Cellular and molecular mechanisms of prion protein function
Final Report Abstract
Growing evidence indicates that the cellular prion protein (PrPC) triggers neurotoxic signals during early stages of prion and Alzheimer’s disease. However, neither the mechanistic basis nor the complexity of such signals is well understood. The present project aimed at elucidating molecular mechanisms of PrP function via a combination of biochemical and cell biological methods in zebrafish embryos. Using standard procedures, we generated antibodies against zebrafish PrPs and used them to confirm the expression of PrP-2 in developing neurons (via immunostaning) as well as to detect the potential conversion of fish PrPs upon exposure to mouse scrapie (via the proteinase K assay). In addition, we analyzed the signaling properties of chimeric PrP molecules with different C-terminal tails. Expression of these proteins in Drosophila S2 cells and zebrafish embryos showed that the specificity of PrP intracellular signals is partly determined by the sequence of its GPI-anchor attachment site. Thus, compared to other GPI-tails and transmembrane domains, those of PrP and Thy-1 shared enhanced activities in cell spreading and SFK activation assays. In addition, our PrP gain- and loss-of-function analyses in zebrafish embryos revealed that the corresponding phenotypes are mediated by the SFKs Fyn and Yes. Hence, activation of SFKs by PrP slows down their degradation and leads to increased SFK signaling. Our data strongly suggest that, during gastrulation, SFKs phosphorylate adherens junction complexes, thereby preventing their endocytosis and degradation. Therefore, PrP is a positive regulator of SFK activity and E-cadherin-based adhesion. Using these functional readouts in zebrafish embryos, we tested the in vivo activity of mouse PrP pathogenic mutants and concluded that their neurotoxicity is not likely to involve loss-of-function but rather subversion or gain-of-function mechanisms. Further mutational analyses showed that the roles of mouse and zebrafish PrPs in cell contact formation, intracellular signaling and cell adhesion largely rely on its globular domain, and that the repetitive region modulates this activity. Interestingly, exposure of primary zebrafish blastocytes to aβ oligomers triggered the PrP/SFK/E-cadherin pathway, indicating that zebrafish embryos can be used as a tool to study downstream signaling events triggered by the interaction between aβ oligomers and PrP in vivo. Altogether, these experiments improve our mechanistic understanding of the roles of PrP in development, physiology and disease, and set the stage for future work aimed at drug discovery and the identification of therapeutic targets for neurodegenerative disease.
Publications
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2013. Conserved roles of the prion protein domains on subcellular localization and cell-cell adhesion. PLoS ONE, 8(7): e70327
Solis GP, Radon Y, Sempou E, Jechow K, Stuermer CAO and Málaga-Trillo E
