Proteolytic processing of the prion protein (PrPC) by endogenous proteases generates bioactive membrane-bound and soluble fragments which may help to explain the pleiotropic roles of this protein in the nervous system and in brain diseases. Shedding of almost full-length PrPC into the extracellular space by the metalloprotease ADAM10 is of peculiar relevance since soluble PrP stimulates axonal outgrowth and is protective in neurodegenerative conditions such as Alzheimer’s and prion disease. However, molecular determinates and mechanisms regulating the shedding of PrP are entirely unknown. We produced an antibody recognizing the neo-epitope of shed PrP generated by ADAM10 in biological samples and used it to study structural and mechanistic aspects affecting the shedding. For this, we investigated genetically modified cellular and murine models by biochemical and morphological approaches. We show that the novel antibody specifically detects shed PrP in cell culture supernatants and murine brain. We demonstrate that ADAM10 is the exclusive sheddase of PrPC in the nervous system and reveal that the glycosylation state and type of membrane-anchorage of PrPC severely affect its shedding. Furthermore, we provide evidence that PrP shedding can be modulated by pharmacological inhibition and stimulation and present data suggesting that shedding is a relevant part of a compensatory network ensuring PrPC homeostasis of the cell. With the new antibody, our study introduces a new tool to reliably investigate PrP-shedding. In addition, this study provides novel and important insight into the regulation of this cleavage event, which is likely to be relevant for diagnostic and therapeutic approaches even beyond neurodegeneration.

The cellular prion protein, designated PrP^C, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrP^C into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrP^C remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrP^C is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrP^C-null mice (Prnp^0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp^0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp^0/0 mice from the lethal infection with IAV. Moreover, Prnp^0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPΔOR)/Prnp^0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrP^C has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp^0/0 and Tg(PrPΔOR)/Prnp^0/0 lungs than in WT lungs. It is thus conceivable that PrP^C functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrP^C in protection against IAV infection, and suggest that PrP^C might be a novel target molecule for anti-influenza therapeutics. Influenza A virus (IAV) is an enveloped, negative sense, single-stranded RNA virus, causing seasonal epidemic outbreaks of influenza. Anti-influenza agents targeting viral molecules, such as neuraminidase inhibitors, are currently available. However, these agents have accelerated emergence of mutant IAVs that are resistant to these agents among human populations. Development of new types of anti-influenza agents is awaited. We show that the cellular prion protein PrP^C has a protective role against lethal infection with IAVs through the octapeptide repeat (OR) region by abrogating lung epithelial cell apoptosis induced by reactive oxygen species (ROS) in infected lungs. We also show that PrP^C could reduce ROS in IAV-infected lungs through the OR region by maintaining Cu ion homeostasis and thereby activating Cu/Zn-dependent superoxide dismutase, SOD1. These results highlight the protective role of PrP^C in IAV infection. Elucidation of the exact mechanism underlying the PrP^C-mediated protection against IAV infection would be important for further understanding the pathogenesis of IAV infection and could be useful for development of new types of anti-influenza therapeutics.

Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrP^Sc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrP^Sc, in brains through constitutive conformational conversion of the cellular prion protein, PrP^C, into PrP^Sc. However, the cellular mechanism by which PrP^Sc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrP^Sc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrP^C and PrP^Sc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrP^Sc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrP^Sc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrP^Sc accumulation in prion-infected cells. The negative role of sortilin in PrP^Sc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrP^Sc in their brains. Interestingly, sortilin was reduced in prion-infected cells and mouse brains. Treatment of prion-infected cells with lysosomal inhibitors, but not proteasomal inhibitors, increased the levels of sortilin. Moreover, sortilin was reduced following PrP^Sc becoming detectable in cells after infection with prions. These results indicate that PrP^Sc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrP^Sc accumulation of itself could impair the sortilin-mediated sorting of PrP^C and PrP^Sc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrP^Sc in prion-infected cells. Once prions consisting mainly of PrP^Sc infect hosts, they constitutively propagate in their brains. Progressive production of PrP^Sc through the constitutive conformational conversion of PrP^C into PrP^Sc underlies prion propagation. However, the mechanism enabling progressive production of PrP^Sc in prion-infected cells remains unknown. We here found that the VPS10P sorting receptor sortilin is involved in degradation of PrP^C and PrP^Sc in infected cells by binding to both molecules and subsequently trafficking them to the lysosomal protein degradation pathway. Interestingly, we also found that degradation of sortilin was stimulated in lysosomes in prion-infected cells possibly as a result of the sortilin-PrP^C or -PrP^Sc complexes being trafficked to lysosomes. Our findings indicate that PrP^Sc itself impairs the sortilin-mediated degradation of PrP^C and PrP^Sc by stimulating degradation of sortilin in lysosomes. This eventually results in progressive production of PrP^Sc in prion-infected cells by increasing the opportunity of PrP^C to convert into PrP^Sc and by accumulating the already produced PrP^Sc. This mechanism was confirmed in sortilin-KO mice infected with prions. The mice had exacerbated prion disease with earlier accumulation of PrP^Sc in their brains.

Misfolding of the cellular prion protein, PrP^C, into the amyloidogenic isoform, PrP^Sc, 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 PrP^C into PrP^Sc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrP^C into PrP^Sc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrP^Sc were different from those of RML and 22L laboratory prions-evoked PrP^Sc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrP^Sc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrP^C into PrP^Sc and formation of infectious prions.

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Δ25-50) or octapeptide repeat (Δ51-90) region alone could not impair the antagonistic function against doppel.