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

Gian Mario Cosseddu,Michele Angelo Di Bari,Joaquin Castilla,Gabriele Vaccari,Romolo Nonno,Natalia Fernandez-Borges,Umberto Agrimi,Cecilia Bucalossi,Neil A Mabbott

doi:10.1371/journal.ppat.1002370

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.

Highlights

Transmissible Spongiform Encephalopathies (TSEs) are progressive and fatal neurodegenerative disorders that include scrapie of sheep, bovine spongiform encephalopathy (BSE) of cattle and Creutzfeldt-Jakob disease (CJD) of humans [1]
In an attempt to transpose to an in vitro system the particular sensitivity of the vole model to human and animal Transmissible Spongiform Encephalopathies (TSEs), we first explored the suitability of vole brain homogenate as a substrate for protein misfolding cyclic amplification (PMCA)
In an attempt to transpose to an in vitro system the plasticity shown by the bank vole model in the transmission of a variety of human and animal TSEs [28,29,30], we explored the suitability of vole brain homogenate as a substrate for PMCA

Summary

Introduction

Transmissible Spongiform Encephalopathies (TSEs) are progressive and fatal neurodegenerative disorders that include scrapie of sheep, bovine spongiform encephalopathy (BSE) of cattle and Creutzfeldt-Jakob disease (CJD) of humans [1]. The prion hypothesis postulates that the causal agent, the prion, consists only of proteins without nucleic acid genome [1]. Alternative hypotheses postulate the presence of a small nucleic acids genome [2], evidences for this are still lacking. The virino hypothesis proposes that the causal agent is an informational hybrid between the agent genome and host conformationally altered PrP [3]. The accumulation in the central nervous system of a post-translationally altered isoform (PrPSc) of the cellular prion protein (PrPC) is the key event in TSE pathogenesis [1], the relationships between PrPSc and infectivity are not definitively clear and evidences for high titers of TSE infectivity associated with extremely low levels of PrPSc have been reported [9]. The process proceeds thereafter in an autocatalytic manner, leading PrPSc aggregates to grow by including new PrPC monomers [1,12]

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