHF6* are deleted, Tau244-372 does lose its ability to form fibrils in SH-SY5Y cells, and the replacement of PHF6 and PHF6* with an unrelated amyloidogenic sequence IFQINS from human lysozyme does rescue the fibril-forming ability of Tau244-372 in SH-SY5Y cells. By contrast, insertion of a non-fibril forming peptide GGGGGG does not drive the disabled Tau244-372 to misfold in SH-SY5Y cells. Furthermore, as revealed by quantum dots based probes combined with annexin V staining, annexin V-FITC apoptosis detection assay, and immunofluorescence, fibril-forming motifs are essential and sufficient for early apoptosis of living SH-SY5Y cells induced by abnormal aggregation of Tau244-372. Our results suggest that fibril-forming motifs could be the determinants of Tau protein tending to misfold in living cells, thereby inducing neuronal apoptosis and causing the initiation and development of AD.
阿尔茨海默病（AD）的一个病理特征是神经元内错误折叠的高磷酸化微管相关蛋白Tau的积累，形成神经原纤维缠结，并导致突触功能障碍和神经元死亡。在此，我们在一个可诱导细胞模型中研究人Tau244 - 372片段的序列依赖性异常聚集。共聚焦激光扫描显微镜、蛋白质免疫印迹和免疫金电子显微镜结果表明，纤维形成基序对于刚果红诱导的SH - SY5Y神经母细胞瘤细胞中Tau244 - 372的异常聚集至关重要且足够：当它的两个纤维形成片段PHF6和PHF6被删除时，Tau244 - 372在SH - SY5Y细胞中确实失去了形成纤维的能力，而用人溶菌酶中一个不相关的淀粉样生成序列IFQINS替换PHF6和PHF6确实挽救了Tau244 - 372在SH - SY5Y细胞中的纤维形成能力。相比之下，插入一个非纤维形成肽GGGGGG不会促使失活的Tau244 - 372在SH - SY5Y细胞中错误折叠。此外，基于量子点的探针结合膜联蛋白V染色、膜联蛋白V - FITC凋亡检测分析和免疫荧光结果表明，纤维形成基序对于Tau244 - 372异常聚集诱导的活SH - SY5Y细胞早期凋亡至关重要且足够。我们的结果表明，纤维形成基序可能是Tau蛋白在活细胞中倾向于错误折叠的决定因素，从而诱导神经元凋亡并导致AD的发生和发展。
