Effect of transition metals (Mn, Cu, Fe) and deoxycholic acid (DA) on the conversion of PrPC to PrPres.

The PMCA (protein misfolding cyclic amplification) technique has been shown to drive the amplification of misfolded prion protein by PrP(Sc) seeds during several cycles of incubation-sonication. Here, we report that cyclic amplification of normal hamster brain homogenates treated with a number of transition metals (manganese [Mn], copper [Cu], and iron [Fe]) leads to conversion of PrP(C) into protease-resistant PrP(res). The efficiency of PrP(res) formation and the glycoforms induced by Mn were different from those obtained by Cu and Fe. Previous results have shown higher Mn and lower Cu levels in the affinity-purified PrP(Sc) from the brain of prion diseases compared with normal hamster brain homogenates. We focused on Mn because we observed higher levels of Mn in whole brain, mitochondria, and scrapie-associated fibril-enriched fractions from the brains of animals with prion disease. In the presence of minute quantities of Mn-induced PrP(res) template with a large amount of PrP(C), PrP(res) amplification is observed. A metal chelater, EDTA reverses the effect of Mn on PrP(res) amplification, suggesting that Mn may play a role in the formation of PrP(res). It has been proposed that metal-catalyzed oxidation of PrP leads to the oxidation of amino acids and extensive aggregation of oxidized PrP. Carboxyl acids such as deoxycholic acid (DA) are oxidized molecules produced by 3' oxidation pathway. In in vitro studies, the potent effect of Mn on PrP(res) amplification is augmented by DA in a dose-dependent manner. On the basis of the evidence of the elevated Mn levels in scrapie-associated fibril (SAF)-enriched preparations from the brains of animals with prion disease, Mn-loaded PrP and oxidized molecules such as carboxyl acids may contribute to the formation of the scrapie isoform of PrP in prion diseases.