Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrP^C into its pathogenic isoform PrP^{Sc 1}. PrP^C is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrP^Sc forms detergent-insoluble aggregates and is partially resistant to PK^2-6. The conversion of PrP^C to PrP^Sc is known to involve a conformational transition of α-helical to β-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrP^Sc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain⁷.Using a combination of biophysical and biochemical approaches, we identified insoluble PrP^C aggregates (designated iPrP^C) from uninfected mammalian brains and cultured neuronal cells^{8, 9}. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms¹⁰, and PK-treatment. The combination of these approaches isolates not only insoluble PrP^Sc and PrP^C aggregates but also soluble PrP^C oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrP^Sc from infected brains and iPrP^C from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrP^C are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrP^Sc that shares the immunoreactive behavior and fragmentation with iPrP^{C 11, 12}. Moreover, we recently demonstrated that iPrP^C is the main species that interacts with amyloid-β protein in Alzheimer disease¹³. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer''s disease¹³, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.