Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK.
Oxford Parkinson's Disease Centre, University of Oxford, South Parks Road, Oxford, OX1 3QX, UK.
Acta Neuropathol Commun. 2021 Jan 28;9(1):18. doi: 10.1186/s40478-020-01117-y.
Multimerization is a key process in prion-like disorders such as Alzheimer's disease (AD), since it is a requirement for self-templating tau and beta-amyloid amyloidogenesis. AT8-immunohistochemistry for hyperphosphorylated tau is currently used for the diagnosis and staging of tau pathology. Given that tau-tau interactions can occur in the absence of hyperphosphorylation or other post-translational modifications (PTMs), the direct visualization of tau multimerization could uncover early pathological tau multimers.
Here, we used bimolecular fluorescent complementation, rapamycin-dependent FKBP/FRB-tau interaction and transmission electron microscopy to prove the in vitro specificity of tau-proximity ligation assay (tau-PLA). We then analyzed MAPT KO and P301S transgenic mice, and human hippocampus and temporal isocortex of all Braak stages with tau-PLA and compared it with immunohistochemistry for the diagnostic antibody AT8, the early phosphorylation-dependent AT180, and the conformational-dependent antibody MC1. Finally, we performed proteinase-K treatment to infer the content of amyloidogenic beta-sheet fold.
Our novel tau-proximity ligation assay (tau-PLA) directly visualized tau-tau interactions in situ, and exclusively recognized tau multimers but not monomers. It elicited no signal in MAPT KO mouse brains, but extensively labelled P301S transgenic mice and AD brain. Two groups of structures were detected, a previously unreported widespread small-sized diffuse pathology and large, neurofibrillary-like lesions. Tau-PLA-labelled diffuse pathology appeared from the earliest Braak stages, mostly unaccompanied by tangle-like tau-immunohistochemistry, being significantly more sensitive than any small-sized dot-/thread-like pathology labelled by AT180-, AT8- and MC1-immunohistochemistry in most regions quantified at stages 0-II. Tau-PLA-labelled diffuse pathology was extremely sensitive to Proteinase-K, in contrast to large lesions.
Tau-PLA is the first method to directly visualize tau multimers both in vitro and in situ with high specificity. We find that tau multimerization appears extensively from the earliest presymptomatic Braak stages as a previously unreported type of diffuse pathology. Importantly, in our study multimerization is the earliest detectable molecular event of AD tau pathology. Our findings open a new window to the study of early tau pathology, with potential implications in early diagnosis and the design of therapeutic strategies.
多聚化是淀粉样蛋白样病变（如阿尔茨海默病）中的关键过程，因为它是自模板化 tau 和β-淀粉样蛋白淀粉样形成的要求。目前，用于 tau 病理学诊断和分期的是 AT8-免疫组化检测磷酸化 tau。鉴于 tau-tau 相互作用可以在没有过度磷酸化或其他翻译后修饰（PTM）的情况下发生，tau 多聚体的直接可视化可能会揭示早期病理性 tau 多聚体。
在这里，我们使用双分子荧光互补、雷帕霉素依赖性 FKBP/FRB-tau 相互作用和透射电子显微镜来证明 tau 接近连接测定（tau-PLA）的体外特异性。然后，我们分析了 MAPT KO 和 P301S 转基因小鼠以及人类海马体和颞叶皮质的所有 Braak 阶段的 tau-PLA，并将其与诊断抗体 AT8、早期磷酸依赖性 AT180 和构象依赖性抗体 MC1 的免疫组化进行了比较。最后，我们进行了蛋白酶 K 处理，以推断淀粉样β-折叠结构的含量。
我们的新型 tau 接近连接测定（tau-PLA）直接在原位可视化 tau-tau 相互作用，并且仅特异性识别 tau 多聚体而不是单体。它在 MAPT KO 小鼠脑中没有产生信号，但广泛标记 P301S 转基因小鼠和 AD 大脑。检测到两组结构，一组是以前未报道的广泛的小尺寸弥散性病变，另一组是大的神经纤维样病变。tau-PLA 标记的弥散性病变从最早的 Braak 阶段开始出现，大多数情况下没有缠结样 tau 免疫组化伴随，在大多数量化的阶段 0-II 区域，比任何小尺寸点状/线状病理学都更敏感，这些病理学由 AT180-、AT8-和 MC1-免疫组化标记。tau-PLA 标记的弥散性病变对蛋白酶 K 非常敏感，而大病变则不然。
tau-PLA 是第一个具有高度特异性的在体外和原位直接可视化 tau 多聚体的方法。我们发现 tau 多聚化从最早的无症状性 Braak 阶段广泛出现，是一种以前未报道的弥散性病变类型。重要的是，在我们的研究中，多聚化是 AD tau 病理学中最早可检测到的分子事件。我们的发现为早期 tau 病理学的研究开辟了一个新的窗口，可能对早期诊断和治疗策略的设计具有重要意义。
