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Probing neuroactive intrinsically disordered protein and membrane interactions with NMR and nanobowls

Abstract

Dementia affects 50 million people around the world, with 10 million new cases every year. The hunt for causes and therapies of dementia causing neurodegenerative diseases (NDDs) have gone hand in hand. An important direction for therapeutic development is to combat the aberrant amyloid aggregation of intrinsically disordered proteins (IDPs). The consensus in the field is that oligomeric intermediates formed during amyloid aggregation are the disease-causing species via their lipid membrane interactions. The heterogeneity and transient nature of intermediates have stymied efforts to study structures, membrane interactions and the mechanism by which therapies act. To this end, this dissertation has three objectives, (i)understand structural features and membrane interactions of intermediates; (ii)develop methods to isolate oligomers from cells in their native forms; (iii)develop efficient therapeutic delivery solutions. First, we employ NMR spectroscopy to resolve atomic scale structural features of a synthetic preparation of pre-fibrillar α-synuclein (αS)-intermediate, an IDP implicated in Parkinson’s disease. We find that the αS-intermediate displays a fold very similar to the fibril, although with distinct lipid interactions and side-chain arrangements. This observation is interesting in the context of the anti-parallel transition to parallel cross β-sheets which is perceived as an important step in initiating amyloid aggregation. Next, to establish accurate structure-function correlations, we propose the use of silica nanobowls to scavenge and purify membrane bound amyloid aggregates from neuronal cultures. We demonstrate with amyloid-β (Aβ) aggregates, an IDP implicated in Alzheimer’s disease, that atleast their aggregation driving domains are conserved by this method and the amount of non-amyloid contaminants is minimal. Lastly, to address the concern of minimizing side-effects and increasing efficiency of therapeutic delivery, we explore the use of magnetically modified nanobowls for targeted delivery to neurons. The three projects in this work advance the understanding of the pathological interaction of amyloid oligomers and membranes and aid in efforts to study and modulate oligomers in their native environments.

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