Central residues in prion protein PrPC are crucial for its conversion into the pathogenic isoform.

Conformational conversion of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases. However, the conversion mechanism remains to be elucidated. Here, we generated Tg(PrPΔ91-106)-8545/Prnp0/0 mice, which overexpress mouse PrP lacking residues 91-106. We showed that none of the mice became sick after intracerebral inoculation with RML, 22L, and FK-1 prion strains nor accumulated PrPScΔ91-106 in their brains except for a small amount of PrPScΔ91-106 detected in one 22L-inoculated mouse. However, they developed disease around 85 days after inoculation with bovine spongiform encephalopathy (BSE) prions with PrPScΔ91-106 in their brains. These results suggest that residues 91-106 are important for PrPC conversion into PrPSc in infection with RML, 22L, and FK-1 prions but not BSE prions. We then narrowed down the residues 91-106 by transducing various PrP deletional mutants into RML- and 22L-infected cells and identified that PrP mutants lacking residues 97-99 failed to convert into PrPSc in these cells. Our in vitro conversion assay also showed that RML, 22L, and FK-1 prions did not convert PrPΔ97-99 into PrPScΔ97-99, but BSE prions did. We further found that PrP mutants with proline residues at positions 97 to 99 or charged residues at positions 97 and 99 completely or almost completely lost their converting activity into PrPSc in RML- and 22L-infected cells. These results suggest that the structurally flexible and noncharged residues 97-99 could be important for PrPC conversion into PrPSc following infection with RML, 22L, and FK-1 prions but not BSE prions.

Ethanolamine Is a New Anti-Prion Compound.

Prion diseases are a group of fatal neurodegenerative disorders caused by accumulation of proteinaceous infectious particles, or prions, which mainly consist of the abnormally folded, amyloidogenic prion protein, designated PrPSc. PrPSc is produced through conformational conversion of the cellular isoform of prion protein, PrPC, in the brain. To date, no effective therapies for prion diseases have been developed. In this study, we incidentally noticed that mouse neuroblastoma N2a cells persistently infected with 22L scrapie prions, termed N2aC24L1-3 cells, reduced PrPSc levels when cultured in advanced Dulbecco's modified eagle medium (DMEM) but not in classic DMEM. PrPC levels remained unchanged in prion-uninfected parent N2aC24 cells cultured in advanced DMEM. These results suggest that advanced DMEM may contain an anti-prion compound(s). We then successfully identified ethanolamine in advanced DMEM has an anti-prion activity. Ethanolamine reduced PrPSc levels in N2aC24L1-3 cells, but not PrPC levels in N2aC24 cells. Also, oral administration of ethanolamine through drinking water delayed prion disease in mice intracerebrally inoculated with RML scrapie prions. These results suggest that ethanolamine could be a new anti-prion compound.

Neurotropic influenza A virus infection causes prion protein misfolding into infectious prions in neuroblastoma cells.

Misfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.

Vaporized Hydrogen Peroxide and Ozone Gas Synergistically Reduce Prion Infectivity on Stainless Steel Wire.

Prions are infectious agents causing prion diseases, which include Creutzfeldt-Jakob disease (CJD) in humans. Several cases have been reported to be transmitted through medical instruments that were used for preclinical CJD patients, raising public health concerns on iatrogenic transmissions of the disease. Since preclinical CJD patients are currently difficult to identify, medical instruments need to be adequately sterilized so as not to transmit the disease. In this study, we investigated the sterilizing activity of two oxidizing agents, ozone gas and vaporized hydrogen peroxide, against prions fixed on stainless steel wires using a mouse bioassay. Mice intracerebrally implanted with prion-contaminated stainless steel wires treated with ozone gas or vaporized hydrogen peroxide developed prion disease later than those implanted with control prion-contaminated stainless steel wires, indicating that ozone gas and vaporized hydrogen peroxide could reduce prion infectivity on wires. Incubation times were further elongated in mice implanted with prion-contaminated stainless steel wires treated with ozone gas-mixed vaporized hydrogen peroxide, indicating that ozone gas mixed with vaporized hydrogen peroxide reduces prions on these wires more potently than ozone gas or vaporized hydrogen peroxide. These results suggest that ozone gas mixed with vaporized hydrogen peroxide might be more useful for prion sterilization than ozone gas or vaporized hydrogen peroxide alone.

Strain-Dependent Prion Infection in Mice Expressing Prion Protein with Deletion of Central Residues 91-106.

Conformational conversion of the cellular prion protein, PrPC, into the abnormally folded isoform, PrPSc, is a key pathogenic event in prion diseases. However, the exact conversion mechanism remains largely unknown. Transgenic mice expressing PrP with a deletion of the central residues 91-106 were generated in the absence of endogenous PrPC, designated Tg(PrP∆91-106)/Prnp0/0 mice and intracerebrally inoculated with various prions. Tg(PrP∆91-106)/Prnp0/0 mice were resistant to RML, 22L and FK-1 prions, neither producing PrPSc∆91-106 or prions in the brain nor developing disease after inoculation. However, they remained marginally susceptible to bovine spongiform encephalopathy (BSE) prions, developing disease after elongated incubation times and accumulating PrPSc∆91-106 and prions in the brain after inoculation with BSE prions. Recombinant PrP∆91-104 converted into PrPSc∆91-104 after incubation with BSE-PrPSc-prions but not with RML- and 22L-PrPSc-prions, in a protein misfolding cyclic amplification assay. However, digitonin and heparin stimulated the conversion of PrP∆91-104 into PrPSc∆91-104 even after incubation with RML- and 22L-PrPSc-prions. These results suggest that residues 91-106 or 91-104 of PrPC are crucially involved in prion pathogenesis in a strain-dependent manner and may play a similar role to digitonin and heparin in the conversion of PrPC into PrPSc.