MPI 1 Mechanism and Prevention of Scrapie Prion Protein Formation

Our project was designed to elucidate the molecular pathways underlying the transition of the Prion protein (PrPC) into a pathogenic conformation, the neurotoxic effects mediated by misfolded PrP conformers, and to develop potential therapeutic strategies. The project combined in vitro and in vivo approaches, ranging from experiments with pure proteins to the analysis of neuronal cells and infected mice. Using a range of methods from biochemistry, biophysics and cell biology, the following specific aims were addressed: Aim 1: To identify domains involved in PrP misfolding and PrPSc propagation Within the framework of the grant we took different approaches to provide mechanistic insight into the conformational transition of PrPC into a pathogenic conformation. First, we created a set of PrP mutants to systematically identify domains involved in maturation and misfolding of PrPC. Second, we analyzed different pathogenic PrP mutants, linked to inherited prion diseases in humans. Third, we performed a detailed analysis of the activity of suramin, an anti-prion compound that was previously identified in a close collaboration with Prof. Schätzl. This comprehensive analysis revealed the following results: - Helix 1 has a bipartite function in the maturation and aggregation of PrP. Formation of complex glycosylated PrP is critically dependent on a membrane anchor (Winklhofer et al., 2003, J Biol Chem). - The C-terminal domain of the prion protein is necessary and sufficient for import into the endoplasmic reticulum (Heske et al., 2004, J Biol Chem). - Pathogenic mutations located in the hydrophobic core of the prion protein interfere with folding and attachment of the glycosylphosphatidylinositol anchor (Kiachopoulos et al., 2005, J Biol Chem). - A quality control pathway for aberrant PrP conformers is present at the plasma membrane (Kiachopoulos et al., 2004, Traffic). - Polarized sorting of PrPC is compromised by pathogenic mutations within the internal hydrophobic domain and by Doppel (Dpl), a neurotoxic paralog of PrPC (Uelhoff et al., 2005, J Biol Chem). Aim 2: To elucidate the role of posttranslational modifications in the formation of PrPSc Mature PrPC is characterized by two N-linked glycans of complex structure and a glycosylphosphatidylinositol (GPI) anchor, raising the question of how propagation of pathogenic PrPSc might be influenced by these posttranslational modifications. Using our scrapie-infected mouse neuroblastoma cells we could show that the complex glycans are dispensable for cellular trafficking of PrPC, however high mannose glycoforms of PrPC are preferred substrates for the conversion into PrPSc (Winklhofer et al., 2003, Traffic). In collaboration with Prof. Oesterhelt and Prof. Engelhard, recombinant (r)PrP expressed in and purified from bacteria was fused with a double palmitoylated and fluorescently labeled peptide to yield lipidated, fluorescent rPrPPalm. In vitro and cell culture assays revealed incorporation of rPrPPalm into lipid vesicles and into the plasma membrane of live neuronal cells (Olschewski et al., 2007, submitted). Aim 3: To analyze neurotoxic effects of PrP accumulation in the cytosol A transgenic mouse model indicated that PrP present in the cytosolic compartment has a neurotoxic potential. We could now show that two pathogenic mutants, Q160Stop and W145Stop, which are linked to inherited prion diseases in humans, are partially located in the cytosol and induce apoptosis (Heske et al., 2004, J Biol Chem; Rambold et al., 2006, Mol Cell Biol). Moreover, we established a yeast and a mammalian cell culture model to demonstrate and analyze the cytotoxic potential of PrP in the cytosolic compartment. Our work revealed that association of the anti-apoptotic protein Bcl-2 with misfolded Prion protein is linked to the toxic potential of cytoPrP. Furthermore, we could provide evidence for a protective role of molecular chaperones (Heller et al., 2003, J Biol Chem; Rambold et al., 2006, Mol CellBiol). Aim 4: To develop strategies to interfere with the propagation of infectious prions We have previously employed scrapie-infected N2a cells as a model to screen for and analyze compounds with anti-prion activity. In a close collaboration with Prof. Kretzschmar and Prof. Tavan we used our cell culture model to re-screen compounds identified in an in vitro anti-aggregation assay. New compounds with anti-prion activity were identified (Bertsch et al., 2005, J Virol), leading to a collaborative patent application ( PCT/EP2005/005614).


Launching date




Funded by

Bavarian State Ministry for Environmental Affairs and Consumer Protection