One of the characteristics of prions is their ability to infect some species but not others and prion resistant species have been of special interest because of their potential in deciphering the determinants for susceptibility. Previously, we developed different in vitro and in vivo models to assess the susceptibility of species that were erroneously considered resistant to prion infection, such as members of the Leporidae and Equidae families. Here we undertake in vitro and in vivo approaches to understand the unresolved low prion susceptibility of canids. Studies based on the amino acid sequence of the canine prion protein (PrP), together with a structural analysis in silico, identified unique key amino acids whose characteristics could orchestrate its high resistance to prion disease. Cell- and brain-based PMCA studies were performed highlighting the relevance of the D163 amino acid in proneness to protein misfolding. This was also investigated by the generation of a novel transgenic mouse model carrying this substitution and these mice showed complete resistance to disease despite intracerebral challenge with three different mouse prion strains (RML, 22L and 301C) known to cause disease in wild-type mice. These findings suggest that dog D163 amino acid is primarily, if not totally, responsible for the prion resistance of canids. Detection of individuals or whole species resistant to any infectious disease is vital to understand the determinants of susceptibility and to develop appropriate therapeutic and preventative strategies. Canids have long been considered resistant to prion infection given the absence of clinical disease despite exposure to the causal agent. Through extensive analysis of the canine prion protein we have detected a key amino acid that might be responsible for their universal resistance to prion disease. Using in vitro and in vivo models we demonstrated that the presence of this residue confers resistance to prion infection when introduced to susceptible animals, opening the way to develop a new therapeutic approach against these, at present, untreatable disorders.

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. Prion disorders are a group of devastating neurodegenerative diseases caused by an aberrantly folded isoform of the endogenous prion protein. The molecular mechanisms by which this proteinaceous pathogen is able to propagate in the central nervous system and cause neuronal death are poorly understood, partially due to the difficulties elucidating the three-dimensional structure of the aggregation-prone aberrant isoform or prion. Obtaining sufficient amounts of highly pure and homogeneous prions for high-resolution structural studies has been limited until now due to technical reasons. Here, we present a novel method for the production of large amounts of highly infectious recombinant prions suitable for solid state Nuclear Magnetic Resonance imaging, which could help to unveil the molecular pathogenesis of these particular pathogens.

Misfolded prion protein aggregates (PrP^Sc) show remarkable structural diversity and are associated with highly variable disease phenotypes. Similarly, other proteins, including amyloid-β, tau, α-synuclein, and serum amyloid A, misfold into distinct conformers linked to different clinical diseases through poorly understood mechanisms. Here we use mice expressing glycophosphatidylinositol (GPI)-anchorless prion protein, PrP^C, together with hydrogen-deuterium exchange coupled with mass spectrometry (HXMS) and a battery of biochemical and biophysical tools to investigate how post-translational modifications impact the aggregated prion protein properties and disease phenotype. Four GPI-anchorless prion strains caused a nearly identical clinical and pathological disease phenotype, yet maintained their structural diversity in the anchorless state. HXMS studies revealed that GPI-anchorless PrP^Sc is characterized by substantially higher protection against hydrogen/deuterium exchange in the C-terminal region near the N-glycan sites, suggesting this region had become more ordered in the anchorless state. For one strain, passage of GPI-anchorless prions into wild type mice led to the emergence of a novel strain with a unique biochemical and phenotypic signature. For the new strain, histidine hydrogen-deuterium mass spectrometry revealed altered packing arrangements of β-sheets that encompass residues 139 and 186 of PrP^Sc. These findings show how variation in post-translational modifications may explain the emergence of new protein conformations in vivo and also provide a basis for understanding how the misfolded protein structure impacts the disease.

Prions are considered the best example to prove that the biological information can be transferred protein to protein through a conformational change. The term “prion-like” is used to describe molecular mechanisms that share similarities with the mammalian prion protein self-perpetuating aggregation and spreading characteristics. Since prions are presumably composed only of protein and are infectious, the more similar the mechanisms that occur in the different neurodegenerative diseases, the more these processes will resemble an infection.In vitroandin vivoexperiments carried out during the last decade in different neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's diseases (PD), and amyotrophic lateral sclerosis (ALS) have shown a convergence toward a unique mechanism of misfolded protein propagation. In spite of the term “infection” that could be used to explain the mechanism governing the diversity of the pathological processes, other concepts as “seeding” or “de novoinduction” are being used to describe thein vivopropagation and transmissibility of misfolded proteins. The current studies are demanding an extended definition of “disease-causing agents” to include those already accepted as well as other misfolded proteins. In this new scenario, “seeding” would be a type of mechanism by which an infectious agent can be transmitted but should not be used to define a whole “infection” process.

Prion diseases are a group of rapidly progressing neurodegenerative disorders caused by the misfolding of the endogenous prion protein (PrP^C) into a pathogenic form (PrP^Sc). This process, despite being the central event underlying these disorders, remains largely unknown at a molecular level, precluding the prediction of new potential outbreaks or interspecies transmission incidents. In this work, we present a method to generate bona fide recombinant prions de novo, allowing a comprehensive analysis of protein misfolding across a wide range of prion proteins from mammalian species. We study more than 380 different prion proteins from mammals and classify them according to their spontaneous misfolding propensity and their conformational variability. This study aims to address fundamental questions in the prion research field such as defining infectivity determinants, interspecies transmission barriers or the structural influence of specific amino acids and provide invaluable information for future diagnosis and therapy applications.

Very solid evidence suggests that the core of full length PrP^Sc is a 4-rung β-solenoid, and that individual PrP^Sc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrP^Sc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions ~116/118, 133–134, 141, 152–153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrP^Sc. Such sites likely define loops and/or borders of β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrP^Sc (recPrP^Sc). The term recPrP^Sc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrP^Sc species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrP^Sc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular ~89/97-152, were detected in some recPrP^Sc preparations; similar fragments are characteristic of atypical strains of brain-derived PrP^Sc. Our results suggest a shared architecture of recPrP^Sc and brain PrP^Sc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrP^Sc. Furthermore, recombinant PrP^Sc offers exciting opportunities for structural studies unachievable with brain-derived PrP^Sc. PrP^Sc Prions propagate by inducing the refolding of the natively folded normal cellular prion protein (PrP^C) into the prion conformation in brain and other mammalian tissues (wild-type). Understanding the structure of PrP^Sc is essential to understanding how PrP^Sc prions propagate. The secondary structure of PrP^C is composed of four α-helical regions, two very small β-sheets and random coil, while PrP^Sc is composed entirely of β-sheets and random coil. The β-sheets of PrP^Sc wind four times to form a spring-like 4-rung β-solenoid. The rungs are connected by stretches of random coil. We have used proteinase K (PK), to cleave these random coil connecters, allowing us to identify the location of the more PK-resistant β-strand stretches within wild-type PrP^Sc. In this work, we use recombinant PrP^Sc, (in vitro generated infectious prions) to show that patterns of PK cleavage for recombinant and wild-type PrP^Sc are very similar, indicating that they share a common architecture. This also means that recombinant PrP^Sc is a true surrogate for wild-type PrP^Sc. Since recombinant PrP^Sc is derived from recombinant PrP, future structural studies employing specific amino acid or stable-isotope labeled amino acid substitutions are easily achievable. Such substitutions are essential for NMR studies.