Deciphering the tau phosphorylation code

阿尔茨海默病(AD)是一种神经退行性疾病，其特征是斑块由Aβ组成，并且 含有微管相关蛋白tau的神经原纤维缠结(NFT)。牛磺酸的病理密切相关 有神经元退化和认知缺陷。由于tau的丢失可以保护A-β诱导的神经毒性， Tau被认为是Aβ的致病下游效应因子，从而导致神经元损伤。此外， 聚集的tau能以类普里子的方式繁殖，启动一种自我延续的毒性级联反应。为了这些 原因、针对tau的方法和新的治疗机制是必不可少的。蛋白的过度磷酸化 Tau是所有tauopy病的一致特征，这表明它可能是发病机制中的必经步骤，因此 合理的调制目标。磷酸化使tau从微管中解离并促进tau聚集 形成成对的螺旋状细丝，进一步堆积形成NFTs。Tau是自然展开的，在正常情况下 条件几乎没有聚集的趋势；因此，NFT中过度磷酸化的tau是 神经退行性变，了解磷酸化tau的病因和模式是必不可少的。这项分析的一个复杂之处在于，存在超过80个潜在的tau磷酸化位点。由于这一问题的复杂性，tau蛋白“过度磷酸化”的定义并不准确。此外，tau在联结和稳态形式的突触可塑性中都发挥着重要作用，但对其磷酸化知之甚少。 在这些途径中涉及的事件。对高兴奋的动态平衡反应特别有趣，因为这 异常过度活动类型在早期AD中被观察到，并可能与发病机制有关。 在这里，我们将使用无偏质量谱(MS)来执行tau磷酸化的全面图谱 在特定生理条件下以及在疾病模型中的模式。将磷酸化地图编译成 Tau修饰的“图谱”将开始破译在生理学中支配tau功能的磷酸化密码。 和病理学。在目标1中，我们将使用定性和定量的MS方法来定义tau的磷酸化 培养的海马神经元不同形式突触可塑性的模式，观察时间进程 观察每种范式后随时间的变化。我们还将使用特定的激酶抑制剂 上述刺激方案有助于识别内源性tau激酶和信号通路。在AIM 2、我们将使用MS来鉴定家族性AD人源化双重敲入模型中的tau磷酸化模式。 P301L-tau作为额颞叶痴呆的主要交互作用模型，我们将在 人源化tau基因敲入小鼠。我们将研究不同的年龄，以了解最早的tau修饰和 疾病进展的概况，以及雄性和雌性小鼠，以阐明疾病严重程度的性别差异。 确定tau的生理调节和磷酸化密码对于理解基础知识具有重要意义 涉及突触可塑性的机制。此外，这项工作将有助于指导新的治疗方法。 用于靶向AD和其他牛磺酸疾病中的致病tau。

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by plaques comprised of Aβ, and neurofibrillary tangles (NFTs) containing the microtubule associated protein tau. Tau pathology closely correlates with neuronal degeneration and cognitive deficits. As the loss of tau protects against Aβ-induced neurotoxicity, tau is thought to act as a pathogenic downstream effector of Aβ to induce neuronal injury. Furthermore, aggregated tau can propagate in a prion-like manner to initiate a self-perpetuating toxic cascade. For these reasons, tau-directed approaches and novel mechanisms of treatment are essential. Hyperphosphorylation of tau is a consistent feature of all tauopathies, suggesting it may be obligatory step in pathogenesis and hence a rational target of modulation. Phosphorylation dissociates tau from microtubules and promotes tau aggregation into paired helical filaments that further accumulate to form NFTs. Tau is natively unfolded, and under normal conditions has little tendency to aggregate; therefore hyperphosphorylated tau in NFTs is a prominent sign of neurodegeneration, and understanding the etiology and pattern of phosphorylated tau is essential. A complication in this analysis is that there are over 80 potential tau phosphorylation sites. Due to the complexity of this problem, tau “hyperphosphorylation” is not precisely defined. Additionally, tau plays important roles during both associative and homeostatic forms of synaptic plasticity, but little is known regarding the phosphorylation events involved in these pathways. Homeostatic responses to hyperexcitation are of particular interest as this type of aberrant overactivity is observed in early stage AD and may be relevant for the initiation of pathogenesis. Here, we will use unbiased mass spectrometry (MS) to perform comprehensive mapping of tau phosphorylation patterns during specific physiological conditions as well as in disease models. Compiling phosphomaps into an “atlas” of tau modifications will begin to decipher the phosphorylation code that governs tau function in physiology and pathology. In Aim 1, we will use qualitative and quantitative MS approaches to define tau phosphorylation patterns during different forms of synaptic plasticity in cultured hippocampal neurons, examining a time course after each paradigm to observe time-dependent changes. We will also use specific kinase inhibitors with the above stimulation protocols to facilitate identification of endogenous tau kinases and signaling pathways. In Aim 2, we will use MS to identify tau phosphorylation patterns in a humanized double knock-in model of familial AD. As a primary tauopathy model of frontotemporal dementia, we will use P301L-tau in the background of humanized tau knock-in mice. We will examine different ages to understand the earliest tau modifications and profile of disease progression, as well as male vs. female mice to elucidate sex differences in disease severity. Defining the physiological regulation and phosphorylation code of tau is highly significant for understanding basic mechanisms involved in synaptic plasticity. Furthermore, this work will help guide new therapeutic approaches for targeting pathogenic tau in AD and other tauopathies.