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

神经纤维缠结，由高度磷酸化的微管相关的细胞内聚集体组成 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。 新的证据表明，细胞外小泡(EV，如外周小体和微泡)， 病理tau作为细胞间的载体，传播tau病理。当务之急是找到 脑源性EV的分子基础，可能关键地调节神经元对EV的摄取和EV的聚集 EV和/或受体神经元中的tau蛋白。我们最近建立了分离电动汽车的方法。 人类大脑样本并成功地进行了蛋白质组学分析。我们发现选择性分子 从EV蛋白质组学数据集能够区分人类AD-EV和健康对照(CTRL)-EV 机器学习分析的准确率为88%，证实了AD-EV分子的致病特性。 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种子 FRET传感器tau种子试验与CTRL-EV的活性比较EV分子亚群显示 与tau播种活动显著相关。这个拟议的项目将加强这些初步成果，并 发现tauopathy中调节tau聚集及其种子的汇聚或特定机制 通过蛋白质组学和脑源性EV样本的生物学检测来摄取EV。为了满足这一点 挑战，我们组建了一个多学科的调查团队，他们有很好的记录 在生物学(Ikezu)、蛋白质组学(Emily I)以及生物工程和生物信息学分析(Issado)方面的成就。 在Aim1中，我们将检查240个新的脑标本(40个AD，40个CTE，40个LBD，40个PSP，40个)的EV样本 CBD和40CTRL)，用于基于精密质谱学的蛋白质组学和tau相互作用，并分析 这些数据集由机器学习方法实现。目标2将检查tau的传播效率，使用 从5种不同牛磺酸疾病和对照病例的同一供体中分离到的tau纤维、寡聚体和EVS 基于FRET的tau接种试验和原代培养小鼠皮质摄取EV的体内外研究 体外培养的神经元。EV相关tau的进一步表征将以生化和显微镜为基础 分析它们的构象和翻译后的变化。我们将评估tau的差异 应用我们的最新技术研究不同脑病患者脑组织内注射tau种子后的增殖 建立了小鼠模型。AIM3将确定最有可能参与EV摄取和tau的候选分子 通过蛋白质组数据集(目标1)和生物数据集(目标2)的生物信息学分析播种活动。我们 然后将测试已识别的分子在EV摄取、tau种子活动和神经元中的功能作用 体外激发活性。候选分子将成为基因沉默或拮抗剂的特定靶点。 它们在体外和体内抑制tau增殖的治疗潜力。成功确定责任人 Tau传播的分子将成为了解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.