The unique phenotypic characteristics of mammalian prions are thought to be
The unique phenotypic characteristics of mammalian prions are thought to be encoded in the conformation of pathogenic prion proteins (PrPSc). the competitive selection of those with lower initial conformational stability. In serial propagation with a nonglycosylated mutant PrPC substrate the dominant PrPSc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to its lowest stability. Cumulatively the data show that SERK1 sporadic Creutzfeldt-Jakob disease PrPSc is not a single conformational entity but a dynamic collection of two distinct populations of particles. This implies the co-existence of different prions whose adaptation and evolution are governed by the selection of progressively less stable faster replicating PrPSc conformers. (1-3) all provide compelling evidence that prion diseases are caused by the accumulation of an aberrantly folded isoform of the prion protein termed PrPSc (4). Variations in prions which cause remarkably different disease phenotypes in the same host are referred to as strains (5 6 For several decades the existence of distinct prion strains that can be passaged indefinitely has polarized the scientific community and was offered as an argument for the existence of a prion-specific genome. Subsequently extraordinary progress in the past decade has produced convincing experimental evidence indicating that the species of prion is encoded in the primary amino acid sequence of PrPSc (6) and that prion strain characteristics are encoded in the self-replicating conformation of PrPSc (7-10). These phenotypic characteristics may undergo mutation in cloned cells but the molecular mechanism responsible for this phenomenon remained elusive in Bay 65-1942 HCl the absence of informative nucleic acid (10). Although recent important experiments with synthetic and rodent-adapted laboratory prions suggest that structural plasticity of PrPSc is a key factor in adaptation and evolution the exact conformational mechanism and relevancy of these observations to wild prions causing natural human prion diseases have not been established (11-13). The extensive phenotypic heterogeneity of the most frequent human prion disease sporadic Creutzfeldt-Jakob disease (sCJD) (14) is currently understood as a Bay 65-1942 HCl complex interplay between polymorphisms in the gene and different PrPSc conformers (6 14 Because the conformations of PrPSc vary in different prion strains the broad spectrum of distinct PrPSc conformers recently found in different cases of sCJD using sensitive biophysical techniques implies that sCJD is caused by a broad array of distinct prions (5 15 16 Furthermore the frequent and perhaps universal presence of both the 21-kDa (type 1) and 19-kDa (type 2) unglycosylated fragments of protease-resistant (r) PrPSc in sCJD (17-21) indicates the co-occurrence of markedly different PrPSc conformers often in the same anatomical structure in the same brain. Apart from challenging the validity of the clinicopathological classification of sCJD based on Bay 65-1942 HCl gene polymorphism and Western blot patterns of type 1 or type 2 rPrPSc (14 22 these findings raise some fundamental questions. (coding region was performed as described (26-28). Patients lacked pathogenic mutations in the and had no history of Bay 65-1942 HCl familial diseases or known exposure to prion agents. These cases underwent additional detailed WB analyses of the PrPSc so that we could ascertain the accuracy of their original classification and confirm that the same brain homogenate analyzed by CDI contained mixed type 1 + 2 PrPSc(129M). Coronal Bay 65-1942 HCl sections of human brain tissues were obtained at autopsy and stored at ?80 °C. Three 200-350-mg cuts of frontal (superior and more posterior middle gyri) or occipital cortex were taken from each brain and used for molecular analyses. Brain Homogenates Slices of tissues weighing 200-350 mg were first homogenized to a final 15% (w/v) concentration in calcium- and magnesium-free PBS pH 7.4 by three 75-s cycles with Mini-beadbeater 16 Cell Disrupter (Biospec Bartlesville OK). The homogenates were then diluted to a final 5% (w/v) in 1% (v/v) Sarkosyl in PBS pH 7.4 and rehomogenized. After clarification at 500 × for 5 min 1 aliquot of the supernatant was treated with protease inhibitors (0.5 mm PMSF and aprotinin and leupeptin at 5 μg/ml respectively). The second aliquot was treated with 50 μg/ml of proteinase K (Amresco Solon OH) for 1 h at 37 °C and shaking 600 rpm on an Eppendorf Thermomixer (Eppendorf Hauppauge NY).