Accumulation of phosphorylated tau is a key pathological feature of Alzheimer’s disease. Phosphorylated tau accumulation causes synaptic impairment, neuronal dysfunction and formation of neurofibrillary tangles. The pathological actions of phosphorylated tau are mediated by surrounding neuronal proteins; however, a comprehensive understanding of the proteins that phosphorylated tau interacts with in Alzheimer’s disease is surprisingly limited. Therefore, the aim of this study was to determine the phosphorylated tau interactome. To this end, we used two complementary proteomics approaches: (i) quantitative proteomics was performed on neurofibrillary tangles microdissected from patients with advanced Alzheimer’s disease; and (ii) affinity purification-mass spectrometry was used to identify which of these proteins specifically bound to phosphorylated tau. We identified 542 proteins in neurofibrillary tangles. This included the abundant detection of many proteins known to be present in neurofibrillary tangles such as tau, ubiquitin, neurofilament proteins and apolipoprotein E. Affinity purification-mass spectrometry confirmed that 75 proteins present in neurofibrillary tangles interacted with PHF1-immunoreactive phosphorylated tau. Twenty-nine of these proteins have been previously associated with phosphorylated tau, therefore validating our proteomic approach. More importantly, 34 proteins had previously been associated with total tau, but not yet linked directly to phosphorylated tau (e.g. synaptic protein VAMP2, vacuolar-ATPase subunit ATP6V0D1); therefore, we provide new evidence that they directly interact with phosphorylated tau in Alzheimer’s disease. In addition, we also identified 12 novel proteins, not previously known to be physiologically or pathologically associated with tau (e.g. RNA binding protein HNRNPA1). Network analysis showed that the phosphorylated tau interactome was enriched in proteins involved in the protein ubiquitination pathway and phagosome maturation. Importantly, we were able to pinpoint specific proteins that phosphorylated tau interacts with in these pathways for the first time, therefore providing novel potential pathogenic mechanisms that can be explored in future studies. Combined, our results reveal new potential drug targets for the treatment of tauopathies and provide insight into how phosphorylated tau mediates its toxicity in Alzheimer’s disease.

中文翻译：

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人类阿尔茨海默病大脑中的磷酸化 tau 相互作用组。

磷酸化 tau 的积累是阿尔茨海默病的一个关键病理特征。磷酸化的 tau 积累会导致突触损伤、神经元功能障碍和神经原纤维缠结的形成。磷酸化 tau 的病理作用是由周围的神经元蛋白介导的；然而，对磷酸化 tau 在阿尔茨海默病中相互作用的蛋白质的全面了解却出人意料地有限。因此，本研究的目的是确定磷酸化的 tau 相互作用组。为此，我们使用了两种互补的蛋白质组学方法：（i）对晚期阿尔茨海默病患者显微解剖的神经原纤维缠结进行定量蛋白质组学；(ii) 亲和纯化-质谱法用于鉴定这些蛋白质中的哪些与磷酸化 tau 特异性结合。我们在神经原纤维缠结中鉴定了 542 种蛋白质。这包括大量检测已知存在于神经原纤维缠结中的许多蛋白质，例如 tau、泛素、神经丝蛋白和载脂蛋白 E。亲和纯化-质谱法证实，存在于神经原纤维缠结中的 75 种蛋白质与 PHF1 免疫反应性磷酸化 tau 相互作用。其中 29 种蛋白质以前与磷酸化的 tau 蛋白相关，因此验证了我们的蛋白质组学方法。更重要的是，之前有 34 种蛋白质与总 tau 相关，但尚未与磷酸化 tau 直接相关（例如突触蛋白 VAMP2、液泡 ATP 酶亚基 ATP6V0D1）；因此，我们提供了新的证据，证明它们与阿尔茨海默病中的磷酸化 tau 蛋白直接相互作用。此外，我们还鉴定了 12 种新型蛋白质，以前不知道在生理或病理上与 tau 相关（例如 RNA 结合蛋白 HNRNPA1）。网络分析表明，磷酸化的 tau 相互作用组富含参与蛋白质泛素化途径和吞噬体成熟的蛋白质。重要的是，我们能够首次确定磷酸化 tau 与这些途径中相互作用的特定蛋白质，因此提供了可以在未来研究中探索的新的潜在致病机制。结合起来，我们的结果揭示了治疗 tau 蛋白病的新潜在药物靶点，并提供了关于磷酸化 tau 蛋白如何介导其在阿尔茨海默病中的毒性的见解。