Abstract
Prion replication is believed to consist of two components, a growth or elongation of infectious isoform of the prion protein (PrPSc) particles and their fragmentation, a process that provides new replication centers. The current study introduced an experimental approach that employs Protein Misfolding Cyclic Amplification with beads (PMCAb) and relies on a series of kinetic experiments for assessing elongation rates of PrPSc particles. Four prion strains including two strains with short incubation times to disease (263K and Hyper) and two strains with very long incubation times (SSLOW and LOTSS) were tested. The elongation rate of brain-derived PrPSc was found to be strain-specific. Strains with short incubation times had higher rates than strains with long incubation times. Surprisingly, the strain-specific elongation rates increased substantially for all four strains after they were subjected to six rounds of serial PMCAb. In parallel to an increase in elongation rates, the percentages of diglycosylated PrP glycoforms increased in PMCAb-derived PrPSc comparing to those of brain-derived PrPSc. These results suggest that PMCAb selects the same molecular features regardless of strain initial characteristics and that convergent evolution of PrPSc properties occurred during in vitro amplification. These results are consistent with the hypothesis that each prion strain is comprised of a variety of conformers or ‘quasi-species’ and that change in the prion replication environment gives selective advantage to those conformers that replicate most effectively under specific environment.
Highlights
Prion diseases are a group of fatal age-dependent neurodegenerative maladies that can either arise spontaneously or via transmission of a prion infectious agent [1]
What are the rate limiting steps in Protein Misfolding Cyclic Amplification (PMCA)/ Protein Misfolding Cyclic Amplification with beads (PMCAb)? Is a PrPSc population transformed during PMCA/ PMCAb? Does PMCA/ PMCAb selectively amplify PrPSc particles with certain physical features? While previous studies focused on characterization of strain-specific conformational stability and aggregation states of PrPSc [24,25,26,27,28], the current study introduces an experimental approach for assessing the dynamic properties of PrPSc and, its elongation rate
In standard PMCAb format, the sonication cycles are separated by 30 minute incubation intervals, during which PrPSc particles grew in size
Summary
Prion diseases are a group of fatal age-dependent neurodegenerative maladies that can either arise spontaneously or via transmission of a prion infectious agent [1]. Significant difficulties in developing an experimental system for amplification of prion infectivity in vitro have been inflaming debate about the biochemical nature of the prion infectious agent. PMCA has been employed for assessing the cross-species transmission barrier [8,9], elucidating strain adaptation and interference [9,10], exploring cofactors involved in prion replication [11,12,13,14] and developing ultrasensitive prion detection and titration assays [15,16,17,18,19]
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