Abstract
The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Here, we use cryo-electron tomography (cryo-ET) to image neuronal α-Syn inclusions in situ at molecular resolution. We show that inclusions seeded by α-Syn aggregates produced recombinantly or purified from patient brain consist of α-Syn fibrils crisscrossing a variety of cellular organelles. Using gold-labeled seeds, we find that aggregate seeding is predominantly mediated by small α-Syn fibrils, from which cytoplasmic fibrils grow unidirectionally. Detailed analysis of membrane interactions revealed that α-Syn fibrils do not contact membranes directly, and that α-Syn does not drive membrane clustering. Altogether, we conclusively demonstrate that neuronal α-Syn inclusions consist of α-Syn fibrils intermixed with membranous organelles, and illuminate the mechanism of aggregate seeding and cellular interaction.
Highlights
The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component
Cryo-electron tomography is ideally suited to test these new ideas, as it can reveal the molecular architecture of protein aggregates at high resolution within neurons pristinely preserved by vitrification[22,23,24]
Primary mouse neurons were cultured on electron microscopy (EM) grids, transduced with GFP-α-Syn and incubated with recombinant α-Syn preformed fibrils (PFFs; Supplementary Fig. 1a, b)
Summary
The molecular architecture of α-Synuclein (α-Syn) inclusions, pathognomonic of various neurodegenerative disorders, remains unclear. α-Syn inclusions were long thought to consist mainly of α-Syn fibrils, but recent reports pointed to intracellular membranes as the major inclusion component. Primary mouse neurons were cultured on EM grids, transduced with GFP-α-Syn and incubated with recombinant α-Syn preformed fibrils (PFFs; Supplementary Fig. 1a, b). As reported, seeding of neurons led to the formation of GFP-α-Syn inclusions that were positive for Lewy body markers, including phospho-α-Syn (Ser129) and p62 (Supplementary Fig. 1c, d).
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