Table_1_Simple, Single-Shot Phosphoproteomic Analysis of Heat-Stable Tau Identifies Age-Related Changes in pS235- and pS396-Tau Levels in Non-human Primates.XLSX

Age is the most significant risk factor for Alzheimer’s disease (AD), and understanding its role in specific aspects of AD pathology will be critical for therapeutic development. Neurofibrillary tangles composed of hyperphosphorylated tau are a quintessential hallmark of AD. To study age-related changes in tau phosphorylation, we developed a simple, antibody-free approach for single shot analysis of tau phosphorylation across the entire protein by liquid-chromatography tandem mass spectrometry. This methodology is species independent; thus, while initially developed in a rodent model, we utilized this technique to analyze 36 phosphorylation sites on rhesus monkey tau from the prefrontal cortex (PFC), a region vulnerable to AD-linked degeneration. Data are available via ProteomeXchange with identifier PXD027971. We identified novel, age-related changes in tau phosphorylation in the rhesus monkey PFC and analyzed patterns of phosphorylation change across domains of the protein. We confirmed a significant increase and positive correlation with age of phosphorylated serine 235 tau and phosphorylated serine 396 tau levels in an expanded cohort of 14 monkeys. Histology showed robust labeling for tau phosphorylated at these sites in vulnerable layer III pyramidal cells in the PFC. The results presented in this study suggest an important role of the natural aging process in tau phosphorylation in rhesus monkey.

年龄是阿尔茨海默病（AD）最显著的危险因素，阐明其在AD病理特定环节中的作用，对治疗药物的研发具有关键意义。由过度磷酸化tau蛋白构成的神经原纤维缠结，是AD的典型病理标志。为研究tau蛋白磷酸化的年龄相关性变化，我们开发了一种简便、无需抗体的方法，可通过液相色谱-串联质谱法对完整tau蛋白的磷酸化状态进行单次分析。该方法具有物种非依赖性：尽管最初是在啮齿动物模型中建立的，但我们利用该技术分析了来自前额叶皮层（PFC）的恒河猴tau蛋白的36个磷酸化位点——前额叶皮层是易受AD相关变性损伤的脑区。研究数据可通过蛋白质组交换数据库（ProteomeXchange）获取，数据集标识符为PXD027971。我们在恒河猴前额叶皮层中发现了tau蛋白磷酸化的新型年龄相关性变化，并分析了该蛋白各结构域的磷酸化变化模式。在纳入14只恒河猴的扩大队列中，我们证实磷酸化丝氨酸235 tau与磷酸化丝氨酸396 tau的水平随年龄显著升高，且与年龄呈正相关。组织学分析显示，在前额叶皮层易受损伤的第三层锥体细胞中，上述两个磷酸化位点的tau蛋白呈现强阳性标记。本研究结果表明，自然衰老过程对恒河猴tau蛋白的磷酸化具有重要调控作用。