Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF.
Ultrasensitive tau biosensor cells detect no seeding in Alzheimer's disease CSF.
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DOI：
10.1186/s40478-021-01185-8
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发表时间：
2021-05-26
影响因子：
7.1
通讯作者：
Diamond MI
中科院分区：
文献类型：
--
作者：
Hitt BD;Vaquer-Alicea J;Manon VA;Beaver JD;Kashmer OM;Garcia JN;Diamond MI
Tau蛋白形成自我复制组装（种子），可能是阿尔茨海默病（AD）和相关Tau病病理进展的基础。重组蛋白制剂和脑匀浆中的播种已经用“生物传感器”细胞系进行了量化，这些细胞系表达带有疾病相关突变（P301S）融合到互补荧光蛋白上的tau。通过荧光共振能量转移（FRET）评分阳性的细胞中诱导聚集的定量是通过细胞成像或流式细胞术完成的。一些研究小组使用各种方法报道了死前脑脊液（CSF）中的种子活性，但这些发现尚未得到广泛复制。为了解决这个问题，我们基于tau表达创建了两种改进的基于fret的生物传感器细胞系，称为版本2低（v2L）和版本2高（v2H）。我们确定v2H细胞对AD衍生的tau种子的敏感性比我们的原始细胞系高100倍，并且结合免疫沉淀可靠地检测到含有100原子的重组tau原纤维或来自AD脑匀浆的32 pg总蛋白的样品中的种子。我们检测了11名临床诊断为AD的受试者的死前脑脊液，其中9名经脑脊液生物标志物证实。我们在v2H细胞中使用免疫沉淀结合种子检测，在任何样本中均未检测到tau种子。因此，我们无法证实先前关于AD患者脑脊液中tau种子活性的报道。尽管如此，下一代超灵敏的tau生物传感器可能对研究界有用，以尽可能敏感和特异性地量化tau病理学。在线版本包含补充材料，可在10.1186/s40478-021-01185-8获得。
Tau protein forms self-replicating assemblies (seeds) that may underlie progression of pathology in Alzheimer’s disease (AD) and related tauopathies. Seeding in recombinant protein preparations and brain homogenates has been quantified with “biosensor” cell lines that express tau with a disease-associated mutation (P301S) fused to complementary fluorescent proteins. Quantification of induced aggregation in cells that score positive by fluorescence resonance energy transfer (FRET) is accomplished by cell imaging or flow cytometry. Several groups have reported seeding activity in antemortem cerebrospinal fluid (CSF) using various methods, but these findings are not yet widely replicated. To address this question, we created two improved FRET-based biosensor cell lines based on tau expression, termed version 2 low (v2L) and version 2 high (v2H). We determined that v2H cells are ~ 100-fold more sensitive to AD-derived tau seeds than our original lines, and coupled with immunoprecipitation reliably detect seeding from samples containing as little as 100 attomoles of recombinant tau fibrils or ~ 32 pg of total protein from AD brain homogenate. We tested antemortem CSF from 11 subjects with a clinical diagnosis of AD, 9 confirmed by validated CSF biomarkers. We used immunoprecipitation coupled with seed detection in v2H cells and detected no tau seeding in any sample. Thus we cannot confirm prior reports of tau seeding activity in the CSF of AD patients. This next generation of ultra-sensitive tau biosensors may nonetheless be useful to the research community to quantify tau pathology as sensitively and specifically as possible. The online version contains supplementary material available at 10.1186/s40478-021-01185-8.
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影响因子：
4.3
作者：
DeVos SL;Corjuc BT;Oakley DH;Nobuhara CK;Bannon RN;Chase A;Commins C;Gonzalez JA;Dooley PM;Frosch MP;Hyman BT
通讯作者：
Hyman BT
改善绿色和红色荧光蛋白的亮度和光稳定性，用于活细胞成像和FRET报告。
影响因子：
4.6
作者：
Bajar BT;Wang ES;Lam AJ;Kim BB;Jacobs CL;Howe ES;Davidson MW;Lin MZ;Chu J
通讯作者：
Chu J
影响因子：
12.7
作者：
Cicognola, Claudia;Brinkmalm, Gunnar;Hoglund, Kina
通讯作者：
Hoglund, Kina
影响因子：
1.2
作者：
Furman, Jennifer L.;Holmes, Brandon B.;Diamond, Marc I.
通讯作者：
Diamond, Marc I.
影响因子：
29
作者：
Gibbons GS;Lee VMY;Trojanowski JQ
通讯作者：
Trojanowski JQ