Tau-Mediated Regulation and Dysregulation of Protein Phosphatase 1

Tau是一种微管相关蛋白，在阿尔茨海默病中异常磷酸化并聚集 疾病（AD）和AD相关痴呆，导致其功能受损。无疾病改善治疗 存在这些疾病，使其成为重要的医疗保健优先事项。虽然我们几十年前就知道 一些修饰与散发性疾病密切相关，tau蛋白的突变直接导致 遗传性退行性tau蛋白病，病理性tau蛋白参与的精确分子途径， 导致神经退化的原因尚不清楚。这种知识差距仍然是一个重大和关键的问题 这一建议旨在解决。该领域目前的想法表明，疾病相关的tau蛋白修饰 通过破坏微管和/或通过增强tau聚集来发挥毒性。然而，我们的证据表明， 其他人支持另一种解释。也就是说，tau的作用是调节信号通路和tau 毒性是由于这一功能的失常。利用从鱿鱼巨轴突中分离的轴浆， 在tau蛋白的N端发现了一个功能性信号基序，称为磷酸酶激活结构域 (PAD)激活蛋白磷酸酶-1（PP 1）依赖的信号级联。我们还发现， 已知的tau的病理变化（例如磷酸化和寡聚化）改变了tau的结构， 该途径的异常PAD依赖性活化和轴突毒性。我们建议集中精力确定 tau蛋白是否正常调节神经元中的PP 1依赖性功能和tau蛋白的PP 1依赖性通路 毒性我们的中心假设是，通过PP 1依赖性机制，tau蛋白通常调节神经元 功能和健康，但在疾病中，tau通过以下异常引起轴突、突触和/或神经元毒性： 这个机制。在目标1中，我们将测试tau和PP 1内的特定基序对于蛋白质的表达至关重要的假设。 与tau蛋白的相互作用和激活PP 1。我们将使用点突变/缺失的组合， 绘制tau和PP 1亚型中的特定结构域，这些结构域是tau结合和激活PP 1的能力的基础。在目标2中， 我们将检验tau蛋白与PP 1一起定位于特定亚细胞区室的假设， PP 1依赖性通路。在这里，我们将使用新的人tau-KI小鼠原代神经元培养物和 超分辨率显微镜、亚细胞级分和其他蛋白质-蛋白质相互作用测定的组合， 确定神经元tau和PP 1相互作用的位置。此外，我们将敲除tau蛋白，以确定其功能 与神经元中多个PP 1依赖性通路的关系。在目标3中，我们将检验疾病 tau蛋白的形式通过PP 1依赖性机制驱动毒性。我们将使用一种新的tau预形成的原纤维接种 在hTau-KI小鼠以及PS19突变体tau小鼠系中的模型，以提供关键的体内翻译 深入了解tau与PP 1的关系以及tau疾病形式如何导致PP 1依赖性毒性。在一起， 拟议中的研究将填补我们知识上的关键空白，并将通过确定 tau在调节神经元中的PP 1中的重要生理和病理作用。

Tau is a microtubule-associated protein that is abnormally phosphorylated and aggregates in Alzheimer’s disease (AD) and AD-related dementias, leading to its impaired functionality. No disease modifying treatment exists for these diseases, making them a significant healthcare priority. Though we have known for decades that several modifications are intimately associated with sporadic disease and mutations in tau directly cause inherited degenerative tauopathies, the precise molecular pathways engaged by pathological tau proteins to actively cause neurodegeneration are unknown. This knowledge gap remains a significant and critical problem that this proposal aims to address. Current thinking in the field suggests that disease-related tau modifications exert toxicity by disruption of microtubules and/or by enhancing tau aggregation. However, evidence from our group and others supports an alternative explanation. That is, tau acts to regulate signaling pathways and tau toxicity is due to an aberration of this function. Using the isolated axoplasm from squid giant axons, we discovered a functional signaling motif in the N-terminus of tau called the phosphatase-activating domain (PAD) that activates a protein phosphatase-1 (PP1)-dependent signaling cascade. We also identified that known pathological changes in tau (e.g. phosphorylation and oligomerization) alter tau’s structure leading to aberrant PAD-dependent activation of this pathway and axonal toxicity. We propose to focus on determining whether tau normally regulates PP1-dependent functions in neurons and on PP1-dependent pathways of tau toxicity. Our central hypothesis is that, through a PP1-dependent mechanism, tau normally regulates neuronal function and health, but in disease tau causes axonal, synaptic and/or neuronal toxicity through aberrations of this mechanism. In Aim 1, we will test the hypothesis that specific motifs within tau and PP1 are critical for the interaction with and activation of PP1 by tau proteins. We will use a combination of point mutations/deletions to map the specific domains in tau and PP1 isoforms that underlie tau’s ability to bind and activate PP1. In Aim 2, we will test the hypothesis that tau localizes with PP1 in specific subcellular compartments where it regulates PP1-dependent pathways. Here, we will use novel human tau-KI mouse primary neuron cultures and a combination of super-resolution microscopy, subcellular fraction and other protein-protein interaction assays to determine where in neurons tau and PP1 interact. Also, we will knockdown tau to determine its functional relationship to multiple PP1-dependent pathways in neurons. In Aim 3, we will test the hypothesis that disease forms of tau drive toxicity via PP1-dependent mechanisms. We will use a novel tau pre-formed fibril seeding model in the hTau-KI mice as well as the PS19 mutant tau mouse line to provide the critical in vivo translational insights for the tau-PP1 relationship and how disease forms of tau lead to PP1-dependent toxicity. Together, the proposed studies will fill critical gaps in our knowledge and will produce a significant impact by identifying important physiological and pathological roles for tau in regulating PP1 in neurons.