RESUMO
Scrapie is a fatal, transmissible neurodegenerative disease that affects sheep and goats. Replication of PrPSc in the lymphoid tissue allows for the scrapie agent to be shed into the environment. Brain and retropharyngeal lymph node (RPLN) from a sheep inoculated with the classical scrapie agent was used to compare infectivity of these tissues. Nine Cheviot sheep were used in this study, randomly assigned into two groups based on inocula. Group one (n = 4) received 1 mL of 10% brain homogenate and consisted of all VRQ/VRQ PRNP genotypes. Group two (n = 5) had three sheep receive 1 mL of a 10% RPLN homogenate (13-7), and two sheep receive 0.5 mL of a 10% RPLN homogenate (13-7) because of availability. Sheep in group two were also VRQ/VRQ genotyped. Brain and lymph tissues were tested by histopathology, immunohistochemistry, western blot, enzyme immunoassay, and conformational stability for PrPSc accumulation. Both groups displayed clinical signs of ataxia, moribund, head tremors, circling, and lethargy prior to euthanizing at an average of 16.2 mpi (months post inoculation) (group one) or 19.56 mpi (group two). Additionally, brainstem tissue from both groups displayed the same apparent molecular mass by western blot examination. Spongiform lesion profiling and PrPSc accumulation in brain and lymph tissues were similar in both groups. Conformational stability results displayed no significant difference in obex or RPLN tissue. Overall, these data suggest lymph nodes containing the classical scrapie agent are infectious to sheep, aiding in the understanding of sheep scrapie transmission.
Assuntos
Encéfalo , Linfonodos , Proteínas PrPSc , Scrapie , Animais , Scrapie/transmissão , Scrapie/patologia , Ovinos , Linfonodos/patologia , Encéfalo/patologia , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Imuno-Histoquímica/veterinária , GenótipoRESUMO
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
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 result from a drastic conformational change of the host-encoded cellular prion protein (PrP), leading to the formation of ß-sheet-rich, insoluble, and protease-resistant self-replicating assemblies (PrPSc). The cellular and molecular mechanisms involved in spontaneous prion formation in sporadic and inherited human prion diseases or equivalent animal diseases are poorly understood, in part because cell models of spontaneously forming prions are currently lacking. Here, extending studies on the role of the H2 α-helix C terminus of PrP, we found that deletion of the highly conserved 190HTVTTTT196 segment of ovine PrP led to spontaneous prion formation in the RK13 rabbit kidney cell model. On long-term passage, the mutant cells stably produced proteinase K (PK)-resistant, insoluble, and aggregated assemblies that were infectious for naïve cells expressing either the mutant protein or other PrPs with slightly different deletions in the same area. The electrophoretic pattern of the PK-resistant core of the spontaneous prion (ΔSpont) contained mainly C-terminal polypeptides akin to C1, the cell-surface anchored C-terminal moiety of PrP generated by natural cellular processing. RK13 cells expressing solely the Δ190-196 C1 PrP construct, in the absence of the full-length protein, were susceptible to ΔSpont prions. ΔSpont infection induced the conversion of the mutated C1 into a PK-resistant and infectious form perpetuating the biochemical characteristics of ΔSpont prion. In conclusion, this work provides a unique cell-derived system generating spontaneous prions and provides evidence that the 113 C-terminal residues of PrP are sufficient for a self-propagating prion entity.
Assuntos
Sequência de Aminoácidos , Proteínas PrPSc , Doenças Priônicas , Agregação Patológica de Proteínas , Deleção de Sequência , Animais , Linhagem Celular , Humanos , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Coelhos , Ovinos , SolubilidadeRESUMO
Prion diseases, Alzheimer's disease and Parkinson's disease (PD) are fatal degenerative disorders that share common neuropathological and biochemical features, including the aggregation of pathological protein conformers. Lymphocyte activation gene 3 (Lag3, also known as CD223) is a member of the immunoglobulin superfamily of receptors expressed on peripheral immune cells, microglia and neurons, which serves as a receptor for α-synuclein aggregates in PD. Here we examined the possible role of Lag3 in the pathogenesis of prion diseases. Through quantitative real-time PCR and RNA-sequencing, we found that the expression levels of Lag3 were relatively low in the adult mouse brains, yet its expression was increased after prion infection. However, we failed finding significant differences regarding the incubation time, PrPSc load, neurodegeneration, astrocyte and microglia reactions and inflammatory gene expression between the Lag3 knockout mice and wild-type littermate controls after prion infection. We conclude that loss of Lag3 has no significant influence on prion disease pathogenesis. Considering that Lag3 is an immune checkpoint receptor, our results suggest that immune checkpoint inhibition (an increasingly prevalent therapeutic modality against many types of cancer) might not exert positive or negative effects on the progression of prion diseases.
Assuntos
Antígenos CD/genética , Encéfalo/patologia , Proteínas PrPSc/genética , Scrapie/genética , Animais , Antígenos CD/imunologia , Astrócitos/imunologia , Astrócitos/patologia , Encéfalo/imunologia , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Progressão da Doença , Regulação da Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Camundongos Knockout , Microglia/imunologia , Microglia/patologia , Neurônios/imunologia , Neurônios/patologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Proteínas PrPSc/imunologia , Proteínas PrPSc/patogenicidade , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Scrapie/imunologia , Scrapie/mortalidade , Scrapie/patologia , Transdução de Sinais , Análise de Sobrevida , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Proteína do Gene 3 de Ativação de LinfócitosRESUMO
Neuroinflammation is recognized as one of the obligatory pathogenic features of neurodegenerative diseases including Alzheimer's, Parkinson's or prion diseases. In prion diseases, space and time correlations between deposition of disease-associated, pathogenic form of the prion protein or PrPSc and microglial-mediated neuroinflammation has been established. Yet, it remains unclear whether activation of microglia is triggered directly by a contact with PrPSc, and what molecular features of PrPSc microglia sense and respond to that drive microglia to inflammatory states. The current study asked the questions whether PrPSc can directly trigger activation of microglia and whether the degree of microglia response depends on the nature of terminal carbohydrate groups on the surface of PrPSc particles. PrPSc was purified from brains of mice infected with mouse-adapted prion strain 22L or neuroblastoma N2a cells stably infected with 22L. BV2 microglial cells or primary microglia were cultured in the presence of purified 22L. We found that exposure of BV2 cells or primary microglia to purified PrPSc triggered proinflammatory responses characterized by an increase in the levels of TNFα, IL6, nitric oxide (NO) and expression of inducible Nitric Oxide Synthase (iNOS). Very similar patterns of inflammatory response were induced by PrPSc purified from mouse brains and neuroblastoma cells arguing that microglia response is independent of the source of PrPSc. To test whether the microglial response is mediated by carbohydrate epitopes on PrPSc surface, the levels of sialylation of PrPSc N-linked glycans was altered by treatment of purified PrPSc with neuraminidase. Partial cleavage of sialic acid residues was found to boost the inflammatory response of microglia to PrPSc. Moreover, transient degradation of Iκßα observed upon treatment with partially desialylated PrPSc suggests that canonical NFκB activation pathway is involved in inflammatory response. The current study is the first to demonstrate that PrPSc can directly trigger inflammatory response in microglia. In addition, this work provides direct evidence that the chemical nature of the carbohydrate groups on PrPSc surface is important for microglial activation.
Assuntos
Inflamação/imunologia , Microglia/imunologia , Proteínas PrPSc/imunologia , Doenças Priônicas/imunologia , Animais , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Carboidratos/imunologia , Epitopos/imunologia , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Interleucina-6/genética , Camundongos , Microglia/metabolismo , Microglia/patologia , Ácido N-Acetilneuramínico/imunologia , Óxido Nítrico/genética , Óxido Nítrico Sintase Tipo II/genética , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Cultura Primária de Células , Doenças Priônicas/genética , Doenças Priônicas/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Cultured cells are valuable models to study prion infections at the cellular level. Unfortunately, the vast majority of cell lines are resistant to the propagation of prion agents. The rabbit epithelial RK13 cell line is among the few cell lines permissive to prion infection. When genetically engineered to express heterologous PrP proteins, RK13 cells become permissive to several strains of prions from various animal species. Here, we describe the generation of stable RK13 cell clones expressing a heterologous PrP protein in an inducible manner, the establishment and maintenance of chronically infected cultures, and the selection of cell clones suitable for cell-based titration of prions.
Assuntos
Células Epiteliais/metabolismo , Efeito Fundador , Immunoblotting/métodos , Proteínas PrPSc/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Técnicas de Cultura de Células , Linhagem Celular , Células Clonais , Clonagem Molecular , Endopeptidase K/química , Células Epiteliais/patologia , Expressão Gênica , Humanos , Camundongos , Plasmídeos/química , Plasmídeos/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Dobramento de Proteína , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Prion neurotoxicity has been modeled in vitro using synthetic peptides derived from the PrPC sequence. The major region of neurotoxicity has been localized to the hydrophobic domain located in the middle of the PrP protein. Neurotoxicity assays are typically performed on cultured mouse cerebellar neurons derived from neonatal pups, and cell viability can be monitored by assays including MTT or MTS, cell death by LDH release, or apoptosis by caspase cleavage assays. These neurotoxicity studies have been useful in identifying cofactors, such as PrPC and metals, as modulators of PrP peptide-mediated neurotoxicity. Given the biosafety issues associated with handling and purifying infectious prions, the use of synthetic peptides, which display a dependence upon PrPC expression for toxicity, as per the PrPSc agent for infectivity, supports the relevance of using these synthetic peptides for understanding PrP-mediated neurotoxicity.
Assuntos
Apoptose/efeitos dos fármacos , Bioensaio , Neurônios/efeitos dos fármacos , Peptídeos/toxicidade , Proteínas PrPSc/genética , Proteínas da Gravidez/genética , Animais , Animais Recém-Nascidos , Sobrevivência Celular/efeitos dos fármacos , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Expressão Gênica , Interações Hidrofóbicas e Hidrofílicas , L-Lactato Desidrogenase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Malondialdeído/análise , Malondialdeído/metabolismo , Camundongos , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/síntese química , Proteínas PrPSc/metabolismo , Proteínas PrPSc/toxicidade , Proteínas da Gravidez/metabolismo , Cultura Primária de Células , Domínios Proteicos , RatosRESUMO
Proteins associated with neurodegenerative diseases are highly pleiomorphic and may adopt an all-α-helical fold in one environment, assemble into all-ß-sheet or collapse into a coil in another, and rapidly polymerize in yet another one via divergent aggregation pathways that yield broad diversity of aggregates' morphology. A thorough understanding of this behaviour may be necessary to develop a treatment for Alzheimer's and related disorders. Unfortunately, our present comprehension of folding and misfolding is limited for want of a physicochemical theory of protein secondary and tertiary structure. Here we demonstrate that electronic configuration and hyperconjugation of the peptide amide bonds ought to be taken into account to advance such a theory. To capture the effect of polarization of peptide linkages on conformational and H-bonding propensity of the polypeptide backbone, we introduce a function of shielding tensors of the Cα atoms. Carrying no information about side chain-side chain interactions, this function nonetheless identifies basic features of the secondary and tertiary structure, establishes sequence correlates of the metamorphic and pH-driven equilibria, relates binding affinities and folding rate constants to secondary structure preferences, and manifests common patterns of backbone density distribution in amyloidogenic regions of Alzheimer's amyloid ß and tau, Parkinson's α-synuclein and prions. Based on those findings, a split-intein like mechanism of molecular recognition is proposed to underlie dimerization of Aß, tau, αS and PrPC, and divergent pathways for subsequent association of dimers are outlined; a related mechanism is proposed to underlie formation of PrPSc fibrils. The model does account for: (i) structural features of paranuclei, off-pathway oligomers, non-fibrillar aggregates and fibrils; (ii) effects of incubation conditions, point mutations, isoform lengths, small-molecule assembly modulators and chirality of solid-liquid interface on the rate and morphology of aggregation; (iii) fibril-surface catalysis of secondary nucleation; and (iv) self-propagation of infectious strains of mammalian prions.
Assuntos
Elétrons , Modelos Moleculares , Agregados Proteicos , Dobramento de Proteína , Multimerização Proteica , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Humanos , Concentração de Íons de Hidrogênio , Proteínas PrPSc/química , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Domínios Proteicos , Estabilidade Proteica , Estrutura Secundária de Proteína , alfa-Sinucleína/química , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Proteínas tau/química , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Prion disease-associated retinal degeneration is attributed to PrP-scrapie (PrPSc), a misfolded isoform of prion protein (PrPC) that accumulates in the neuroretina. However, a lack of temporal and spatial correlation between PrPSc and cytotoxicity suggests the contribution of host factors. We report retinal iron dyshomeostasis as one such factor. PrPC is expressed on the basolateral membrane of retinal-pigment-epithelial (RPE) cells, where it mediates uptake of iron by the neuroretina. Accordingly, the neuroretina of PrP-knock-out mice is iron-deficient. In RPE19 cells, silencing of PrPC decreases ferritin while over-expression upregulates ferritin and divalent-metal-transporter-1 (DMT-1), indicating PrPC-mediated iron uptake through DMT-1. Polarization of RPE19 cells results in upregulation of ferritin by ~10-fold and ß-cleavage of PrPC, the latter likely to block further uptake of iron due to cleavage of the ferrireductase domain. A similar ß-cleavage of PrPC is observed in mouse retinal lysates. Scrapie infection causes PrPSc accumulation and microglial activation, and surprisingly, upregulation of transferrin despite increased levels of ferritin. Notably, detergent-insoluble ferritin accumulates in RPE cells and correlates temporally with microglial activation, not PrPSc accumulation, suggesting that impaired uptake of iron by PrPSc combined with inflammation results in retinal iron-dyshomeostasis, a potentially toxic host response contributing to prion disease-associated pathology.
Assuntos
Ferro/metabolismo , Proteínas Priônicas/metabolismo , Retina/metabolismo , Animais , Transporte Biológico , Cricetinae , Modelos Animais de Doenças , Feminino , Expressão Gênica , Homeostase , Humanos , Camundongos , Camundongos Knockout , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Proteínas Priônicas/química , Proteínas Priônicas/genética , ProteóliseRESUMO
Prion disease is a fatal neurodegenerative disease that may result from the conversion of normal cellular prion protein (PrPC) to the pathogenic scrapie PrP isoform (PrPSc), however, how proliferation of prion leads to neuronal apoptosis is still not clear. In this study, to explore the role of the endoplasmic reticulum (ER) in prion diseases, we engineered the KDEL ER-retention motif to the C-terminus of PrPC and studied its effect on N2A cell toxicity. The KDEL retention signal led to the accumulation of PrP in the ER, and KDEL signal could effectively deplete PrP from the cell surface and trap PrP in the ER/Cis-Golgi compartment. PrPC molecules were delayed in their transit along the early pathway of the secretory compartment, however, they did not aggregate, and were not resistant to Proteinase K (PK) or become detergent-insoluble. Moreover, we found that the ER was not the site where PrP became detergent-insoluble and acquired PK resistance. In addition, an MTT assay indicated cells expressing PrPC/N2A were sensitive to proteasome inhibition, but not N2A cells expressing PrPKDEL. Our findings suggest that the ER is not a compartment in which wild type PrPC is able to initiate aggregation, protease resistance or other scapie-like properties of PrP.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Motivos de Aminoácidos , Animais , Apoptose , Linhagem Celular Tumoral , Endopeptidase K/química , Retículo Endoplasmático/efeitos dos fármacos , Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Leupeptinas/farmacologia , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Engenharia de Proteínas , Transporte Proteico , Proteólise/efeitos dos fármacosRESUMO
Glycosylphosphatidylinositol (GPI) anchoring of the prion protein (PrPC) influences PrPC misfolding into the disease-associated isoform, PrPres, as well as prion propagation and infectivity. GPI proteins are found in cholesterol- and sphingolipid-rich membrane regions called rafts. Exchanging the GPI anchor for a nonraft transmembrane sequence redirects PrPC away from rafts. Previous studies showed that nonraft transmembrane PrPC variants resist conversion to PrPres when transfected into scrapie-infected N2a neuroblastoma cells, likely due to segregation of transmembrane PrPC and GPI-anchored PrPres in distinct membrane environments. Thus, it remained unclear whether transmembrane PrPC might convert to PrPres if seeded by an exogenous source of PrPres not associated with host cell rafts and without the potential influence of endogenous expression of GPI-anchored PrPC To further explore these questions, constructs containing either a C-terminal wild-type GPI anchor signal sequence or a nonraft transmembrane sequence containing a flexible linker were expressed in a cell line derived from PrP knockout hippocampal neurons, NpL2. NpL2 cells have physiological similarities to primary neurons, representing a novel and advantageous model for studying transmissible spongiform encephalopathy (TSE) infection. Cells were infected with inocula from multiple prion strains and in different biochemical states (i.e., membrane bound as in brain microsomes from wild-type mice or purified GPI-anchorless amyloid fibrils). Only GPI-anchored PrPC supported persistent PrPres propagation. Our data provide strong evidence that in cell culture GPI anchor-directed membrane association of PrPC is required for persistent PrPres propagation, implicating raft microdomains as a location for conversion. IMPORTANCE: Mechanisms of prion propagation, and what makes them transmissible, are poorly understood. Glycosylphosphatidylinositol (GPI) membrane anchoring of the prion protein (PrPC) directs it to specific regions of cell membranes called rafts. In order to test the importance of the raft environment on prion propagation, we developed a novel model for prion infection where cells expressing either GPI-anchored PrPC or transmembrane-anchored PrPC, which partitions it to a different location, were treated with infectious, misfolded forms of the prion protein, PrPres We show that only GPI-anchored PrPC was able to convert to PrPres and able to serially propagate. The results strongly suggest that GPI anchoring and the localization of PrPC to rafts are crucial to the ability of PrPC to propagate as a prion.
Assuntos
Expressão Gênica , Microdomínios da Membrana , Proteínas PrPC/genética , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Príons/metabolismo , Animais , Linhagem Celular , Técnicas de Inativação de Genes , Proteínas de Membrana , Camundongos , Proteínas PrPSc/química , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Isoformas de Proteínas , Transporte ProteicoRESUMO
Prion diseases are devastating neurodegenerative disorders with no known cure. One strategy for developing therapies for these diseases is to identify compounds that block conversion of the cellular form of the prion protein (PrPC) into the infectious isoform (PrPSc). Most previous efforts to discover such molecules by high-throughput screening methods have utilized, as a read-out, a single kind of cellular assay system: neuroblastoma cells that are persistently infected with scrapie prions. Here, we describe the use of an alternative cellular assay based on suppressing the spontaneous cytotoxicity of a mutant form of PrP (Δ105-125). Using this assay, we screened 75,000 compounds, and identified a group of phenethyl piperidines (exemplified by LD7), which reduces the accumulation of PrPSc in infected neuroblastoma cells by >90% at low micromolar doses, and inhibits PrPSc-induced synaptotoxicity in hippocampal neurons. By analyzing the structure-activity relationships of 35 chemical derivatives, we defined the pharmacophore of LD7, and identified a more potent derivative. Active compounds do not alter total or cell-surface levels of PrPC, and do not bind to recombinant PrP in surface plasmon resonance experiments, although at high concentrations they inhibit PrPSc-seeded conversion of recombinant PrP to a misfolded state in an in vitro reaction (RT-QuIC). This class of small molecules may provide valuable therapeutic leads, as well as chemical biological tools to identify cellular pathways underlying PrPSc metabolism and PrPC function.
Assuntos
Piperidinas/química , Piperidinas/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Proteínas PrPSc/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Linhagem Celular Tumoral , Células HEK293 , Humanos , Proteínas PrPSc/genéticaRESUMO
Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly ß-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases.
Assuntos
Neurônios/metabolismo , Proteínas PrPC/química , Proteínas PrPSc/química , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/patologia , Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Neurônios/patologia , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Vesículas Transportadoras/patologia , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
UNLABELLED: Phenotypic diversity in prion diseases can be specified by prion strains in which biological traits are propagated through an epigenetic mechanism mediated by distinct PrP(Sc) conformations. We investigated the role of host-dependent factors on phenotypic diversity of chronic wasting disease (CWD) in different host species that express the same prion protein gene (Prnp). Two CWD strains that have distinct biological, biochemical, and pathological features were identified in transgenic mice that express the Syrian golden hamster (SGH) Prnp. The CKY strain of CWD had a shorter incubation period than the WST strain of CWD, but after transmission to SGH, the incubation period of CKY CWD was â¼150 days longer than WST CWD. Limited proteinase K digestion revealed strain-specific PrP(Sc) polypeptide patterns that were maintained in both hosts, but the solubility and conformational stability of PrP(Sc) differed for the CWD strains in a host-dependent manner. WST CWD produced PrP(Sc) amyloid plaques in the brain of the SGH that were partially insoluble and stable at a high concentration of protein denaturant. However, in transgenic mice, PrP(Sc) from WST CWD did not assemble into plaques, was highly soluble, and had low conformational stability. Similar studies using the HY and DY strains of transmissible mink encephalopathy resulted in minor differences in prion biological and PrP(Sc) properties between transgenic mice and SGH. These findings indicate that host-specific pathways that are independent of Prnp can alter the PrP(Sc) conformation of certain prion strains, leading to changes in the biophysical properties of PrP(Sc), neuropathology, and clinical prion disease. IMPORTANCE: Prions are misfolded pathogenic proteins that cause neurodegeneration in humans and animals. Transmissible prion diseases exhibit a spectrum of disease phenotypes and the basis of this diversity is encoded in the structure of the pathogenic prion protein and propagated by an epigenetic mechanism. In the present study, we investigated prion diversity in two hosts species that express the same prion protein gene. While prior reports have demonstrated that prion strain properties are stable upon infection of the same host species and prion protein genotype, our findings indicate that certain prion strains can undergo dramatic changes in biological properties that are not dependent on the prion protein. Therefore, host factors independent of the prion protein can affect prion diversity. Understanding how host pathways can modify prion disease phenotypes may provide clues on how to alter prion formation and lead to treatments for prion, and other, human neurodegenerative diseases of protein misfolding.
Assuntos
Encéfalo/patologia , Genótipo , Fragmentos de Peptídeos/química , Placa Amiloide/patologia , Proteínas PrPSc/química , Doença de Emaciação Crônica/patologia , Animais , Encéfalo/metabolismo , Cricetulus , Cervos , Epigênese Genética , Humanos , Injeções Intraventriculares , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/genética , Placa Amiloide/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Conformação Proteica , Estabilidade Proteica , Proteólise , Solubilidade , Especificidade da Espécie , Doença de Emaciação Crônica/genética , Doença de Emaciação Crônica/metabolismoRESUMO
Polo-like kinases (PLKs) family has long been known to be critical for cell cycle and recent studies have pointed to new dimensions of PLKs function in the nervous system. Our previous study has verified that the levels of PLK3 in the brain are severely downregulated in prion-related diseases. However, the associations of PLKs with prion protein remain unclear. In the present study, we confirmed that PrP protein constitutively interacts with PLK3 as determined by both in vitro and in vivo assays. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. Overexpression of PLK3 did not affect the level of wild-type PrP, but significantly decreased the levels of the mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed to be independent of its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrPs. Moreover, PLK3 overexpression in a scrapie infected cell line caused notable reduce of PrP(Sc) level in a dose-dependent manner, but had minimal effect on the expression of PrP(C) in its normal partner cell line. Our findings here confirmed the molecular interaction between PLK3 and PrP and outlined the regulatory activity of PLK3 on the degradation of abnormal PrPs, even its pathogenic isoform PrP(Sc). We, therefore, assume that the recovery of PLK3 in the early stage of prion infection may be helpful to prevent the toxic accumulation of PrP(Sc) in the brain tissues.
Assuntos
Doenças Priônicas/patologia , Príons/genética , Príons/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Linhagem Celular , Cricetinae , Células HEK293 , Humanos , Mesocricetus , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transfecção , Proteínas Supressoras de TumorRESUMO
The cellular prion protein (PrP(C)) is an ubiquitously expressed glycoprotein that is most abundant in the central nervous system. It is thought to play a role in many cellular processes, including neuroprotection, but may also contribute to Alzheimer's disease and some cancers. However, it is best known for its central role in the prion diseases, such as Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. These protein misfolding diseases can be sporadic, acquired, or genetic and are caused by refolding of endogenous PrP(C) into a beta sheet-rich, pathogenic form, PrP(Sc). Once prions are present in the central nervous system, they increase and spread during a long incubation period that is followed by a relatively short clinical disease phase, ending in death. PrP molecules can be broadly categorized as either 'good' (cellular) PrP(C) or 'bad' (scrapie prion-type) PrP(Sc), but both populations are heterogeneous and different forms of PrP(C) may influence various cellular activities. Both PrP(C) and PrP(Sc) are localized predominantly at the cell surface, with the C-terminus attached to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor and both can exist in cleaved forms. PrP(C) also has cytosolic and transmembrane forms, and PrP(Sc) is known to exist in a variety of conformations and aggregation states. Here, we discuss the roles of different PrP isoforms in sickness and in health, and show the subcellular distributions of several forms of PrP that are particularly relevant for PrP(C) to PrP(Sc) conversion and prion-induced pathology in the hippocampus.
Assuntos
Espaço Intracelular/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Animais , Humanos , Espaço Intracelular/genética , Proteínas PrPC/genética , Proteínas PrPSc/genética , Transporte ProteicoRESUMO
Prion diseases are neurodegenerative disorders characterized by the accumulation of an abnormal isoform of the protease-insensitive isoform (PrPSc) of prion protein. Human prion protein fragment 106126 [PrP (106126)] contains most of the pathological characteristics associated with PrPSc. Although a number of compounds have been identified to inhibit PrP accumulation or dissolve fibrils and aggregates in vitro, there is currenlty no treatment available for these progressive neurodegenerative diseases. Baicalein, the dried root of Scutellaria baicalensis (S. baicalensis) Georgi (known as Huang-qin in traditional Chinese medicine) has been reported to exert neuroprotective effects on neurodegenerative diseases. In the present study, we investigated the effects of baicalein on the development of prion diseases using SH-SY5Y and SK-N-SH cells in vitro. We found that baicalein protected the cells against PrPinduced neuronal cell death by inhibiting the production of reactive oxygen species (ROS) and mitochondrial dysfunction using ROS detection assay and MTP assay. We demonstrated that baicalein treatment regulated the phosphorylation of c-Jun N-terminal kinase (JNK) by using western blot analysis and Annexin V assay. Our data suggest that baicalein has potential for use as a therapeutic drug for the treatment of various neurodegenerative diseases, including prion diseases.
Assuntos
Antioxidantes/farmacologia , Flavanonas/farmacologia , MAP Quinase Quinase 4/metabolismo , Neurônios/enzimologia , Proteínas PrPSc/metabolismo , Anexina A5/genética , Anexina A5/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Humanos , MAP Quinase Quinase 4/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/patologia , Proteínas PrPSc/genética , Doenças Priônicas/dietoterapia , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , Doenças Priônicas/patologia , Espécies Reativas de OxigênioRESUMO
The structural details of the essential entity of prion disease, fibril prion protein (PrP(Sc)), are still elusive despite the large body of evidence supporting the prion hypothesis. Five major working models of PrP(Sc) structure, which are not compatible with each other, have been proposed. However, no systematic evaluation has been performed on those models. We devised a method that combined systematic point mutation with threading on knowledge-based amino acid potentials. A comprehensive mutation experiment was performed on mouse prion protein, and the PrP(Sc) conversion efficiency of each mutant was examined. The models were evaluated based on the mutation data by using the threading method. Although the data turned out to be rather more consistent with the models that assumed a conversion of the N-terminal region of core PrP into a ß helix than with others, substantial modifications were also required to further improve the current model based on recent experimental results.
Assuntos
Modelos Moleculares , Proteínas PrPSc/química , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Neurônios/citologia , Neurônios/metabolismo , Mutação Puntual , Proteínas PrPSc/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , TransfecçãoRESUMO
UNLABELLED: A new type of antiprion compound, Gly-9, was found to inhibit abnormal prion protein formation in prion-infected neuroblastoma cells, in a prion strain-independent manner, when the cells were treated for more than 1 day. It reduced the intracellular prion protein level and significantly modified mRNA expression levels of genes of two types: interferon-stimulated genes were downregulated after more than 2 days of treatment, and the phosphodiesterase 4D-interacting protein gene, a gene involved in microtubule growth, was upregulated after more than 1 day of treatment. A supplement of interferon given to the cells partly restored the abnormal prion protein level but did not alter the normal prion protein level. This interferon action was independent of the Janus activated kinase-signal transducer and activator of transcription signaling pathway. Therefore, the changes in interferon-stimulated genes might be a secondary effect of Gly-9 treatment. However, gene knockdown of phosphodiesterase 4D-interacting protein restored or increased both the abnormal prion protein level and the normal prion protein level, without transcriptional alteration of the prion protein gene. It also altered the localization of abnormal prion protein accumulation in the cells, indicating that phosphodiesterase 4D-interacting protein might affect prion protein levels by altering the trafficking of prion protein-containing structures. Interferon and phosphodiesterase 4D-interacting protein had no direct mutual link, demonstrating that they regulate abnormal prion protein levels independently. Although the in vivo efficacy of Gly-9 was limited, the findings for Gly-9 provide insights into the regulation of abnormal prion protein in cells and suggest new targets for antiprion compounds. IMPORTANCE: This report describes our study of the efficacy and potential mechanism underlying the antiprion action of a new antiprion compound with a glycoside structure in prion-infected cells, as well as the efficacy of the compound in prion-infected animals. The study revealed involvements of two factors in the compound's mechanism of action: interferon and a microtubule nucleation activator, phosphodiesterase 4D-interacting protein. In particular, phosphodiesterase 4D-interacting protein was suggested to be important in regulating the trafficking or fusion of prion protein-containing vesicles or structures in cells. The findings of the study are expected to be useful not only for the elucidation of cellular regulatory mechanisms of prion protein but also for the implication of new targets for therapeutic development.