Unraveling the key to the resistance of canids to prion diseases

Abstract

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.

Dogs are resistant to prion infection, due to the presence of aspartic or glutamic acid at position 163 of their prion protein

“…These studies revealed that feline PrP was the most similar in terms of amino acid sequence and as domestic cats are susceptible to at least three known prion strains (BSE, CWD, and CJD) the six amino acid difference between canine and feline PrP was studied in detail. The E/D polymorphism in position 163 was highlighted due to its almost exclusive presence in the canidae family and chosen as the most likely candidate for canine resistance to prion disease . NMR assessment of dog’s PrP C structure also pointed out the unique charge distribution posed by the presence of D in position 163 .…”

“…Considerable amounts of data have been generated suggesting that canine PrP C is highly resistant to conformation change to PrP res compared to prion susceptible species. 22,32,36,37,41,42 Our group has now established, both in vivo and in vitro, that the amino acid residue in position 163 is the key determinant 32,33 and that an aspartic or a glutamic acid in this position (or equivalent in other species) is what conferred resistance to prion infection in the models in which those proteins were expressed. 32,34 Furthermore, these PrP C that are highly resistant to conformation change showed a dominant negative effect when co-expressed with WT mouse PrP.…”

“…22,32,36,37,41,42 Our group has now established, both in vivo and in vitro, that the amino acid residue in position 163 is the key determinant 32,33 and that an aspartic or a glutamic acid in this position (or equivalent in other species) is what conferred resistance to prion infection in the models in which those proteins were expressed. 32,34 Furthermore, these PrP C that are highly resistant to conformation change showed a dominant negative effect when co-expressed with WT mouse PrP. 32 Prior to the present report, evidence confirming that murine models expressing WT canine PrP C were resistant to infection by a panel of prion isolates from different species was lacking and we have addressed this by both bioassays and in vitro propagation experiments.…”

“…Sequence alignment studies of the PRNP gene identified the presence of either glutamic (E) or aspartic (D) acids (both negatively charged amino acids) in position 163 in dogs PrP when compared to cats and these might be responsible for the differing resistance of the two species with respect to susceptibility to BSE and CWD . Furthermore, mouse PRNP with substitutions equivalent to the canine amino acid residues proved to be resistant to conversion to PrP res both in vitro, by means of recombinant PrP‐based PMCA, and in vivo in two different transgenic mouse models with asparagine (N) to aspartic acid substitution at position 158 (N158D) . Additionally, mouse PRNP with this canid substitution provided a protective dominant negative effect by inhibiting PrP C conversion in transgenic chimeras co‐expressing WT mouse PRNP .…”

“…26 Furthermore, mouse PRNP with substitutions equivalent to the canine amino acid residues proved to be resistant to conversion to PrP res both in vitro, by means of recombinant PrP-based PMCA, and in vivo in two different transgenic mouse models with asparagine (N) to aspartic acid substitution at position 158 (N158D). 32 Additionally, mouse PRNP with this canid substitution provided a protective dominant negative | 3971 VIDAL et AL. effect by inhibiting PrP C conversion in transgenic chimeras co-expressing WT mouse PRNP.…”