Identification of protective prion protein residues with flies: insights into the dog PrP-N158D substitution. Diego E. Rincon Limas¹, Jonatan Sanchez-Garcia¹, Yan Zhang¹, Joaquin Castilla², Pedro Fernandez-Funez¹. 1) Dept Neurology, University of Florida, Gainesville, FL; 2) CIC bioGUNE, Bizkaia, Spain.

   The central event in the pathogenesis of all forms of prion disease involves a conversion of the host-encoded cellular prion protein PrPc to its pathogenic conformer PrPsc. However, the molecular mechanisms that regulate this conformational conversion are mostly unknown. A clue to understanding the structure and conformational dynamics of PrP has come from the dog, a rare mammal resistant to prion diseases. A comparative study identified a charged amino acid (PrPD158) in dog PrP that is not conserved in other mammals susceptible to prion diseases (PrPN158). Unfortunately, little is known about how this residue affects PrP structure. We hypothesized that altering the charge of the loop connecting Helix 1 and the first beta-sheet could affect the stability of the globular domain and, thus, the toxicity of PrP. To determine the stabilizing effect of Asp158, we compared transgenic flies expressing wild type (MoPrP) and mutant (MoPrP-N158D) mouse PrP. We first observed that the MoPrP-N158D protein is more stable than MoPrP since significantly lower levels of mRNA lead to comparable levels of protein, suggesting that MoPrP is actively degraded in flies. We have shown before that MoPrP accumulates disease-specific PrP isoforms by immunoprecipitation with the 15B3 conformational antibody. However, flies expressing MoPrP-N158D do not accumulate 15B3-specifc conformations, indicating its higher conformational stability. Finally, whereas expression of MoPrP in motor neurons induced aggressive locomotor dysfunction in climbing assays, flies expressing MoPrP-N158D were similar to control flies, supporting the lack of toxicity. These results demonstrate that Asp158 exerts a key stabilizing activity on PrP and prevents formation of disease-specific PrP isoforms. Altogether, our data indicate that residue PrPN158 might be a target for anti-prion therapies and that Drosophila is an ideal system to genetically dissect fundamental, unknown aspects of PrP-associated pathology.