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)和与AD相关的痴呆，导致其功能受损。无疾病修饰性治疗 这些疾病的存在，使它们成为重要的医疗保健优先事项。尽管我们几十年前就知道 几个基因突变与散发性疾病密切相关，tau基因突变直接导致 遗传性退行性tauopathy，病理性tau蛋白参与的精确分子通路 主动导致神经退行性变的原因尚不清楚。这一知识差距仍然是一个重大而关键的问题。 这项提案旨在解决的问题。目前该领域的想法表明，与疾病相关的tau修饰 通过破坏微管和/或通过增强tau聚集来施加毒性。然而，来自我们的证据 Group和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异常造成的。利用分离的鱿鱼巨大轴突的轴浆，我们 在tau的N端发现了一个功能信号基序，称为磷酸酶激活结构域 (PAD)激活依赖于蛋白磷酸酶-1(PP1)的信号级联反应。我们还确认， 已知的tau的病理变化(如磷酸化和寡聚化)改变tau的结构导致 该通路的异常PAD依赖激活和轴突毒性。我们建议集中精力确定 Tau是否正常调节神经元中PP1依赖的功能和tau依赖的PP1通路 毒性。我们的中心假设是，通过PP1依赖的机制，tau正常地调节神经元 功能和健康，但在疾病中，tau通过以下途径引起轴突、突触和/或神经元毒性 这个机制。在目标1中，我们将检验这样的假设，即tau和pp1中的特定基序对 Tau蛋白与PP1的相互作用和活化。我们将使用点突变/缺失的组合来 映射tau和PP1亚型中的特定结构域，这些结构域是tau结合和激活PP1能力的基础。在目标2中， 我们将检验这样一种假设，即tau与pp1一起定位在它调节的特定亚细胞间隔中。 PP1依赖的通路。在这里，我们将使用新型的人类tau-ki小鼠原代神经元培养和 结合超分辨显微镜、亚细胞分数和其他蛋白质-蛋白质相互作用分析 确定tau和PP1在神经元中相互作用的位置。此外，我们将敲除tau以确定其功能 与神经元中多条依赖PP1的通路的关系。在目标3中，我们将检验疾病的假设 Tau通过依赖PP1的机制驱动毒性的形式。我们将使用一种新型的tau预成型纤维播种。 在hTau-Ki小鼠模型以及PS19突变tau小鼠品系中提供关键的体内翻译 对tau-PP1关系的洞察以及tau的疾病形式如何导致PP1依赖的毒性。一起， 拟议的研究将填补我们知识的关键空白，并将产生重大影响，通过确定 Tau在调节神经元PP1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.