Abstract
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 brain(7). 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 forms(10), 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 disease(13). In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease(13), suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.