Alpha-Synuclein (alpha-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinsons disease (PD) and multiple systems atrophy (MSA). alpha-Syn aggregates spread throughout the brain during disease progression, suggesting mechanisms of intercellular seeding. Formation of alpha-Syn amyloid fibrils is observed in vitro and fibrillar alpha-Syn has been purified from patient brains, but recent reports questioned whether disease-relevant alpha-Syn aggregates are fibrillar in structure. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like alpha-Syn inclusions in situ at molecular resolution. We show that the inclusions consist of alpha-Syn fibrils crisscrossing a variety of cellular organelles such as the endoplasmic reticulum (ER), mitochondria and autophagic structures, without interacting with membranes directly. Neuronal inclusions seeded by recombinant or MSA patient-derived alpha-Syn aggregates have overall similar architecture, although MSA-seeded fibrils show higher structural flexibility. Using gold-labeled seeds we find that aggregate nucleation is predominantly mediated by alpha-Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal alpha-Syn inclusions contain alpha-Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.

中文翻译：

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神经元α-突触核蛋白包涵体的原位结构

α-突触核蛋白（α-Syn）聚集是毁灭性神经退行性疾病的标志，包括帕金森病（PD）和多系统萎缩（MSA）。在疾病发展过程中，α-Syn聚集体散布在整个大脑中，表明存在细胞间播种的机制。在体外观察到α-Syn淀粉样蛋白原纤维的形成，并且已经从患者的大脑中纯化了原纤维α-Syn，但是最近的报道质疑与疾病相关的α-Syn聚集体在结构上是否为原纤维。在这里，我们使用低温电子断层扫描（cryo-ET）在分子分辨率下原位成像神经元路易体样α-Syn夹杂物。我们显示，内含物由α-Syn原纤维组成，这些原纤维交叉穿过各种细胞器，例如内质网（ER），线粒体和自噬结构，而不直接与膜相互作用。尽管通过MSA接种的原纤维显示出更高的结构柔韧性，但通过重组或MSA患者来源的α-Syn聚集体接种的神经元内含物总体上具有相似的结构。使用金标记的种子，我们发现聚集成核作用主要由α-Syn寡聚体介导，且原纤维从种子单向生长。我们的结果最终证明，神经元的α-Syn内含物包含与细胞膜混合的α-Syn原纤维，并阐明了聚集核的机制。使用金标记的种子，我们发现聚集成核作用主要由α-Syn寡聚体介导，且原纤维从种子单向生长。我们的结果最终证明，神经元的α-Syn内含物包含与细胞膜混合的α-Syn原纤维，并阐明了聚集核的机制。使用金标记的种子，我们发现聚集成核作用主要由α-Syn寡聚体介导，且原纤维从种子单向生长。我们的结果最终证明，神经元的α-Syn内含物包含与细胞膜混合的α-Syn原纤维，并阐明了聚集核的机制。