Prion diseases are fatal neurodegenerative disorders and are linked with the conversion of the cellular isoform of the prion protein (PrP(C)) into the abnormal beta-sheet-rich isoform. It is widely accepted that the soluble oligomers of beta-PrP are neurotoxic and that they are more pathologically significant. To unravel the molecular mechanism under the conversion process, it is critical to identify the factors that can promote the conversion from PrP(C) to the beta-oligomers. By recording circular dichroism spectra and performing a size-exclusion HPLC assay, we found that the conformation of the recombinant human prion protein (rPrP(C)) was converted from an alpha-helical conformation into beta-sheet oligomers under a macromolecular crowding condition. The soluble beta-oligomers of rPrP were resistant to proteinase K digestion and could bind to the dyes thioflavin T and 8-anilino-1-naphthalene sulfonate. Furthermore, by the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, we showed that the soluble beta-oligomers were neurotoxic. These results suggest that macromolecular crowding, which has not been considered before, is a key intracellular factor in the formation of soluble neurotoxic beta-oligomers in prion diseases.
Autophagy plays an important role in targeting cellular proteins, protein aggregates and organelles for degradation for cell survival. Autophagy dysfunction has been extensively described in neurodegenerative conditions linked to protein misfolding and aggregation. However, the role of autophagy in the prion disease process is unclear. Here, we show that when expressed in mouse neuroblastoma N2a cells, cytoplasmic PrP (cyPrP) aggregates lead to endoplasmic reticulum stress (ER stress), activation of reticulon 3 (RTN3), impairment of ubiquitin-proteasome system (UPS), induction of autophagy and apoptosis. RTN3 belongs to the reticulon family with the highest expression in the brain and RTN3 is often activated under ER stress. To assess the function of RTN3 in pathological conditions involving cyPrP protein misfolding, we knocked down the expression of RTN3 in cyPrP-transfected cells; unexpectedly, the inhibition of expression of RTN3 enhances the induction of autophagy resulted from cyPrP aggregates, and the process is mediated by the enhanced interaction between Bcl-2 and Beclin1 promoted by RTN3, which enhances Bcl-2-mediated inhibition of Beclin 1-dependent autophagy. Furthermore, down-regulation of RTN3 promoted the clearance of cyPrP aggregates, allowed the activity of the UPS to resume and alleviated ER stress; ultimately, apoptosis due to the cyPrP aggregates was inhibited. Together, these data suggest that RTN3 negatively regulates autophagy to block the clearance of cyPrP aggregates and provide a clue regarding the potential to induce autophagy for the treatment of prion disease and other neurodegenerative diseases such as Parkinson disease (PD), Alzheimer disease (AD) and Huntington disease (HD).
Prion diseases are fatal neurodegenerative disorder associated with the conversion of the cellular isoform of the prion protein (PrP(C)) into the infectious scrapie isoform (PrP(Sc)). Deposition of misfolded prion proteins (PrP) on certain regions of brain can result in prion diseases. As a membrane-bound chaperone of the endoplasmic reticulum (ER), calnexin ensures the proper folding and quality control of newly synthesized proteins. Using purified components in vitro, calnexin associated with many proteins and suppresses their thermal aggregation effectively. We for the first time analyzed PrP-calnexin interaction. The immunoprecipitation, confocal microscope and native polyacrylamide-gel electrophoresis results indicated that calnexin could bind PrP both in vitro and in vivo. The turbidity result showed that calnexin could supress thermal aggregation of PrP. MTT, flow cytometry (FCM) and caspase activity studies demonstrated that calnexin prevent caspase-3-mediated cytotoxicity induced by PrP. These results implied that calnexin is potentially beneficial for the resistance of prion diseases.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.