Alzheimer’s disease (AD) is a neurodegenerative disorder without a cure or prevention to date. Hyperphosphorylated tau forms the neurofibrillary tangles (NFTs) that correlate well with the progression of cognitive impairments. Animal studies demonstrated the pathogenic role of hyperphosphorylated tau. Understanding how abnormal phosphorylation renders a normal tau prone to form toxic fibrils is key to delineating molecular pathology and to developing efficacious drugs for AD. Production of a tau bearing the disease-relevant hyperphosphorylation and molecular characters is a pivotal step. Here, we report the preparation and characterization of a recombinant hyperphosphorylated tau (p-tau) with strong relevance to disease. P-tau generated by the PIMAX approach resulted in phosphorylation at multiple epitopes linked to the progression of AD neuropathology. In stark contrast to unmodified tau that required an aggregation inducer, and which had minimal effects on cell functions, p-tau formed inducer-free fibrils that triggered a spike of mitochondrial superoxide, induced apoptosis, and caused cell death at sub-micromolar concentrations. P-tau-induced apoptosis was suppressed by inhibitors for reactive oxygen species. Hyperphosphorylation apparently caused rapid formation of a disease-related conformation. In both aggregation and cytotoxicity, p-tau exhibited seeding activities that converted the unmodified tau into a cytotoxic species with an increased propensity for fibrillization. These characters of p-tau are consistent with the emerging view that hyperphosphorylation causes tau to become an aggregation-prone and cytotoxic species that underlies diffusible pathology in AD and other tauopathies. Our results further suggest that p-tau affords a feasible tool for Alzheimer’s disease mechanistic and drug discovery studies.

阿尔茨海默病 (AD) 是一种神经退行性疾病，迄今为止尚无治愈或预防方法。过度磷酸化的 tau 形成与认知障碍进展密切相关的神经原纤维缠结 (NFT)。动物研究证明了过度磷酸化 tau 的致病作用。了解异常磷酸化如何使正常 tau 易于形成有毒原纤维是描绘分子病理学和开发有效的 AD 药物的关键。具有与疾病相关的过度磷酸化和分子特征的 tau 蛋白的产生是关键的一步。在这里，我们报告了与疾病密切相关的重组过度磷酸化 tau (p-tau) 的制备和表征。PIMAX 方法产生的 P-tau 导致与 AD 神经病理学进展相关的多个表位的磷酸化。与需要聚集诱导剂且对细胞功能影响最小的未修饰 tau 形成鲜明对比，p-tau 形成不含诱导剂的原纤维，触发线粒体超氧化物的峰值，诱导细胞凋亡，并在亚微摩尔浓度下导致细胞死亡。P-tau 诱导的细胞凋亡被活性氧抑制剂抑制。过度磷酸化显然导致疾病相关构象的快速形成。在聚集和细胞毒性方面，p-tau 表现出接种活性，将未修饰的 tau 转化为具有增加的原纤维化倾向的细胞毒性物质。p-tau 的这些特征与新出现的观点一致，即过度磷酸化导致 tau 成为一种易于聚集和细胞毒性的物质，是 AD 和其他 tau 病中弥漫性病理学的基础。我们的结果进一步表明 p-tau 为阿尔茨海默病机制和药物发现研究提供了可行的工具。