Although several studies demonstrate prion-like properties of Tau fibrils, the effect of size in the seeding capacity of these aggregates is not fully understood. The aim of this study is to characterize Tau seeds by their size and seeding capacity. Tau aggregates were isolated from postmortem AD brain tissue and separated from low molecular weight species by sucrose gradient ultracentrifugation. Biochemical characterization of the different fractions was done by non-reducing Western blotting and aggregate-specific immuno-assays using in house developed anti-Tau monoclonal antibodies, including PT76 which binds to an epitope close to the microtubule-binding domain and, hence, also to K18. Seeding efficiency was then assessed in HEK293 cells expressing K18 FRET sensors. We observed that upon sonication of Tau aggregates different size-distributed tau aggregates are obtained. In biochemical assays, these forms show higher signals than the non-sonicated material in some aggregation-specific Tau assays. This could be explained by an increased epitope exposure of the smaller aggregates created by the sonication. By analyzing human brain derived and recombinant (K18) Tau aggregates in a cellular FRET assay, it was observed that, in the absence of transfection reagent, sonicated aggregates showed higher aggregation induction. Preparations also showed altered profiles on native PAGE upon sonication and we could further separate different aggregate species based on their molecular weight via sucrose gradients. This study further elucidates the molecular properties regarding relative aggregate size and seeding efficiency of sonicated vs. non-sonicated high molecular weight Tau species. This information will provide a better knowledge on how sonication, a commonly used technique in the field of study of Tau aggregation, impacts the aggregates. In addition, the description of PT76-based aggregation specific assay is a valuable tool to quantify K18 and human AD Tau fibrils.

中文翻译：

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体外和体内Tau原纤维的大小分布和（Pro249-Ser258）表位暴露的比较

尽管几项研究表明Tau原纤维具有病毒样性质，但这些聚集体的接种量对大小的影响尚不完全清楚。这项研究的目的是通过大小和播种能力来表征Tau种子。从死后AD脑组织中分离出Tau聚集体，并通过蔗糖梯度超速离心从低分子量物质中分离出Tau聚集体。通过内部研发的抗Tau单克隆抗体（包括与结合在微管结合域附近的表位结合的PT76）进行的非还原Western印迹和聚集体特异性免疫测定，对不同部分进行了生化表征。到K18。然后评估表达K18 FRET传感器的HEK293细胞的播种效率。我们观察到，在超声处理Tau聚集体时，会获得不同大小的tau聚集体。在生化测定中，在某些聚集特异性Tau测定中，这些形式显示出比未超声处理的物质更高的信号。这可以通过超声处理产生的较小聚集体的表位暴露增加来解释。通过在细胞FRET分析中分析人脑衍生和重组（K18）Tau聚集体，可以观察到，在不存在转染试剂的情况下，超声处理的聚集体显示出更高的聚集诱导作用。超声处理后，天然PAGE上的制剂也显示出变化的轮廓，我们可以根据其分子量通过蔗糖梯度进一步分离不同的聚集物种。这项研究进一步阐明了相对于未超声处理的高分子量Tau菌种的相对聚集体大小和接种效率的分子特性。此信息将提供有关Tau聚集研究领域中常用的超声处理如何影响聚集体的更好知识。此外，基于PT76的聚集特异性测定的描述是定量K18和人AD Tau原纤维的有价值的工具。