The Investigations Of Prion's Propagation In Peripheral Tissues And NADPH Enhances The Replication Of PrP^Sc With PMCA

Transmissible spongiform encephalopathies(TSEs),or prion diseases,are transmissible neurodegenerative disorders of protein conformation.This group of diseases such as Creutzfeldt-Jakob disease(CJD),fatal familial insomnia(FFI), Gerstmann-Straussler-Scheinker syndrome(GSS),bovine spongiform encephalopathy(BSE),sheep and goat scrapie et al,are caused by a unique infectious agent that seems to be able to propagate without any nucleic acid,termed prions. Prions are considered to consist mainly of a misfolded scrapie-associated,aggregated protease-resistant isoform of the cellular prion protein(PrP^C) and the pathological isoform presented in the tissues of infected individuals is called PrP^Sc.The investigation on this convesion is considered to be crucial to verify the mechanism of prion disease and to prevent or cure TSEs.In this study,a methodology,namely protein misfolding cyclic amplification(PMCA),was utilized to reveal the propagation ability of prions across various tissues and accessory pyridine nucleotides facilitated prion's propagation in vitro.To verify the propagating ability of PrP^Sc,various tissues were spiked PrP^Sc and subjected to PMCA.The results showed the PrP^Sc from hamsters' brain tissues of scrapie agent 263K could replicate efficiently in spleen and muscle tissues like in brain,but failed in kidney and intestine.The main biochemical properties of the PMCA-generated PrP^Sc were similar as the brain-derived prions,such as PK-resistance,the glycosylated patterns as well as ratios.Moreover,the agents that could destroy the infectivity of PrP^Sc in bioassays,i.e.sodium hydroxide and thermal inactivation,were capable of abolishing the replicative activities of PMCA-derived PrP^Sc.This study provides reliable data that the PrP^Sc from TSEs-infected brains can utilize the PrP^C from non-neural tissues for its propagation.Similarity of the replicative ability in PMCA in vitro and the infectivity in vivo for evaluation of the biochemical features and inactivating effectiveness of TSEs agent highlights the possibility to use PMCA instead of time-consumed animal challenge.To verify whether the reduced pyridine nucleotides,for example,reduced nicotinamide adenine dinucleotide phosphate(NADPH),could facilitate propagation of PrP^Sc in vitro.It showed that PrP^Sc can propagate more efficiently when NADPH was present in the PMCA reaction.This facilitation revealed a significant dose-dependent relationship and abolished when NADPH had been oxidized. Reduced nicotinamide adenine dinucleotide(NADH),an analogue of NADPH,could also perform this enhancement,whereas other analogues,including NADP,NAD and ascorbate failed.Recombinant hamster full-length PrP(23-231)was also utilized in PMCA reactions to determine the direct function of NADPH.The results demonstrated that NADPH could promote the recombinant PrP conversion into proteinase K-resistant isoform,but scrapie strain 263K could not convert human recombinant PrP(23-230) in PMCA.We conclude that reduced pyridine nucleotide can efficiently facilitate the propagation of prions in vitro and this activity of enhancement is also limited by species barrier.More importantly,this phenomenon reveals a potential mechanism that reduced pyridine nucleotide plays an accessory factor during the development of TSEs.