In situ architecture of neuronal alpha-Synuclein inclusions

-Synuclein (-Syn) aggregation is a hallmark of devastating neurodegenerative disorders including Parkinsons disease (PD) and multiple systems atrophy (MSA)1,2. -Syn aggregates spread throughout the brain during disease progression2, suggesting mechanisms of intercellular seeding. Formation of -Syn amyloid fibrils is observed in vitro3,4 and fibrillar -Syn has been purified from patient brains5,6, but recent reports questioned whether disease-relevant -Syn aggregates are fibrillar in structure7-9. Here we use cryo-electron tomography (cryo-ET) to image neuronal Lewy body-like -Syn inclusions in situ at molecular resolution. We show that the inclusions consist of -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 -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 -Syn oligomers, with fibrils growing unidirectionally from the seed. Our results conclusively demonstrate that neuronal -Syn inclusions contain -Syn fibrils intermixed with cellular membranes, and illuminate the mechanism of aggregate nucleation.