Bandyopadhyay Bhaswati, Li Guibin, Yin Haishan, Kuret Jeff
Center for Molecular Neurobiology, the Department of Molecular and Cellular Biochemistry, Ohio State University, Columbus 43210, USA.
J Biol Chem. 2007 Jun 1;282(22):16454-64. doi: 10.1074/jbc.M700192200. Epub 2007 Apr 11.
Intracellular aggregation of the microtubule-associated protein tau into filamentous inclusions is a defining characteristic of Alzheimer disease. Because appearance of tau-aggregate bearing lesions correlates with both cognitive decline and neurodegeneration, it has been hypothesized that tau aggregation may be directly toxic to cells that harbor them. Testing this hypothesis in cell culture has been complicated by the resistance of full-length tau isoforms to aggregation over experimentally tractable time periods. To overcome this limitation, a small-molecule agonist of the tau aggregation reaction, Congo red, was used to drive aggregation within HEK-293 cells expressing full-length tau isoform htau40. Formation of detergent-insoluble aggregates was both time and agonist concentration dependent. At 10 microM Congo red, detergent-insoluble aggregates appeared with pseudo-first order kinetics and a half-life of approximately 5 days. By 7 days in culture, total tau levels increased 2-fold, with approximately 30% of total tau converted into detergent-insoluble aggregates. Agonist addition also led to rapid losses in the tubulin binding activity of tau, although tau was not hyperphosphorylated as judged by occupancy of phosphorylation sites Ser396/Ser404. Tau aggregation was associated with decreased viability as detected by ToPro-3 uptake. The results, which establish a new approach for analysis of tau aggregation in cells independent of tau hyperphosphorylation, suggest that conformational changes associated with aggregation are incompatible with microtubule binding, and that toxicity associated with intracellular tau aggregation is not acute but develops over a period of days.
微管相关蛋白tau在细胞内聚集成丝状内含物是阿尔茨海默病的一个决定性特征。由于携带tau聚集物的病变出现与认知能力下降和神经退行性变均相关，因此有人提出tau聚集可能对含有它们的细胞具有直接毒性。在细胞培养中测试这一假设一直很复杂，因为全长tau异构体在实验可控的时间段内难以聚集。为了克服这一限制，使用tau聚集反应的小分子激动剂刚果红来驱动表达全长tau异构体htau40的HEK-293细胞内的聚集。去污剂不溶性聚集体的形成既依赖时间也依赖激动剂浓度。在10 microM刚果红存在下，去污剂不溶性聚集体以准一级动力学出现，半衰期约为5天。培养7天时，总tau水平增加了2倍，约30%的总tau转化为去污剂不溶性聚集体。添加激动剂还导致tau的微管蛋白结合活性迅速丧失，尽管根据磷酸化位点Ser396/Ser404的占据情况判断tau没有过度磷酸化。通过ToPro-3摄取检测发现，tau聚集与细胞活力下降有关。这些结果建立了一种独立于tau过度磷酸化分析细胞内tau聚集的新方法，表明与聚集相关的构象变化与微管结合不相容，并且细胞内tau聚集相关的毒性不是急性的，而是在数天内逐渐发展的。
