The Size and Stability of Infectious Prion Aggregates Fluctuate Dynamically during Cellular Uptake and Disaggregation

Prion diseases arise when PrP^Sc, an aggregated, infectious, and insoluble conformer of the normally soluble mammalian prion protein, PrP^C, catalyzes the conversion of PrP^C into more PrP^Sc, which then accumulates in the brain leading to disease. PrP^Sc is the primary, if not sole, component of the infectious prion. Despite the stability and protease insensitivity of PrP^Sc aggregates, they can be degraded after cellular uptake. However, how cells disassemble and degrade PrP^Sc is poorly understood. In this work, we analyzed how the protease sensitivity and size distribution of PrP^Sc aggregates from two different mouse-adapted prion strains, 22L, that can persistently infect cells and 87V, that cannot, changed during cellular uptake. We show that within the first 4 h following uptake large PrP^Sc aggregates from both prion strains become less resistant to digestion by proteinase K (PK) through a mechanism that is dependent upon the acidic environment of endocytic vesicles. We further show that during disassembly, PrP^Sc aggregates from both strains become more resistant to PK digestion through the apparent removal of protease-sensitive PrP^Sc, with PrP^Sc from the 87V strain disassembled more readily than PrP^Sc from the 22L strain. Taken together, our data demonstrate that the sizes and stabilities of PrP^Sc from different prion strains change during cellular uptake and degradation, thereby potentially impacting the ability of prions to infect cells.