Spatial organization and conformational ensembles of amyloids inside cells

Project Summary The formation of intracellular inclusions containing fibrillar aggregates of alpha-synuclein (α-syn) is a hallmark of neurodegenerative diseases such as Lewy body dementia, Parkinson’s disease, and multiple systems atrophy. An intrinsically disordered monomer, α-syn, can assemble into various fibrillar forms influenced by its environment. Understanding the conformations that α-syn adopts in health and disease is crucial for developing diagnostic and therapeutic tools. Although advances in structural biology have accelerated the characterization of isolated amyloid fibrils, it is essential to know the α-syn structures in biological settings, as isolated fibrils may not represent in vivo conformations. Current methods, such as isolating fibrils from post-mortem tissues or using seeding to amplify fibrils in vitro, might not capture the prevalent in-cell conformations. This project aims to identify the amyloid conformations and their abundances inside cells, which will enhance our understanding of amyloid propagation and aid in identifying biologically relevant structures. We will investigate how different cellular environments affect seeded amyloid propagation by using biosensor cell lines and neurons. In biosensor cell lines expressing wild-type and mutant α-syn, we will introduce α-syn and assess its aggregation into amyloid fibrils upon exposure to seeds. This approach will allow us to compare amyloid propagation in vitro with that inside cells. In neurons, we will study the seeded amyloid propagation of well-characterized α-syn fibrils, considering the influence of the cellular environment on fibril formation and propagation. Understanding how specific cell types affect amyloid propagation is crucial, as different neurodegenerative diseases involve amyloid fibrils accumulating in distinct cell types. Additionally, we will analyze amyloid structures from tissue seeded Lewy body dementia patients. By comparing these structures with known α-syn fibril conformations, we aim to identify unique features and polymorphs relevant to disease pathology. We will use a multi-scale approach with two complementary, state-of-the-art in situ structural techniques: in-cell DNP sensitivity-enhanced NMR spectroscopy and cellular cryo- ET/subtomogram averaging to characterize the conformational ensembles and spatial organization of α-syn amyloids in situ. This comprehensive approach will provide insights into the structural diversity, molecular interactions and organization of α-syn amyloids, as well as their effects on cells, ultimately advancing our understanding of neurodegenerative disease mechanisms and pathology, and informing the development of targeted diagnostics and therapeutics.