Abstract：
Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer\'s disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.
摘要：
tau蛋白的聚集体是几种神经退行性疾病的众所周知的标志，统称为tau病，包括额叶颞叶痴呆和阿尔茨海默病(AD)。监测tau从生理单体到病理寡聚体或聚集体的高通量转化过程，定量方式和在细胞环境中仍然是该领域的主要挑战。鉴定能够干扰那些过程的分子具有高度的治疗兴趣。这里,我们开发了一系列的分子间和分子内tau生物传感器的基础上，高度敏感的纳米亮氨酸酶（Nluc）二元技术（NanoBiT），能够监测病理构象变化和tau在活细胞的自我相互作用。我们的tau生物传感器库可靠地报告了i.微管处生理全长tau的分子接近度；ii.与tau磷酸化相关的tau构象和自我相互作用的变化，以及iii。由重组tau的种子或来自tau病理学小鼠模型的小鼠脑裂解物诱导的tau相互作用。通过比较包含不同tau形式（即全长或短片段，野生型,或疾病相关tau（P301L）变体）进一步了解tau转化过程。提出了用于高通量适用性和鉴定干扰病理性tau转化过程的分子的概念证明数据。这种新型tau生物传感器旨在促进活细胞中病理性tau转化的分子机制的公开，并发现tau相关神经退行性疾病的新药候选物。
