Prion diseases are caused by a misfolding of the cellular prion protein (PrP) to a pathogenic isoform named PrP. Prions exist as strains, which are characterized by specific pathological and biochemical properties likely encoded in the three-dimensional structure of PrP. However, whether cofactors determine these different PrP conformations and how this relates to their specific biological properties is largely unknown. To understand how different cofactors modulate prion strain generation and selection, Protein Misfolding Cyclic Amplification was used to create a diversity of infectious recombinant prion strains by propagation in the presence of brain homogenate. Brain homogenate is known to contain these mentioned cofactors, whose identity is only partially known, and which facilitate conversion of PrP to PrP. We thus obtained a mix of distinguishable infectious prion strains. Subsequently, we replaced brain homogenate, by different polyanionic cofactors that were able to drive the evolution of mixed prion populations toward specific strains. Thus, our results show that a variety of infectious recombinant prions can be generated in vitro and that their specific type of conformation, i.e., the strain, is dependent on the cofactors available during the propagation process. These observations have significant implications for understanding the pathogenesis of prion diseases and their ability to replicate in different tissues and hosts. Importantly, these considerations might apply to other neurodegenerative diseases for which different conformations of misfolded proteins have been described.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE‐derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.

The large chronic wasting disease (CWD)-affected cervid population in the USA and Canada, and the risk of the disease being transmitted to humans through intermediate species, is a highly worrying issue that is still poorly understood. In this case, recombinant protein misfolding cyclic amplification was used to determine, in vitro, the relevance of each individual amino acid on cross-species prion transmission. Others and we have found that the β2–α2 loop is a key modulator of transmission barriers between species and markedly influences infection by sheep scrapie, bovine spongiform encephalopathy (BSE), or elk CWD. Amino acids that differentiate ovine and deer normal host prion protein (PrP C ) and associated with structural rigidity of the loop β2–α2 (S173N, N177T) appear to confer resistance to some prion diseases. However, addition of methionine at codon 208 together with the previously described rigid loop substitutions seems to hide a key in this species barrier, as it makes sheep recombinant prion protein highly susceptible to CWD-induced misfolding. These studies indicate that interspecies prion transmission is not only governed just by the β2–α2 loop amino acid sequence but also by its interactions with the α3-helix as shown by substitution I208M. Transmissible spongiform encephalopathies, characterized by long incubation periods and spongiform changes associated with neuronal loss in the brain, have been described in several mammalian species appearing either naturally (scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, chronic wasting disease in cervids, Creutzfeldt–Jakob disease in humans) or by experimental transmission studies (scrapie in mice and hamsters). Much of the pathogenesis of the prion diseases has been determined in the last 40 years, such as the etiological agent or the fact that prions occur as different strains that show distinct biological and physicochemical properties. However, there are many unanswered questions regarding the strain phenomenon and interspecies transmissibility. To assess the risk of interspecies transmission between scrapie and chronic wasting disease, an in vitro prion propagation method has been used. This technique allows to predict the amino acids preventing the transmission between sheep and deer prion diseases.

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.