Direct evidence of the presence of cross-linked Aβ dimers in the brains of Alzheimer’s disease patients

Brain-derived amyloid-β (Aβ) dimers are associated with Alzheimer´s disease (AD). However, their covalent nature remains controversial. This feature is relevant, as a covalent cross-link would make brain-derived dimers (native dimers) more synaptotoxic than Aβ monomers and would make them suitable candidates for biomarker development. To resolve this controversy, we here present a three-step approach. First, we validated a type of synthetic cross-linked Aβ (CL Aβ) dimers, obtained by means of the photo-induced cross-linking of unmodified proteins (PICUP) reaction, as well-defined mimics of putative native CL Aβ dimers. Second, we used these PICUP CL Aβ dimers as standards to improve the isolation of native Aβ dimers and to develop state-of-the-art mass spectrometry (MS) strategies to allow their characterization. Third, we applied these MS methods to the analysis of native Aβ dimer samples allowing the detection of the CL [Aβ(6-16)]2 peptide comprising a dityrosine cross-link. This result demonstrates the presence of CL Aβ dimers in the brains of patients with AD and opens up avenues for establishing new therapeutic targets and developing novel biomarkers for this disease.

脑源性淀粉样β（amyloid-β, Aβ）二聚体与阿尔茨海默病（Alzheimer's disease, AD）密切相关，但其共价特性仍存在争议。该特性具有重要研究价值：若脑源性二聚体（天然二聚体）存在共价交联，其突触毒性将强于Aβ单体，且可作为开发生物标志物的理想候选对象。为解决这一争议，本文提出一套三步研究方案。首先，我们验证了通过非修饰蛋白光诱导交联（photo-induced cross-linking of unmodified proteins, PICUP）技术制备的合成交联型Aβ（cross-linked Aβ, CL Aβ）二聚体，将其作为推定天然CL Aβ二聚体的明确模拟物。其次，我们以该PICUP制备的CL Aβ二聚体为标准品，优化了天然Aβ二聚体的分离方法，并建立了先进的质谱（mass spectrometry, MS）表征策略。最后，我们将上述MS方法应用于天然Aβ二聚体样品分析，成功检测到含二酪氨酸交联的CL [Aβ(6-16)]₂肽段。该结果证实了AD患者大脑中存在CL Aβ二聚体，为该疾病确立新型治疗靶点及开发新型生物标志物开辟了新路径。