Wiseman James A, Murray Helen C, Faull Richard L M F, Dragunow Michael, Turner Clinton P, Dieriks Birger Victor, Curtis Maurice A
Department of Anatomy and Medical Imaging, University of Auckland, Auckland, New Zealand.
Centre for Brain Research, University of Auckland, Auckland, 1023, New Zealand.
NPJ Parkinsons Dis. 2024 Jan 2;10(1):1. doi: 10.1038/s41531-023-00614-w.
In Parkinson's disease (PD), and other α-synucleinopathies, α-synuclein (α-Syn) aggregates form a myriad of conformational and truncational variants. Most antibodies used to detect and quantify α-Syn in the human brain target epitopes within the C-terminus (residues 96-140) of the 140 amino acid protein and may fail to capture the diversity of α-Syn variants present in PD. We sought to investigate the heterogeneity of α-Syn conformations and aggregation states in the PD human brain by labelling with multiple antibodies that detect epitopes along the entire length of α-Syn. We used multiplex immunohistochemistry to simultaneously immunolabel tissue sections with antibodies mapping the three structural domains of α-Syn. Discrete epitope-specific immunoreactivities were visualised and quantified in the olfactory bulb, medulla, substantia nigra, hippocampus, entorhinal cortex, middle temporal gyrus, and middle frontal gyrus of ten PD cases, and the middle temporal gyrus of 23 PD, and 24 neurologically normal cases. Distinct Lewy neurite and Lewy body aggregate morphologies were detected across all interrogated regions/cases. Lewy neurites were the most prominent in the olfactory bulb and hippocampus, while the substantia nigra, medulla and cortical regions showed a mixture of Lewy neurites and Lewy bodies. Importantly, unique N-terminus immunoreactivity revealed previously uncharacterised populations of (1) perinuclear, (2) glial (microglial and astrocytic), and (3) neuronal lysosomal α-Syn aggregates. These epitope-specific N-terminus immunoreactive aggregate populations were susceptible to proteolysis via time-dependent proteinase K digestion, suggesting a less stable oligomeric aggregation state. Our identification of unique N-terminus immunoreactive α-Syn aggregates adds to the emerging paradigm that α-Syn pathology is more abundant and complex in human brains with PD than previously realised. Our findings highlight that labelling multiple regions of the α-Syn protein is necessary to investigate the full spectrum of α-Syn pathology and prompt further investigation into the functional role of these N-terminus polymorphs.
在帕金森病（PD）及其他α-突触核蛋白病中，α-突触核蛋白（α-Syn）聚集体形成了大量构象和截短变体。大多数用于检测和定量人脑中α-Syn的抗体靶向该140个氨基酸蛋白C末端（第96 - 140位氨基酸）内的表位，可能无法捕获PD中存在的α-Syn变体的多样性。我们试图通过用多种检测α-Syn全长表位的抗体进行标记，来研究PD人脑内α-Syn构象和聚集状态的异质性。我们使用多重免疫组织化学技术，用映射α-Syn三个结构域的抗体同时对组织切片进行免疫标记。在10例PD病例的嗅球、延髓、黑质、海马、内嗅皮质、颞中回和额中回，以及23例PD病例和24例神经正常病例的颞中回中，可视化并定量了离散的表位特异性免疫反应性。在所有检测的区域/病例中均检测到了不同的路易小体神经突和路易小体聚集形态。路易小体神经突在嗅球和海马中最为突出，而黑质、延髓和皮质区域则显示出路易小体神经突和路易小体的混合。重要的是，独特的N末端免疫反应性揭示了以前未被表征的（1）核周、（2）神经胶质（小胶质细胞和星形胶质细胞）和（3）神经元溶酶体α-Syn聚集体群体。这些表位特异性N末端免疫反应性聚集群体通过时间依赖性蛋白酶K消化易受蛋白水解作用，表明其寡聚体聚集状态不太稳定。我们对独特的N末端免疫反应性α-Syn聚集体的鉴定进一步证明了一个新出现的范式，即与之前的认识相比，α-Syn病理学在患有PD的人脑中更为丰富和复杂。我们的研究结果强调，标记α-Syn蛋白的多个区域对于研究α-Syn病理学的全貌是必要的，并促使进一步研究这些N末端多态性的功能作用。
