In the brains of humans and animals affected by prion disorders such as vCJD, BSE, scrapie, and chronic wasting disease, a cellular glycoprotein termed PrPC converts into an insoluble, pathogenic isoform designated PrPSc. Much evidence supports the notion that PrPSc is a crucial component of infectious prions. Although biophysical, biochemical, and in vivo infectivity studies have helped in defining the composition of infectious prions; the cellular mechanisms that mediate the generation of new infectious prions remain unknown. To address this question, the principal investigator has developed and characterized a unique system to amplify PrPSc 10-fold in brain membrane preparations in vitro without detergents or sonication. Consistent with the characteristics of PrPC conversion to PrPSc in vivo, this method this method of PrPSc amplification is species and strain specific, and depends on time, pH, and temperature. Using this system, the principal investigator has discovered that in addition to PrPC and PrPSc, specific host RNA molecules and a free thiolate anion are required for the in vitro amplification. The principal investigator has now been able to purify PrPC in an amplifiable state to carry out these conversion reactions. Based on these observations, the mechanisms underlying the conformational change of PrPC to PrPSc will be defined in three Specific Aims. In Aim 1, novel cofactors required for PrPSc amplification in vitro will be identified. In Aim 2, specific RNA molecules that catalyze PrPSc formation in vitro will be identified, and in Aim 3, the mechanism by which thiolate groups facilitate PrPSc amplification will be determined. These investigations will further our knowledge of the fundamental mechanism underlying prion propagation, and provide new directions in the investigation of neurodegenerative diseases. [unreadable] [unreadable]