Resolving the Structural Conversion, Aggregation and Neurotoxicity of Prion Proteins at the Single Molecule Level

Abstract

Prion protein (PrP) misfolding and oligomerization are key pathogenic events in prion disease. When PrP misfolds, it imposes its structure upon natively folded proteins and templates their aggregation. The consequence of this self-amplifying cycle is an accumulation of toxic PrP aggregates that destroys neurons and invariably kill the organism. Copper exposure has been linked to prion pathogenesis, however, its mechanistic basis is unknown. Here we resolve, with single molecule precision, the molecular determinants of copper mediated PrP misfolding, oligomerization and neurotoxicity. Using a single molecule fluorescence assay, we demonstrate that copper induces PrP monomers to misfold before oligomer assembly; the intrinsically disordered N-terminal region is obligatory for this structural change. Single molecule force spectroscopy measurements show that the misfolded monomers have a 900 fold higher binding affinity which promotes their oligomerization. Using a cell-free seeding assay, we demonstrate that these oligomers, serve as seeds that template amyloid formation. Finally, using brain slice cultures, we show that the oligomers mediate inflammation and degeneration of neuronal tissue. Our study provides the first direct proof that copper misfolds PrP, subsequently promoting oligomerization and cytotoxicity.