Patient-based structural biology of Lewy body dementia using cryo-electron microscopy

ABSTRACT The conversion of soluble, monomeric protein into insoluble, fibrillar amyloid aggregates is a hallmark of numerous neurodegenerative diseases. The major neuropathological hallmarks of Parkinson’s disease and Dementia with Lewy Bodies is the accumulation of α-synuclein proteins in the brain. Investigations into the structure of the tau filament core isolated from brains of patients have elucidated the molecular composition of the pathologically relevant filament cores implicit to the pathogenesis of Alzheimer’s disease (AD). These results demonstrate the presence of disease-specific strains with specific molecular motifs, such as the β-helix structure that is present in AD filaments, and have highlighted critical features that are innate to the formation of specific amyloid structures, such as polymorphism in the subunit structure and differential interfaces between protofilaments. Recent structural determination has shown that recombinant tau aggregated in the presence of heparin adopts an entirely different fold that is not likely to be relevant to pathology, thereby highlighting the importance of studying the protein aggregates in their endogenous environment. By establishing the methodologies to extract filaments from diseased brains in a highly reproducible manner and by leveraging our skill in cryo-electron microscopy (cryo-EM), we are now uniquely poised to investigate additional and highly relevant pathologically relevant fibrillar forms. Consequently, the objectives of the current project are to characterize the nature and molecular structures of α-synuclein (α-syn) and amyloid-β (Aβ) filaments in the Lewy Body Dementia (LBD) brain to generate novel insight into the etiology, toxicity and spreading of protein aggregates in this disease. Specifically, we will determine and compare the filament heterogeneity found in LBD and assess the ability of these peptides to co-aggregate or interact with one-another. We aim to employ the knowledge gained from the filament cores of each protein to generate robust aggregation models in vitro and in vivo that closely resemble pathology, as well as core-specific antibodies and PET ligands. We will determine the frequency and importance of novel post-translational modifications (PTMs) in the diseased brain through an integrative approach combining cryo-EM with mass-spectrometry, biochemistry, molecular biology and biophysical methods. These methods and insights will inform the design and development of novel strategies to diagnose and treat disease, including next generation PET ligands and antibodies, as well as the development of in vitro and ex vivo models that properly recapitulate the disease itself. Finally, we will employ cryo-electron tomography (cryo-ET) to map the nature of the cell in the presence of these aggregates in their complex environments, as well as where each protein localizes, thereby enabling us to monitor for the interplay of LDB filaments with additional cellular factors known to influence α-syn aggregation, such as specific types of lipids.