α-Synuclein proximity ligation assay (PLA) has proved a sensitive technique for detection of non-Lewy body α-synuclein aggregate pathology. Here, we describe the MJF-14 PLA, a new PLA towards aggregated α-synuclein with unprecedented specificity, using the aggregate-selective α-synuclein antibody MJFR-14-6-4-2 (hereafter MJF-14). Signal in the assay correlates with α-synuclein aggregation in cell culture and human neurons, induced by α-synuclein overexpression or pre-formed fibrils. Co-labelling of MJF-14 PLA and pS129-α-synuclein immunofluorescence in post-mortem cases of dementia with Lewy bodies shows that while the MJF-14 PLA reveals extensive non-inclusion pathology, it is not sensitive towards pS129-α-synuclein-positive Lewy bodies. In Parkinson’s disease brain, direct comparison of PLA and immunohistochemistry with the MJF-14 antibody shows widespread α-synuclein pathology preceding the formation of conventional Lewy pathology. In conclusion, we introduce an improved α-synuclein aggregate PLA to uncover abundant non-inclusion pathology, which deserves future validation with brain bank resources and in different synucleinopathies.

α-突触核蛋白邻近连接试验 （PLA） 已被证明是一种检测非路易体 α-突触核蛋白聚集体病变的敏感技术。在这里，我们描述了 MJF-14 PLA，这是一种针对聚集体 α-突触核蛋白的新型 PLA，具有前所未有的特异性，使用聚集体选择性 α-突触核蛋白抗体 MJFR-14-6-4-2 （以下简称 MJF-14）。检测中的信号与细胞培养物和人神经元中的 α-突触核蛋白聚集相关，由 α-突触核蛋白过表达或预先形成的原纤维诱导。在痴呆路易体的尸检病例中 MJF-14 PLA 和 pS129-α-突触核蛋白免疫荧光的共标记表明，虽然 MJF-14 PLA 揭示了广泛的非包涵体病理学，但它对 pS129-α-突触核蛋白阳性路易体不敏感。在帕金森病的大脑中，PLA 和免疫组织化学与 MJF-14 抗体的直接比较显示，在常规 Lewy 病理学形成之前，存在广泛的 α-突触核蛋白病理学。总之，我们引入了一种改进的 α-突触核蛋白聚集体 PLA 来发现丰富的非包涵体病理，这值得未来用脑库资源和不同的突触核蛋白病进行验证。