Polar substitutions in helix 3 produce toxic, transmembrane isoforms of the Prion protein. Jonatan Sanchez-Garcia¹, Daniela Arbelaez¹, Kurt Jensen¹, Diego Rincon-Limas^1,3, Pedro Fernandez-Funez^1,2,3. 1) Department of Neurology, Univ of Florida, Gainesville, FL; 2) Department of Neuroscience, Center for Movement Disorders and Neurorestoration, University of Florida, Gainesville, FL 32611, USA; 3) Genetics Institute and Center for Translational Research on Neurodegenerative Diseases, University of Florida, Gainesville, FL 32611, USA.

   Prion diseases encompass a diverse group of neurodegenerative conditions characterized by vacuolar degeneration and accumulation of misfolded conformers of the Prion protein (PrP). Although transmission of these disorders are mediated by the protease-resistant scrapie conformation (PrPSc), other PrP isoforms mediate neurodegeneration. To better understand how PrP misfolding leads to neurotoxicity, we introduced polar substitutions in two conserved methionines in helix 3, M205 and M212, in mouse PrP. In vitro studies revealed that these two residues controlled the stability of the globular domain, while oxidation of these Met was proposed to promote PrP conversion in humans and mice. To study the consequence of M205S and M205,212S on PrP biogenesis, folding, and pathogenesis in vivo, we expressed these mutants in Drosophila. We found that, unlike PrP-WT, M205S and M205,212S underwent hyperglycosylation, intracellular accumulation, and widespread conformational changes due to the lack oxidative folding. Surprisingly, PrP-M205S and PrP-M205,212S accumulated as C-terminal transmembrane (Ctm), a topology that had only been described for mutations in the signal peptide and the transmembrane domain and it is linked to prion disease. Finally, PrP-M205,212S not localized in the lipid rafts altering localization of syntaxin and neuroglian in the lipid rafts. These mislocalizations induce abnormal development of axonal projections in the brain and indicate PrP-M205,212S neurodevelopmental toxicity. These results identify the lack of oxidative folding as a key factor in the formation of Ctm PrP, a mechanism that may be relevant in the pathogenesis of several inherited forms of prion diseases.