Various post-translationally modified (PTM) proteoforms of alpha-synuclein (aSyn)—including C-terminally truncated (CTT) and Serine 129 phosphorylated (Ser129-p) aSyn—accumulate in Lewy bodies (LBs) in different regions of the Parkinson’s disease (PD) brain. Insight into the distribution of these proteoforms within LBs and subcellular compartments may aid in understanding the orchestration of Lewy pathology in PD. We applied epitope-specific antibodies against CTT and Ser129-p aSyn proteoforms and different aSyn domains in immunohistochemical multiple labelings on post-mortem brain tissue from PD patients and non-neurological, aged controls, which were scanned using high-resolution 3D multicolor confocal and stimulated emission depletion (STED) microscopy. Our multiple labeling setup highlighted a consistent onion skin-type 3D architecture in mature nigral LBs in which an intricate and structured-appearing framework of Ser129-p aSyn and cytoskeletal elements encapsulates a core enriched in CTT aSyn species. By label-free CARS microscopy we found that enrichments of proteins and lipids were mainly localized to the central portion of nigral aSyn-immunopositive (aSyn+) inclusions. Outside LBs, we observed that 122CTT aSyn+ punctae localized at mitochondrial membranes in the cytoplasm of neurons in PD and control brains, suggesting a physiological role for 122CTT aSyn outside of LBs. In contrast, very limited to no Ser129-p aSyn immunoreactivity was observed in brains of non-neurological controls, while the alignment of Ser129-p aSyn in a neuronal cytoplasmic network was characteristic for brains with (incidental) LB disease. Interestingly, Ser129-p aSyn+ network profiles were not only observed in neurons containing LBs but also in neurons without LBs particularly in donors at early disease stage, pointing towards a possible subcellular pathological phenotype preceding LB formation. Together, our high-resolution and 3D multicolor microscopy observations in the post-mortem human brain provide insights into potential mechanisms underlying a regulated LB morphogenesis.

α-突触核蛋白 (aSyn) 的各种翻译后修饰 (PTM) 蛋白质型 - 包括 C 末端截短 (CTT) 和丝氨酸 129 磷酸化 (Ser129-p) aSyn - 在帕金森病不同区域的路易体 (LBs) 中积累(PD) 大脑。深入了解这些蛋白质在 LB 和亚细胞区室中的分布可能有助于理解 PD 中路易病理学的编排。我们将针对 CTT 和 Ser129-p aSyn 蛋白型和不同 aSyn 结构域的表位特异性抗体应用于 PD 患者和非神经系统老年对照的死后脑组织的免疫组织化学多重标记，使用高分辨率 3D 多色共聚焦和受激发射耗尽（STED）显微镜。我们的多重标记设置突出了成熟黑质 LB 中一致的洋葱皮型 3D 架构，其中 Ser129-p aSyn 和细胞骨架元素的复杂且结构化的框架封装了富含 CTT aSyn 物种的核心。通过无标记 CARS 显微镜，我们发现蛋白质和脂质的富集主要位于黑质 aSyn 免疫阳性 (aSyn+) 包涵体的中心部分。在 LBs 之外，我们观察到 122CTT aSyn+ 斑点位于 PD 和对照大脑神经元细胞质中的线粒体膜上，这表明 122CTT aSyn 在 LBs 之外具有生理作用。相反，在非神经系统对照的大脑中观察到非常有限的 Ser129-p aSyn 免疫反应性，而神经元细胞质网络中 Ser129-p aSyn 的排列是患有（偶发）LB 疾病的大脑的特征。有趣的是，Ser129-p aSyn+ 网络分布不仅在含有 LB 的神经元中观察到，而且在没有 LB 的神经元中也观察到，特别是在疾病早期的供体中，这表明 LB 形成之前可能存在亚细胞病理表型。总之，我们在死后人脑中的高分辨率和 3D 多色显微镜观察提供了对受调节的 LB 形态发生的潜在机制的见解。指向LB形成之前可能的亚细胞病理表型。总之，我们在死后人脑中的高分辨率和 3D 多色显微镜观察提供了对受调节的 LB 形态发生的潜在机制的见解。指向LB形成之前可能的亚细胞病理表型。总之，我们在死后人脑中的高分辨率和 3D 多色显微镜观察提供了对受调节的 LB 形态发生的潜在机制的见解。