Ultra-Efficient PrPSc Amplification Highlights Potentialities and Pitfalls of PMCA Technology

Abstract

In order to investigate the potential of voles to reproduce in vitro the efficiency of prion replication previously observed in vivo, we seeded protein misfolding cyclic amplification (PMCA) reactions with either rodent-adapted Transmissible Spongiform Encephalopathy (TSE) strains or natural TSE isolates. Vole brain homogenates were shown to be a powerful substrate for both homologous or heterologous PMCA, sustaining the efficient amplification of prions from all the prion sources tested. However, after a few serial automated PMCA (saPMCA) rounds, we also observed the appearance of PK-resistant PrPSc in samples containing exclusively unseeded substrate (negative controls), suggesting the possible spontaneous generation of infectious prions during PMCA reactions. As we could not definitively rule out cross-contamination through a posteriori biochemical and biological analyses of de novo generated prions, we decided to replicate the experiments in a different laboratory. Under rigorous prion-free conditions, we did not observe de novo appearance of PrPSc in unseeded samples of M109M and I109I vole substrates, even after many consecutive rounds of saPMCA and working in different PMCA settings. Furthermore, when positive and negative samples were processed together, the appearance of spurious PrPSc in unseeded negative controls suggested that the most likely explanation for the appearance of de novo PrPSc was the occurrence of cross-contamination during saPMCA. Careful analysis of the PMCA process allowed us to identify critical points which are potentially responsible for contamination events. Appropriate technical improvements made it possible to overcome PMCA pitfalls, allowing PrPSc to be reliably amplified up to extremely low dilutions of infected brain homogenate without any false positive results even after many consecutive rounds. Our findings underline the potential drawback of ultrasensitive in vitro prion replication and warn on cautious interpretation when assessing the spontaneous appearance of prions in vitro.

Progress towards structural understanding of infectious sheep PrP-amyloid

“…A gravely polymorphic mixture is the undesirable PMCA outcome. [25][26][27] This lack of homogeneity poses an insurmountable obstacle for latest state of the art solid-state NMR-spectroscopy of a 209-residue protein such as PrP. In contrast, uniform protein denaturation by guanidinhydrochloride (GdnHCl) and urea results in less infectious but homogeneous recPrP-samples (recPrP-amyloid).…”

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

“…Formation of infectivity in PMCA reactions has since been reproduced using other TSE strains (10,25,27,38,40,50). Prolonged rounds of serial PMCA can lead to de novo formation of TSE infectivity in unseeded reactions (5,16,20), but such experiments require extreme care to exclude cross-contamination (19). A recent study showed evidence of high infectivity titers in PMCA reactions with the hyper strain of transmissible mink encephalopathy, although incubation times per infectious unit were longer than those observed for brain-derived infectivity (51).…”

In vitro replication highlights the mutability of prions

“…19 We have previously shown that bank voles are a sensitive and permissive bioassay for many different prion strains, [20][21][22][23] and that these features are reflected when using vole brain homogenates as substrate for prion amplification. 24 Having set up extremely sensitive and controlled conditions for in vitro amplification of prions by using bank vole PrP C as an ultra-efficient substrate, 24 in this study we aimed at investigating the intrinsic mutational ability of prion strains in serial homologous PMCA reactions, in the absence of strong selective constraints such as PrP sequence mismatches, PrP C modifications, RNA-depletion or the addition of drugs. To this aim, we explored the evolutionary potential of 2 prion strains submitted to different passage regimens, i.e., either propagated through large population passages or subjected to repeated bottleneck events.…”