Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

Abstract

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrPC, into a plethora of assemblies, PrPSc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrPSc structures. However, the degree to which intra- and inter-strain PrPSc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrPSc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrPSc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrPSc particles. We found that each strain had a continuum of PrPSc assemblies, with strong correlation between PrPSc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrPSc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrPSc took place at a hydrodynamic radius (Rh) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrPSc subpopulations and demonstrate that while PrPSc quaternary structure is a major contributor to PrPSc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrPSc particles from maintaining proteinase K resistance below an Rh of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.

Cryo-EM of prion strains from the same genotype of host identifies conformational determinants

“…Many low-resolution biochemical and spectroscopic comparisons of preparations of different PrP Sc strains have provided evidence of distinct conformations even when strains are formed from the same polypeptide sequence (e.g. (2,(9)(10)(11)(12)(13)(14)(15)(16)(17)). Moreover, PrP Sc strains have been shown to impose their general conformational attributes onto newly recruited PrP molecules in cell-free conversion and amplification reactions (9,18).…”

Emergence of CWD strains

“…Analysis of the fractions provides information for specific prion preparations regarding hydrodynamic radius allowing one to correlate the relative size of prion particles with infectivity. Cortez et al (2021) demonstrated that two hamster-adapted prion strains with distinct clinical presentation (hyper and drowsy; Bessen and Marsh 1992) differ in the size of the most infectious particle. Interestingly, both PrP Sc aggregates become PK-resistant at the same size.…”

“…On the initial passage, the incubation period was long and western blot analysis showed two different sized PrP Sc migration patterns. Low titer passage then resulted in the adaptation and selection of two different strains (hyperactive, HY; and drowsy, DY) with significantly different incubation periods and two different PrP Sc structures (Bartz et al 2000;Caughey et al 1998;Safar et al 1998;Cortez et al 2021).…”

Transport of Prions in the Peripheral Nervous System: Pathways, Cell Types, and Mechanisms

“…It is unclear how these differences in strain-specific properties of PrP Sc result in differences in the phenotype of disease. Recent work, however, suggests that PrP Sc particle size can influence prion formation efficiency and PrP Sc clearance and that strain-specific ratios of these PrP Sc particle sizes may provide a mechanistic basis for strain-specific rates of prion formation, incubation period of disease, and tissue tropism [ 34 , 35 , 36 ].…”