RESUMO
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.
Assuntos
Doenças Priônicas , Proteínas Priônicas , Príons , Animais , Camundongos , Doenças Priônicas/genética , Proteínas Priônicas/química , Proteínas Priônicas/genética , Príons/patogenicidade , Prolina , Isoformas de Proteínas/genética , Translocação GenéticaRESUMO
The cellular prion protein, PrPC , is a copper-binding protein abundantly expressed in the brain, particularly by neurons, and its conformational conversion into the amyloidogenic isoform, PrPSc , plays a key pathogenic role in prion diseases. However, the role of copper binding to PrPC in prion diseases remains unclear. Here, we fed mice with a low-copper or regular diet and intracerebrally inoculated them with two different mouse-adapted RML scrapie and BSE prions. Mice with a low-copper diet developed disease significantly but only slightly later than those with a regular diet after inoculation with BSE prions, but not with RML prions, suggesting that copper could play a minor role in BSE prion pathogenesis, but not in RML prion pathogenesis. We then generated two lines of transgenic mice expressing mouse PrP with copper-binding histidine (His) residues in the N-terminal domain replaced with alanine residues, termed TgPrP(5H > A)-7342/Prnp0/0 and TgPrP(5H > A)-7524/Prnp0/0 mice, and similarly inoculated RML and BSE prions into them. Due to 2-fold higher expression of PrP(5H > A) than PrPC in wild-type (WT) mice, TgPrP(5H > A)-7524/Prnp0/0 mice were highly susceptible to these prions, compared to WT mice. However, TgPrP(5H > A)-7342/Prnp0/0 mice, which express PrP(5H > A) 1.2-fold as high as PrPC in WT mice, succumbed to disease slightly, but not significantly, later than WT mice after inoculation with RML prions, but significantly so after inoculation with BSE prions. Subsequent secondary inoculation experiments revealed that amino acid sequence differences between PrP(5H > A) and WT PrPSc created no prion transmission barrier to BSE prions. These results suggest that copper-binding His residues in PrPC are dispensable for RML prion pathogenesis but have a minor effect on BSE prion pathogenesis. Taken together, our current results suggest that copper could have a minor effect on prion pathogenesis in a strain-dependent manner through binding to His residues in the N-terminal domain of PrPC .
RESUMO
The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.
Assuntos
Vírus da Influenza A/metabolismo , Pulmão , Macrófagos , Infecções por Orthomyxoviridae , Proteínas PrPC/metabolismo , Transdução de Sinais , Animais , Anticorpos Monoclonais Murinos/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Pulmão/virologia , Macrófagos/metabolismo , Macrófagos/patologia , Macrófagos/virologia , Camundongos , Camundongos Mutantes , Infecções por Orthomyxoviridae/genética , Infecções por Orthomyxoviridae/metabolismo , Infecções por Orthomyxoviridae/patologia , Proteínas PrPC/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Prion diseases are a group of devastating neurodegenerative disorders, which include Creutzfeldt-Jakob disease (CJD) in humans, and scrapie and bovine spongiform encephalopathy (BSE) in animals [...].
Assuntos
Síndrome de Creutzfeldt-Jakob , Encefalopatia Espongiforme Bovina , Doenças Priônicas , Príons , Scrapie , Animais , Encéfalo/metabolismo , Bovinos , Síndrome de Creutzfeldt-Jakob/etiologia , Síndrome de Creutzfeldt-Jakob/patologia , Encefalopatia Espongiforme Bovina/patologia , Doenças Priônicas/etiologia , Doenças Priônicas/patologia , Príons/metabolismo , Scrapie/patologia , OvinosRESUMO
Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.
Assuntos
Síndrome de Creutzfeldt-Jakob/genética , Doença de Gerstmann-Straussler-Scheinker/genética , Influenza Humana/genética , Insônia Familiar Fatal/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Proteínas Priônicas/genética , Animais , Linhagem Celular Tumoral , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patologia , Síndrome de Creutzfeldt-Jakob/virologia , Doença de Gerstmann-Straussler-Scheinker/metabolismo , Doença de Gerstmann-Straussler-Scheinker/patologia , Doença de Gerstmann-Straussler-Scheinker/virologia , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/patogenicidade , Influenza Humana/metabolismo , Influenza Humana/patologia , Influenza Humana/virologia , Insônia Familiar Fatal/metabolismo , Insônia Familiar Fatal/patologia , Insônia Familiar Fatal/virologia , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Neurônios/virologia , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Conformação Proteica , Genética Reversa/métodosRESUMO
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.
Assuntos
Peróxido de Hidrogênio/química , Ozônio/química , Príons , Aço Inoxidável , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/farmacologia , Camundongos , Ozônio/farmacologia , Proteínas PrPC/antagonistas & inibidores , Proteínas PrPC/química , Doenças Priônicas/etiologia , Doenças Priônicas/prevenção & controle , Aço Inoxidável/químicaRESUMO
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.
Assuntos
Encéfalo/metabolismo , Etanolamina/farmacologia , Proteínas PrPSc , Doenças Priônicas , Animais , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos ICR , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/genética , Doenças Priônicas/metabolismoRESUMO
Worldwide spread of influenza A virus (IAV) strains, which are resistant to currently available anti- influenza agents such as viral neuraminidase inhibitors, has encouraged identification of new target molecules for anti-influenza agents. Reactive oxygen species (ROS) causing oxidative stress play a pivotal role in the pathogenesis of lung injuries induced by infection with IAVs, therefore suggesting that anti-oxidative therapeutics targeting cellular molecules could be beneficial against IAV infection without inducing drug-resistant IAV strains. We recently found that the normal cellular prion protein, PrPC, whose conformational conversion into the amyloidogenic isoform, PrPSc, in the brain is a key pathogenic event in prion diseases, is expressed by lung epithelial cells and exerts a protective role against IAV infection in mice by reducing ROS in infected lungs. The Cu content and activity of anti- oxidative enzyme Cu/Zn-superoxide dismutase, or SOD1, were lower in the lungs of PrPC-knockout mice, suggesting that the anti-oxidative activity of PrPC is probably attributable to its function of activating SOD1 through regulating Cu content in lungs. Here, we introduce PrPC as a novel modulator of influenza and its potential implication for anti-oxidative therapies for IAV infection. We also introduce other candidate targets reported for anti- oxidative anti-influenza therapies.
Assuntos
Antivirais/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Proteínas Priônicas/farmacologia , Animais , Antioxidantes/farmacologia , Antivirais/uso terapêutico , Interações Hospedeiro-Patógeno , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/genética , Influenza Humana/metabolismo , Oxirredução/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Replicação Viral/efeitos dos fármacosRESUMO
The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 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 Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPΔOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC 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 Prnp0/0 and Tg(PrPΔOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC 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 PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics.
Assuntos
Proteínas PrPC/metabolismo , Proteínas Priônicas/metabolismo , Animais , Encéfalo/patologia , Cobre/metabolismo , Suscetibilidade a Doenças/metabolismo , Vírus da Influenza A/metabolismo , Vírus da Influenza A/patogenicidade , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Proteínas PrPC/fisiologia , Doenças Priônicas/metabolismo , Proteínas Priônicas/farmacologia , Príons/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The normal cellular isoform of prion protein, designated PrPC, is constitutively converted to the abnormally folded, amyloidogenic isoform, PrPSc, in prion diseases, which include Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. PrPC is a membrane glycoprotein consisting of the non-structural N-terminal domain and the globular C-terminal domain. During conversion of PrPC to PrPSc, its 2/3 C-terminal region undergoes marked structural changes, forming a protease-resistant structure. In contrast, the N-terminal region remains protease-sensitive in PrPSc. Reverse genetic studies using reconstituted PrPC-knockout mice with various mutant PrP molecules have revealed that the N-terminal domain has an important role in the normal function of PrPC and the conversion of PrPC to PrPSc. The N-terminal domain includes various characteristic regions, such as the positively charged residue-rich polybasic region, the octapeptide repeat (OR) region consisting of five repeats of an octapeptide sequence, and the post-OR region with another positively charged residue-rich polybasic region followed by a stretch of hydrophobic residues. We discuss the normal functions of PrPC, the conversion of PrPC to PrPSc, and the neurotoxicity of PrPSc by focusing on the roles of the N-terminal regions in these topics.
Assuntos
Doenças Priônicas/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/metabolismo , Animais , Humanos , Mutação , Doenças Priônicas/genética , Proteínas Priônicas/genética , Conformação Proteica , Domínios ProteicosRESUMO
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.
Assuntos
Encefalopatia Espongiforme Bovina/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Deficiências na Proteostase/genética , Scrapie/genética , Deleção de Sequência , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Clonagem Molecular , Suscetibilidade a Doenças , Encefalopatia Espongiforme Bovina/metabolismo , Encefalopatia Espongiforme Bovina/patologia , Expressão Gênica , Injeções Intraventriculares , Camundongos , Camundongos Transgênicos , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/administração & dosagem , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Scrapie/metabolismo , Scrapie/patologia , Especificidade da EspécieRESUMO
Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrPC into PrPSc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/Prnp0/0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrPScΔOR in their brains. We show here that Tg(PrPΔOR)/Prnp0/0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrPScΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrPC into PrPSc after infection with BSE prions. However, Tg(PrPΔOR)/Prnp0/0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrPScΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/Prnp0/0 mice than PrPSc in control wild-type mice. Taken together, these results indicate that the OR region of PrPC could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions.IMPORTANCE Structure-function relationship studies of PrPC conformational conversion into PrPSc are worthwhile to understand the mechanism of the conversion of PrPC into PrPSc We show here that, by inoculating Tg(PrPΔOR)/Prnp0/0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrPC into PrPSc after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrPScΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrPScΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrPC into PrPSc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions.
Assuntos
Suscetibilidade a Doenças , Encefalopatia Espongiforme Bovina/fisiopatologia , Proteínas PrPC/química , Proteínas PrPC/fisiologia , Doenças Priônicas/fisiopatologia , Príons/patogenicidade , Animais , Encéfalo/patologia , Bovinos , Encefalopatia Espongiforme Bovina/prevenção & controle , Humanos , Camundongos , Camundongos Transgênicos , Oligopeptídeos/química , Oligopeptídeos/genética , Proteínas PrPC/genética , Doenças Priônicas/prevenção & controle , Príons/química , Príons/genética , Deleção de SequênciaRESUMO
Prion diseases are a group of fatal neurodegenerative disorders caused by prions, which consist mainly of the abnormally folded isoform of prion protein, PrPSc. A pivotal pathogenic event in prion disease is progressive accumulation of prions, or PrPSc, in brains through constitutive conformational conversion of the cellular prion protein, PrPC, into PrPSc. However, the cellular mechanism by which PrPSc is progressively accumulated in prion-infected neurons remains unknown. Here, we show that PrPSc is progressively accumulated in prion-infected cells through degradation of the VPS10P sorting receptor sortilin. We first show that sortilin interacts with PrPC and PrPSc and sorts them to lysosomes for degradation. Consistently, sortilin-knockdown increased PrPSc accumulation in prion-infected cells. In contrast, overexpression of sortilin reduced PrPSc accumulation in prion-infected cells. These results indicate that sortilin negatively regulates PrPSc accumulation in prion-infected cells. The negative role of sortilin in PrPSc accumulation was further confirmed in sortilin-knockout mice infected with prions. The infected mice had accelerated prion disease with early accumulation of PrPSc 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 PrPSc becoming detectable in cells after infection with prions. These results indicate that PrPSc accumulation stimulates sortilin degradation in lysosomes. Taken together, these results show that PrPSc accumulation of itself could impair the sortilin-mediated sorting of PrPC and PrPSc to lysosomes for degradation by stimulating lysosomal degradation of sortilin, eventually leading to progressive accumulation of PrPSc in prion-infected cells.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Príons/metabolismo , Animais , Lisossomos/metabolismo , Camundongos , Neurônios/metabolismo , Doenças Priônicas/metabolismo , Transporte Proteico/fisiologiaRESUMO
Prion diseases are progressive fatal neurodegenerative illnesses caused by the accumulation of transmissible abnormal prion protein (PrP). To find treatments for prion diseases, we searched for substances from natural resources that inhibit abnormal PrP formation in prion-infected cells. We found that high-molecular-weight components from insect cuticle extracts reduced abnormal PrP levels. The chemical nature of these components was consistent with that of melanin. In fact, synthetic melanin produced from tyrosine or 3-hydroxy-l-tyrosine inhibited abnormal PrP formation. Melanin did not modify cellular or cell surface PrP levels, nor did it modify lipid raft or cellular cholesterol levels. Neither did it enhance autophagy or lysosomal function. Melanin was capable of interacting with PrP at two N-terminal domains. Specifically, it strongly interacted with the PrP region of amino acids 23 to 50 including a positively charged amino acid cluster and weakly interacted with the PrP octarepeat peptide region of residues 51 to 90. However, the in vitro and in vivo data were inconsistent with those of prion-infected cells. Abnormal PrP formation in protein misfolding cyclic amplification was not inhibited by melanin. Survival after prion infection was not significantly altered in albino mice or exogenously melanin-injected mice compared with that of control mice. These data suggest that melanin, a main determinant of skin color, is not likely to modify prion disease pathogenesis, even though racial differences in the incidence of human prion diseases have been reported. Thus, the findings identify an interaction between melanin and the N terminus of PrP, but the pathophysiological roles of the PrP-melanin interaction remain unclear.IMPORTANCE The N-terminal region of PrP is reportedly important for neuroprotection, neurotoxicity, and abnormal PrP formation, as this region is bound by many factors, such as metal ions, lipids, nucleic acids, antiprion compounds, and several proteins, including abnormal PrP in prion disease and the Aß oligomer in Alzheimer's disease. In the present study, melanin, a main determinant of skin color, was newly found to interact with this N-terminal region and inhibits abnormal PrP formation in prion-infected cells. However, the data for prion infection in mice lacking melanin production suggest that melanin is not associated with the prion disease mechanism, although the incidence of prion disease is reportedly much higher in white people than in black people. Thus, the roles of the PrP-melanin interaction remain to be further elucidated, but melanin might be a useful competitive tool for evaluating the functions of other ligands at the N-terminal region.
Assuntos
Melaninas/metabolismo , Doenças Priônicas/prevenção & controle , Príons/metabolismo , Animais , Linhagem Celular , Melaninas/administração & dosagem , Camundongos , Neurônios/metabolismo , Doenças Priônicas/tratamento farmacológico , Ligação Proteica , Mapeamento de Interação de Proteínas , Análise de SobrevidaRESUMO
The N-terminal polybasic region of the normal prion protein, PrPC, which encompasses residues 23-31, is important for prion pathogenesis by affecting conversion of PrPC into the pathogenic isoform, PrPSc. We previously reported transgenic mice expressing PrP with residues 25-50 deleted in the PrP-null background, designated as Tg(PrP∆preOR)/Prnp 0/0 mice. Here, we produced two new lines of Tg(PrP∆preOR)/Prnp 0/0 mice, each expressing the mutant protein, PrP∆preOR, 1.1 and 1.6 times more than PrPC in wild-type mice, and subsequently intracerebrally inoculated RML and 22L prions into them. The lower expresser showed slightly reduced susceptibility to RML prions but not to 22L prions. The higher expresser exhibited enhanced susceptibility to both prions. No prion transmission barrier was created in Tg(PrP∆preOR)/Prnp 0/0 mice against full-length PrPSc. PrPSc∆preOR accumulated in the brains of infected Tg(PrP∆preOR)/Prnp 0/0 mice less than PrPSc in control wild-type mice, although lower in RML-infected Tg(PrP∆preOR)/Prnp 0/0 mice than in 22L-infected mice. Prion infectivity in infected Tg(PrP∆preOR)/Prnp 0/0 mice was also lower than that in wild-type mice. These results indicate that deletion of residues 25-50 only slightly affects prion susceptibility, the conversion of PrPC into PrPSc, and prion infectivity in a strain-specific way. PrP∆preOR retains residues 23-24 and lacks residues 25-31 in the polybasic region. It is thus conceivable that residues 23-24 rather than 25-31 are important for the polybasic region to support prion pathogenesis. However, other investigators have reported that residues 27-31 not 23-24 are important to support prion pathogenesis. Taken together, the polybasic region might support prion pathogenesis through multiple sites including residues 23-24 and 27-31.
Assuntos
Doenças Priônicas , Proteínas Priônicas/metabolismo , Sequência de Aminoácidos , Animais , Suscetibilidade a Doenças , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Priônicas/genética , Sequências Repetitivas de Aminoácidos , Deleção de SequênciaRESUMO
We prepared ß-sheet-rich recombinant full-length prion protein (ß-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this ß-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to ß-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of ß-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of ß-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing ß-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to ß-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.
Assuntos
Imunoglobulina G/química , Neuroblastoma/metabolismo , Príons/química , Scrapie/metabolismo , Animais , Anticorpos/química , Dicroísmo Circular/métodos , Engenharia Genética/métodos , Humanos , Imuno-Histoquímica/métodos , Camundongos , Microscopia de Força Atômica/métodos , Microscopia Confocal/métodos , Biblioteca de Peptídeos , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de ProteínaRESUMO
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases. Infection by the oral route is assumed to be important, although its pathogenesis is not understood. Using prion protein (PrP) knockout mice, we investigated the sequence of events during the invasion of orally administered PrPs through the intestinal mucosa and the spread into lymphoid tissues and the peripheral nervous system. Orally administered PrPs were incorporated by intestinal epitheliocytes in the follicle-associated epithelium and villi within 1 hour. PrP-positive cells accumulated in the subfollicle region of Peyer's patches a few hours thereafter. PrP-positive cells spread toward the mesenteric lymph nodes and spleen after the accumulation of PrPs in the Peyer's patches. The number of PrP molecules in the mesenteric lymph nodes and spleen peaked at 2 days and 6 days after inoculation, respectively. The epitheliocytes in the follicle-associated epithelium incorporating PrPs were annexin V-positive microfold cells and PrP-positive cells in Peyer's patches and spleen were CD11b-positive and CD14-positive macrophages. Additionally, PrP-positive cells in Peyer's patches and spleen were detected in the vicinity of peripheral nerve fibers in the early stages of infection. These results indicate that orally delivered PrPs were incorporated by microfold cells promptly after challenge and that macrophages might act as a transporter of incorporated PrPs from the Peyer's patches to other lymphoid tissues and the peripheral nervous system.
Assuntos
Encéfalo/metabolismo , Mucosa Intestinal/metabolismo , Macrófagos/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Príons/farmacocinética , Administração Oral , Animais , Imuno-Histoquímica , Intestino Delgado/metabolismo , Linfonodos/metabolismo , Masculino , Mesentério/metabolismo , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/patologia , Doenças Priônicas/etiologia , Príons/administração & dosagem , Baço/metabolismoRESUMO
The cellular isoform of prion protein, designated PrPC, is a membrane glycoprotein expressed most abundantly in the brain, particularly by neurons, and its conformational conversion into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying mechanism in the pathogenesis of prion diseases, a group of neurodegenerative disorders in humans and animals. Most cases of these diseases are sporadic and their aetiologies are unknown. We recently found that a neurotropic strain of influenza A virus (IAV/WSN) caused the conversion of PrPC into PrPSc and the subsequent formation of infectious prions in mouse neuroblastoma cells after infection. These results show that IAV/WSN is the first non-prion pathogen capable of inducing the conversion of PrPC into PrPSc and propagating infectious prions in cultured neuronal cells, and also provide the intriguing possibility that IAV infection in neurons might be a cause of or be associated with sporadic prion diseases. Here, we present our findings of the IAV/WSN-induced conversion of PrPC into PrPSc and subsequent propagation of infectious prions, and also discuss the biological significance of the conversion of PrPC into PrPSc in virus infections.
Assuntos
Orthomyxoviridae , Proteínas PrPC , Doenças Priônicas , Príons , Animais , Encéfalo/patologia , Camundongos , Proteínas PrPC/metabolismo , Doenças Priônicas/metabolismo , Proteínas Priônicas/metabolismo , Príons/metabolismoRESUMO
We first verified that a single chain Fv fragment against prion protein (anti-PrP scFv) was secreted by HEK293T cells and prevented prion replication in infected cells. We then stably expressed anti-PrP scFv in brain-engraftable murine microglial cells and intracerebrally injected these cells into mice before or after infection with prions. Interestingly, the injection before or at an early time point after infection attenuated the infection marginally but significantly prolonged survival times of the mice. These suggest that the ex vivo gene transfer of anti-PrP scFvs using brain-engraftable cells could be a possible immunotherapeutic approach against prion diseases.
Assuntos
Encéfalo/citologia , Microglia/fisiologia , Microglia/transplante , Príons/imunologia , Scrapie/fisiopatologia , Anticorpos de Cadeia Única/imunologia , Animais , Linhagem Celular , Vetores Genéticos , Células HEK293 , Humanos , Camundongos , Microglia/citologia , Príons/patogenicidade , Príons/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Scrapie/terapia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/uso terapêutico , Taxa de SobrevidaRESUMO
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.