Investigations on post-endoplasmic reticulum quality control mechanisms for prion protein aggregates

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Sustaining correct protein folding is critically important for cell survival and several neurodegenerative disorders are associated with the accumulation of misfolded proteins. Among these are prion diseases, which are fatal transmissible disorders of animals and humans. They are unique because they are caused by prions, pathogens which are composed solely of a misfolded isoform of the cellular prion protein PrPc, termed PrPSc. The long-term goals of my group are to elucidate mechanisms of neurodegeneration and to discover novel targets for inhibiting prion propagation and spreading by studying prion-host cell interactions and trafficking of prion proteins. One central question of my research program is how correct folding of PrPc is controlled within neuronal cells. PrPc is a glycoprotein located at the cell surface. Here, PrPc is found in lipid rafts, membrane microdomains with a high content of cholesterol and sphingolipids. Upon recycling and final internalization, PrPc is transported along the endocytic pathway for degradation in acidic vesicles. In mammalian cells, the best characterized protein quality control machinery resides in the endoplasmic reticulum (ER). However, what happens beyond the ER? Are there similar mechanisms existing that ensure protein quality control in post-ER compartments, such as the Golgi network? In my previously published work I described a novel post-ER quality control mechanism for PrPc. I demonstrated that treatment of cultured cells with the naphthyl-urea compound suramin induces PrPc aggregation. These aggregates are intracellulary retained and their transport to the cell surface is prevented. Instead, they are re-routed from the trans-Golgi network (TGN) to lysosomes for degradation. In contrast to pathogenic PrPSc aggregates, they are non-infectious and can be degraded by the cells with a half-life similar to that of PrPc. My further deletion mutant analysis revealed that re-routing, but not aggregation, is dependent on the presence of the pre-octarepeat domain (aa 23-50) of the PrP amino-terminus. Of note, similar observations were made by others for pathogenic PrP mutants. These mutants had a higher propensity to aggregate and were, as the drug-induced PrP aggregates described by myself, intracellularly re-routed to lysosomes for degradation. Furthermore, the protease furin is subjected to post-ER quality control upon aggregation, and aggregates are transported to lysosomes. This indicates that re-routing of protein aggregates from TGN to lysosomes is a cellular mechanism not only relevant for PrP trafficking. In contrast to the well investigated ER protein quality control mechanisms, only little is known about post-ER protein quality control in mammalian cells. The goal of my proposed studies is to use drug-induced PrP aggregates as a model to gain novel mechanistic insights into post-ER quality control in mammalian cells. This will be achieved by three specific aims: 1. to identify proteins that are involved in post-ER quality control by siRNA library screens 2. to analyse the role of glycosylation in recognition of PrP aggregates, and 3. to investigate PrP aggregation in non-drug treated neuronal cells. I expect to obtain new insights into so far under-investigated post-ER quality control mechanisms in mammalian cells. Results obtained from my study will advance the understanding of the cellular capacity to counteract protein misfolding not only of the prion protein, but also of other proteins travelling through the secretory pathway. In a long term, my fundamental research on protein misfolding will result in novel drug targets to improve protein folding which will inspire translational approaches by the applied health research industry.