In vitro amplification of prions from deer and other animal species through the protein misfolding cyclic amplification (PMCA) technique

Abstract

Protein misfolding cyclic amplification (PMCA) is an ultrasensitive, cell-free technique that mimics the conversion of the host cellular prion protein (PrPC) to its pathogenic version (PrPSc) in an accelerated manner. PMCA can amplify minimal quantities of disease-associated prion particles contained in various sample types, including biological and environmental specimens. This is especially relevant for chronic wasting disease (CWD), a prion disease of cervids that currently manifests at epidemic proportions in North America. Considering the ecology of CWD prions, it is imperative to identify their presence in environmental components. Prion replication by PMCA is achieved through multiple incubation/sonication cycles, and PMCA products contain large prion quantities that can be easily visualized by conventional techniques such as dot blots or western blots. Here, we describe an updated protocol to replicate CWD prions by means of the PMCA technology in animal-derived samples and environmental fomites. We also describe newly optimized PMCA protocols to identify CWD-prions in non-cervid, sympatric hosts. Finally, we also explain modifications of this technique to increase its throughput, focused in the screening of small molecule libraries to identify anti-prion molecules. Multiple reports demonstrate that PMCA is able to detect as little as a single prion polymer in specific tissues (e.g. brain). Considering this, this technique has been proposed as a promising method for the diagnosis of prion diseases. In addition, PMCA faithfully replicates the biological information present in the original prion “seeds”, fact that has greatly helped in our understanding of prion pathogenic mechanisms. Importantly, prion diseases remain without cure, and the optimization of PMCA for drug discovery may importantly advance efforts in this direction.