Tau-Fyn Interaction and Alzheimer's Disease

项目概要/摘要 阿尔茨海默病（AD）是全球痴呆症的主要原因，其影响将呈指数级增长 随着人口老龄化。迫切需要新的治疗方法来治疗AD。微管- 相关蛋白Tau已经被大量研究，因为它聚集成神经元缠结， 这是AD的标志之一Tau基因敲除在几种AD模型中具有保护性， 其作为AD治疗靶点的潜力。有趣的是，Tau减少也防止网络过度兴奋， 在这些模型中，其发生在AD的早期并且可能导致神经变性。同样，在一个主要 在神经元培养系统中，Tau减少防止淀粉样蛋白-β（Aβ）毒性和谷氨酸或NMDA诱导的神经元毒性。 兴奋性毒性简而言之，Tau减少在多种系统中具有保护作用，但其机制是 目前尚不清楚。在AD中过度磷酸化的Tau的中央脯氨酸富集区， 几个PxxP基序介导与含SH 3结构域的蛋白质结合，包括非受体 酪氨酸激酶Fyn。Fyn也是网络超兴奋性的重要介质，因为它使AMPA磷酸化 和NMDA受体来加强它们的信号传导并调节树突棘动力学。外源性Aβ 在突触后密度激活Fyn，导致神经元中的NMDAR磷酸化和兴奋性毒性。 各种证据表明Tau与Fyn的相互作用可能是Aβ毒性的关键介导剂。遗传 敲除Tau或Fyn可防止原代神经元中的Aβ毒性，过度磷酸化的Tau具有更高的 Tau在体内介导Fyn向树突的运输，导致Aβ诱导的认知缺陷 和网络过度兴奋。然而，Tau-Fyn相互作用是Aβ毒性的关键介质的想法， 还没有被直接测试，这是这个项目的目标。我的总体假设是陶菲克 相互作用是Aβ诱导的结构和功能异常的关键介质。我来测试一下 假设使用Tau-Fyn相互作用的肽抑制剂Tau-PxxP 5/6，由先前的成员开发， 罗伯森实验室我开发了一种基于邻位连接的测定法来证实Tau-PxxP 5/6抑制内源性 Tau-Fyn原位相互作用，发现它阻止Aβ诱导的神经突变性和膜运输 功能障碍在这里，我将确定Tau-PxxP 5/6是否能阻止Aβ诱导的树突棘形态学缺陷 使用3D形态测量分析和Aβ和gabazine诱导的网络超兴奋性，使用多电极 阵我还将确定Tau的Fyn结合区是否是Tau介导Aβ毒性的关键区域 通过用不同的突变的Tau构建体转导Tau敲除的神经元来防止Fyn结合。此外 在体外研究中，我将确定Tau-PxxP 5/6是否能预防认知缺陷、癫痫样活动和癫痫发作 使用AD的hAPPJ 20小鼠模型进行体内易感性。拟议的工作将提供对 Aβ毒性的分子机制，并可能提供一个有前途的治疗策略，以治疗AD。

Project Summary/Abstract Alzheimer’s disease (AD) is the leading cause of dementia worldwide and its impact will increase exponentially as the population ages. New therapeutic approaches are desperately needed to treat AD. The microtubule- associated protein Tau is has been heavily studies because it aggregates into neurofibrillary tangles within neurons, one of the hallmarks of AD. Genetic knockout of Tau is protective in several models of AD, highlighting its potential as a therapeutic target for AD. Interestingly, Tau reduction also prevents network hyperexcitability, which occurs early in AD and may contribute to neurodegeneration, in these models. Similarly, in a primary neuron culture system, Tau reduction prevents amyloid-β (Aβ) toxicity and glutamate or NMDA-induced excitotoxicity. In short, Tau reduction is protective in a variety of systems, but the mechanism by which it does so is currently unknown. Tau’s central proline-rich region, which is hyperphosphorylated in AD, has several PxxP motifs that mediate binding with SH3 domain–containing proteins including the nonreceptor tyrosine kinase Fyn. Fyn is also an important mediator of network hyperexcitability, as it phosphorylates AMPA and NMDA receptors to strengthen their signaling and regulates dendritic spine dynamics. Exogenous Aβ activates Fyn at the postsynaptic density, leading to NMDAR phosphorylation and excitotoxicity in neurons. Diverse evidence indicates that Tau’s interaction with Fyn could be a critical mediator of Aβ toxicity. Genetic knockout of either Tau or Fyn prevents Aβ toxicity in primary neurons, hyperphosphorylated Tau has a higher affinity for Fyn, and Tau mediates trafficking of Fyn to dendrites in vivo, leading to Aβ-induced cognitive deficits and network hyperexcitability. However, the idea that the Tau-Fyn interaction is a critical mediator of Aβ toxicity has not been directly tested, which is the goal of this project. My overarching hypothesis is that the Tau-Fyn interaction is a critical mediator of Aβ-induced structural and functional abnormalities. I will test this hypothesis using a peptide inhibitor of the Tau-Fyn interaction, Tau-PxxP5/6, developed by previous members of the Roberson lab. I developed a proximity ligation-based assay to confirm that Tau-PxxP5/6 inhibits endogenous Tau-Fyn interaction in situ and found that it prevents Aβ-induced neurite degeneration and membrane trafficking dysfunction. Here, I will determine if Tau-PxxP5/6 prevents Aβ-induced deficits in dendritic spine morphology using 3D morphometric analysis and Aβ- and gabazine-induced network hyperexcitability using multi-electrode arrays. I will also determine if the Fyn-binding region of Tau is the critical region of Tau that mediates Aβ toxicity by transducing Tau knockout neurons with different mutated Tau constructs to prevent Fyn binding. In addition to in vitro studies, I will determine if Tau-PxxP5/6 prevents cognitive deficits, epileptiform activity and seizure susceptibility in vivo using the hAPPJ20 mouse model of AD. The proposed work will provide insights into the molecular mechanisms of Aβ toxicity and could provide a promising therapeutic strategy to treat AD.