Molecular characterization of extracellular vesicles for the spread of misfolded tau protein

神经元缠结，由细胞内过度磷酸化的微管相关蛋白聚集体组成。 蛋白tau（tau）是迄今为止与阿尔茨海默病（AD）临床症状最相关的病理学。 新出现的证据表明，细胞外囊泡（EV，如外来体和微泡），转移 病理性tau蛋白作为载体在细胞之间传播，传播tau蛋白病理。当务之急是找到 脑源性EV的分子基础，这可能是关键的调节EV神经元的摄取和聚集 EV和/或受体神经元中的tau蛋白。我们最近已经建立了分离EV的方法， 人类大脑样本，并成功地进行了他们的蛋白质组学分析。我们发现选择性分子 能够区分人类AD-EV与健康对照（CTRL）-EV， 通过机器学习分析，准确率达到88%，确认了AD-EV分子的致病特征。 此外，我们令人兴奋的初步数据表明，AD-EV具有显著更高的tau播种， 通过FRET传感器tau接种测定与CTRL-EV相比的活性，其中EV分子的子集显示 与tau播种活性显著相关。本拟议项目将巩固这些初步成果， 在介导tau聚集及其播种的tau蛋白病中发现会聚或特异性机制 通过蛋白质组学和脑源性EV样品的生物学检查来进行EV摄取。满足这一 挑战，我们组建了一个多学科的调查小组，他们有着良好的记录， 在生物学（Ikezu）、蛋白质组学（Emili）和生物工程和生物信息学分析（Issador）方面取得了成就。 在目的1中，我们将检测来自240个新脑标本（AD 40个，CTE 40个，LBD 40个，PSP 40个， CBD和40 CTRL）用于基于质谱的蛋白质组学和tau相互作用，并分析 通过机器学习的方法来处理这些数据集。Aim 2将使用以下方法检查tau传播的效率： 从5种不同tau蛋白病的相同供体和对照病例中分离的tau原纤维、寡聚体和EV， 使用基于FRET的tau接种试验和原代培养小鼠皮质EV摄取的体外和体内研究 体外培养的神经元。EV相关的tau蛋白将进一步通过生物化学和基于显微镜的特征来表征。 分析它们的构象和翻译后变化。我们将评估tau的差异 使用我们最近的研究， 建立小鼠模型。Aim 3将鉴定最可能参与EV摄取和tau蛋白的候选分子。 通过对蛋白质组数据集（Aim 1）和生物数据集（Aim 2）的生物信息学分析确定接种活性。我们 然后将测试所鉴定的分子对EV摄取、tau播种活性和神经元的功能作用。 在体外的发射活动。候选分子将被基因沉默或拮抗剂特异性靶向 因为它们在体外和体内阻止tau增殖的治疗潜力。成功识别责任人 tau传播的分子将作为理解EV介导的疾病进展的基础。

Neurofibrillary tangles, composed of intracellular aggregates of hyperphosphorylated microtubule-associated protein tau (tau), are by far the most correlated pathology for clinical symptoms of Alzheimer disease (AD). Emerging evidence suggests that extracellular vesicles (EVs, such as exosomes and microvesicles), transfer pathological tau between cells as vehicles, propagating tau pathology. It is urgently important to find the molecular basis of brain-derived EVs, which may critically regulate EV uptake by neurons and aggregation of tau protein in EVs and/or recipient neurons. We have recently established the method for isolating EVs from human brain samples and successfully performed their proteomic profiling. We found that selective molecules from the EV proteomics datasets were able to differentiate human AD-EV from healthy control (CTRL)-EV with 88% accuracy by machine learning analysis, confirming pathogenic character of AD-EV molecules. Furthermore, our exciting preliminary data have shown that AD-EV have significantly higher tau seeding activity compared to CTRL-EV by FRET sensor tau seeding assay with subsets of EV molecules showing significant association with tau seeding activity. This proposed project will fortify these preliminary results and find the converging or specific mechanisms among tauopathies for mediating tau aggregation and its seeding via EV uptake through proteomics and biological examination of brain-derived EV samples. To meet this challenge, we assembled a multi-disciplinary team of investigators who have a strong record of accomplishments in biologic (Ikezu), proteomic (Emili) and bioengineering and bioinformatic analysis (Issador). In Aim1, we will examine EV samples from 240 new brain specimens (40 AD, 40 CTE, 40 LBD, 40 PSP, 40 CBD and 40 CTRL) for precision mass-spectrometry-based proteomics and tau-interactomes, and analyze those datasets by the machine learning approach. Aim 2 will examine the efficiency of tau propagation using tau fibrils, oligomers and EVs isolated from the same donors of the 5 different tauopathies and control cases in vitro and in vivo using FRET-based tau seeding assay and EV uptake by primary cultured mouse cortical neurons in vitro. EV-associated tau will be further characterized by the biochemical and microscopy-based analysis for their conformational and posttranslational changes. We will evaluate the difference in tau propagation after the intracranial injection of the tau seeds from different tauopathy brains using our recently established mouse models. Aim3 will identify candidate molecules most likely involved in EV uptake and tau seeding activity by bioinformatic analysis of the proteome dataset (Aim 1) and biological datasets (Aim 2). We will then test the functional roles of the identified molecules on EV uptake, tau seeding activities and neuronal firing activities in vitro. The candidate molecules will be specifically targeted by gene silencing or antagonists for their therapeutic potential to halt tau propagation in vitro and in vivo. Successful identification of responsible molecules for tau propagation will serve as a foundation for understanding EV-mediated disease progression.