Holmes Brandon B, Furman Jennifer L, Mahan Thomas E, Yamasaki Tritia R, Mirbaha Hilda, Eades William C, Belaygorod Larisa, Cairns Nigel J, Holtzman David M, Diamond Marc I
Department of Neurology, Hope Center for Neurological Disorders, Knight Alzheimer's Disease Research Center, and.
Division of Oncology, Section of Stem Cell Biology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A. 2014 Oct 14;111(41):E4376-85. doi: 10.1073/pnas.1411649111. Epub 2014 Sep 26.
Transcellular propagation of protein aggregates, or proteopathic seeds, may drive the progression of neurodegenerative diseases in a prion-like manner. In tauopathies such as Alzheimer's disease, this model predicts that tau seeds propagate pathology through the brain via cell-cell transfer in neural networks. The critical role of tau seeding activity is untested, however. It is unknown whether seeding anticipates and correlates with subsequent development of pathology as predicted for a causal agent. One major limitation has been the lack of a robust assay to measure proteopathic seeding activity in biological specimens. We engineered an ultrasensitive, specific, and facile FRET-based flow cytometry biosensor assay based on expression of tau or synuclein fusions to CFP and YFP, and confirmed its sensitivity and specificity to tau (∼ 300 fM) and synuclein (∼ 300 pM) fibrils. This assay readily discriminates Alzheimer's disease vs. Huntington's disease and aged control brains. We then carried out a detailed time-course study in P301S tauopathy mice, comparing seeding activity versus histological markers of tau pathology, including MC1, AT8, PG5, and Thioflavin S. We detected robust seeding activity at 1.5 mo, >1 mo before the earliest histopathological stain. Proteopathic tau seeding is thus an early and robust marker of tauopathy, suggesting a proximal role for tau seeds in neurodegeneration.
蛋白质聚集体(即蛋白病种子)的跨细胞传播可能以朊病毒样方式驱动神经退行性疾病的进展。在诸如阿尔茨海默病等tau蛋白病中,该模型预测tau蛋白种子通过神经网络中的细胞间转移在大脑中传播病理状态。然而,tau蛋白播种活性的关键作用尚未得到验证。目前尚不清楚播种是否如对病原体所预测的那样,先于病理状态的后续发展并与之相关。一个主要限制是缺乏一种强大的检测方法来测量生物样本中的蛋白病播种活性。我们基于tau蛋白或突触核蛋白与CFP和YFP的融合表达,设计了一种超灵敏、特异且简便的基于FRET的流式细胞术生物传感器检测方法,并证实了其对tau蛋白(约300 fM)和突触核蛋白(约300 pM)纤维的敏感性和特异性。该检测方法能够轻松区分阿尔茨海默病与亨廷顿病以及老年对照大脑。然后,我们在P301S tau蛋白病小鼠中进行了详细的时间进程研究,比较了播种活性与tau蛋白病理的组织学标志物,包括MC1、AT8、PG5和硫黄素S。我们在1.5个月时检测到了强大的播种活性,比最早的组织病理学染色早1个月以上。因此,蛋白病性tau蛋白播种是tau蛋白病的一个早期且强大的标志物,表明tau蛋白种子在神经退行性变中起近端作用。