Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation
Tau Trimers Are the Minimal Propagation Unit Spontaneously Internalized to Seed Intracellular Aggregation
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DOI：
10.1074/jbc.m115.652693
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发表时间：
2015-06-12
影响因子：
4.8
通讯作者：
Diamond, Marc I.
中科院分区：
文献类型：
--
作者：
Mirbaha, Hilda;Holmes, Brandon B.;Diamond, Marc I.
Tau淀粉样蛋白组合体从细胞外部向细胞内部传播聚集，这可能介导tauopathy的进展。负责这一活动的Tau组件或“种子”的临界大小目前尚不清楚，但这可能对有效疗法的设计很重要。我们研究了从阿尔茨海默病(AD)脑中纯化的重组Tau重复结构域(RD)和Tau组件，这些组件主要由全长Tau组成。首先对大的RD纤维进行超声波处理，以产生一系列的组装尺寸。我们证实了我们有能力通过尺寸排除层析、荧光相关光谱、交联后的Western印迹和质谱分析来拆分从n=1到>；100个单位的稳定的Tau。所有重组Tau组件都与细胞表面的硫酸乙酰肝素蛋白多糖结合，这是吸收和种植Tau所必需的，因为它们对肝素和氯酸盐的抑制同样敏感。然而，细胞只内化n>；=3个单位的RD组装。接下来，我们分析了AD或对照大脑中的Tau组件。AD大脑包含聚集的物种，而正常大脑以单体为主，没有证据表明有大的组装。HEK293细胞和原代神经元以肝素和氯酸盐敏感的方式自发内化AD脑中n>；=3个单位的Tau。只有来自AD脑的n>；=3单元组件自发地在HEK293细胞中种子细胞内的Tau聚集。这些结果表明，Tau凝聚体从细胞外到细胞内的自发繁殖存在一个明显的最小尺寸(n=3)的Tau种子，而许多较大尺寸的可溶性聚集体则触发了吸收和播种。
Tau amyloid assemblies propagate aggregation from the outside to the inside of a cell, which may mediate progression of the tauopathies. The critical size of Tau assemblies, or "seeds," responsible for this activity is currently unknown, but this could be important for the design of effective therapies. We studied recombinant Tau repeat domain (RD) and Tau assemblies purified from Alzheimer disease (AD) brain composed largely of full-length Tau. Large RD fibrils were first sonicated to create a range of assembly sizes. We confirmed our ability to resolve stable assemblies ranging from n = 1 to > 100 units of Tau using size exclusion chromatography, fluorescence correlation spectroscopy, cross-linking followed by Western blot, and mass spectrometry. All recombinant Tau assemblies bound heparan sulfate proteoglycans on the cell surface, which are required for Tau uptake and seeding, because they were equivalently sensitive to inhibition by heparin and chlorate. However, cells only internalized RD assemblies of n >= 3 units. We next analyzed Tau assemblies from AD or control brains. AD brains contained aggregated species, whereas normal brains had predominantly monomer, and no evidence of large assemblies. HEK293 cells and primary neurons spontaneously internalized Tau of n >= 3 units from AD brain in a heparin-and chlorate-sensitive manner. Only n >= 3-unit assemblies from AD brain spontaneously seeded intracellular Tau aggregation in HEK293 cells. These results indicate that a clear minimum size (n = 3) of Tau seed exists for spontaneous propagation of Tau aggregation from the outside to the inside of a cell, whereas many larger sizes of soluble aggregates trigger uptake and seeding.