Jiang Peizhou, Dickson Dennis W
Department of Neuroscience, Mayo Clinic, 4500 San Pablo Road, Jacksonville, FL, 32224, USA.
Acta Neuropathol Commun. 2025 Mar 8;13(1):53. doi: 10.1186/s40478-025-01969-2.
Identifying protein deposits and associated components is crucial for understanding the pathogenesis of neurodegenerative disorders with intracellular or extracellular deposits. Correlative light and electron microscopy (CLEM) has emerged as a powerful tool to accurately study tissue and cellular pathology by examination of the same target at both microstructural and ultrastructural levels. However, the technical challenges with CLEM have limited its application to neuropathology. Here, we developed a simplified efficient CLEM method and applied it to a cell model that produces a high proportion of α-synuclein (αS) inclusions with immunopositivity to phosphorylated αS and the synaptic vesicle marker SV2A and synaptophysin. This approach incorporates modifications in sample processing and innovative fiducial marking techniques, which enhance antigen preservation and improve target registration, respectively. These advancements achieve an optimal balance in sensitivity, accuracy, efficiency, and cost-effectiveness compared to current CLEM methods employing different strategies. Using this method, we identified and analyzed αS inclusions in cell cultures, as well as various pathological protein deposits in postmortem brain tissues from individuals with a range of neurodegenerative disorders. Our findings replicate recently reported new features of αS pathology and also reveal unrecognized a variety forms of small αS inclusions in human brain, which provide valuable insights into mechanisms underlying Lewy-related pathology. Application of this enhanced CLEM method is a powerful tool in research on neurodegenerative disorders, including αS-opathies.
识别蛋白质沉积物及其相关成分对于理解伴有细胞内或细胞外沉积物的神经退行性疾病的发病机制至关重要。相关光电子显微镜（CLEM）已成为一种强大的工具，可通过在微观结构和超微结构水平上检查同一靶点来准确研究组织和细胞病理学。然而，CLEM的技术挑战限制了其在神经病理学中的应用。在此，我们开发了一种简化高效的CLEM方法，并将其应用于一个细胞模型，该模型产生高比例的α-突触核蛋白（αS）包涵体，对磷酸化αS以及突触囊泡标记物SV2A和突触素呈免疫阳性。这种方法结合了样本处理方面的改进和创新的基准标记技术，分别增强了抗原保存并改善了靶点配准。与采用不同策略的当前CLEM方法相比，这些进展在灵敏度、准确性、效率和成本效益方面实现了最佳平衡。使用这种方法，我们在细胞培养物中识别并分析了αS包涵体，以及来自患有一系列神经退行性疾病个体的死后脑组织中的各种病理性蛋白质沉积物。我们的发现重现了最近报道的αS病理学新特征，还揭示了人脑中未被识别的多种形式的小αS包涵体，这为路易体相关病理学的潜在机制提供了有价值的见解。这种增强的CLEM方法的应用是研究包括αS病在内的神经退行性疾病的有力工具。
