RESUMEN
Transfusion of blood and blood products contaminated with the pathogenic form of prion protein Prp(sc), thought to be the causative agent of variant a Creutzfeldt-Jakob disease (vCJD), may result in serious consequences in recipients with a compromised immune system, for example, as seen in HIV-1 infection. In the present study, we demonstrate that treatment of peripheral blood monocyte-derived macrophages (MDM) with PrP106-126, a synthetic domain of PrP(sc) that has intrinsic functional activities related to the full-length protein, markedly increased their susceptibility to HIV-1 infection, induced cytokine secretion, and enhanced their migratory behavior in response to N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP). Live-cell imaging of MDM cultured in the presence of PrP106-126 showed large cell clusters indicative of cellular activation. Tyrosine kinase inhibitor STI-571, protein kinase C inhibitor K252B, and cyclin-dependent kinase inhibitor olomoucine attenuated PrP106-126-induced altered MDM functions. These findings delineate a previously undefined functional role of PrP106-126-mediated host cell response in promoting HIV-1 pathogenesis.
Asunto(s)
Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Macrófagos/virología , Monocitos/virología , Proteínas PrPSc/farmacología , Células Cultivadas , Infecciones por VIH/metabolismo , Humanos , Proteínas PrPSc/metabolismoRESUMEN
Microtubule-associated protein 2 (MAP2) belongs to the family of heat stable MAPs, which takes part in neuronal morphogenesis, maintenance of cellular architecture and internal organization, cell division and cellular processes. To obtain insight into the possible alteration and the role of MAP2 in transmissible spongiform encephalopathies (TSEs), the MAP2 levels in the brain tissues of agent 263K-infected hamsters and human prion diseases were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, MAP2 levels in the brain tissues of scrapie infected hamsters, a patient with genetic Creutzfeldt-Jakob disease (G114V gCJD) and a patient with fatal familial insomnia (FFI) were almost undetectable. The decline of MAP2 was closely related with prolonged incubation time. Exposure of SK-N-SH neuroblastoma cell line to cytotoxic PrP106-126 peptide significantly down-regulated the cellular MAP2 level and remarkably disrupted the microtubule structure, but did not alter the level of tubulin. Moreover, the levels of calpain, which mediated the degradation of a broad of cytoskeletal proteins, were significantly increased in both PrP106-126 treated SK-N-SH cells and brain tissues of 263K prion-infected hamsters. Our data indicate that the decline of MAP2 is a common phenomenon in TSEs, which seems to occur at an early stage of incubation period. Markedly increased calpain level might contribute to the reduction of MAP2.
Asunto(s)
Encéfalo/metabolismo , Calpaína/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedades por Prión/metabolismo , Enfermedades de los Roedores/metabolismo , Scrapie/metabolismo , Adulto , Secuencia de Aminoácidos , Animales , Western Blotting , Encéfalo/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Síndrome de Creutzfeldt-Jakob/metabolismo , Cricetinae , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Insomnio Familiar Fatal/metabolismo , Mesocricetus , Microscopía Confocal , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas PrPSc/farmacologíaRESUMEN
Many neurodegenerative disorders are induced by protein conformational change. Prion diseases are characterized by protein conformational conversion from a normal cellular form (PrP(C)) to an abnormal scrapie isoform (PrP(Sc)). PrP106-126 is an accepted model for studying the characteristics of PrP(Sc) because they share many biological and physiochemical properties. To understand how metal complexes affect the property of the prion peptide, the present work investigated interactions between Pd complexes and PrP106-126 based on our previous research using Pt and Au complexes to target the peptide. The selected compounds (Pd(phen)Cl(2), Pd(bipy)Cl(2), and Pd(en)Cl(2)) showed strong binding affinity to PrP106-126 and affected the conformation and aggregation of this active peptide in a different binding mode. Our results indicate that it may be the metal ligand-induced spatial effect rather the binding affinity that contributes to better inhibition on peptide aggregation. This finding would prove valuable in helping design and develop novel metallodrugs against prion diseases.
Asunto(s)
Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/farmacología , Paladio/química , Proteínas PrPSc/antagonistas & inhibidores , Priones/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dicroismo Circular , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Neuroblastoma/tratamiento farmacológico , Compuestos Organometálicos/química , Paladio/metabolismo , Proteínas PrPSc/metabolismo , Proteínas PrPSc/farmacología , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/metabolismo , Priones/química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The implication of dendritic cells (DCs) in the peripheral spreading of prions has increased in the last few years. It has been recently described that DCs can transmit prions to primary neurons from the central nervous system. In order to improve the understanding of the earliest steps of prion peripheral neuroinvasion, we studied, using an in vitro model, the effect of exposing primary peripheral neurons to scrapie-infected lymphoid cells. Thanks to this system, there is evidence that bone marrow dendritic cells (BMDCs) are in connection with neurites of peripheral neurons via cytoplasmic extensions. BMDCs are competent to internalize prions independently from the expression of cellular prion protein (PrP(C)) and have the capacity to transmit detergent-insoluble, relatively proteinase K-resistant prion protein (PrP(Sc)) to peripheral neurons after 96 h of coculture. Furthermore, we confirmed the special status of the peripheral nervous system in front of prion diseases. Contrary to central neurons, PrP(Sc) infection does not disturb survival and neurite outgrowth. Our model demonstrates that PrP(Sc)-loaded dendritic cells and peripheral nerve fibers that are included in neuroimmune interfaces can initiate and spread prion neuroinvasion.
Asunto(s)
Células Dendríticas/metabolismo , Sistema Inmunológico/metabolismo , Sistema Nervioso Periférico/metabolismo , Priones/metabolismo , Animales , Comunicación Celular/fisiología , Aumento de la Célula/efectos de los fármacos , Extensiones de la Superficie Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Citoplasma/metabolismo , Células Dendríticas/citología , Fibroblastos/metabolismo , Glicosilación , Antígenos de Histocompatibilidad Clase II/metabolismo , Sistema Inmunológico/citología , Uniones Intercelulares/metabolismo , Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/metabolismo , Neuronas/citología , Neuronas/metabolismo , Sistema Nervioso Periférico/citología , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Proteínas PrPSc/farmacología , Priones/genética , Priones/farmacología , Scrapie/etiología , Scrapie/metabolismoRESUMEN
Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (FDCs) in the GALT. Studies in mice have shown that this accumulation is obligatory for the efficient delivery of the TSE agent to the brain. However, which GALTs are crucial for disease pathogenesis is uncertain. Mice deficient in specific GALT components were used here to determine their separate involvement in scrapie agent neuroinvasion from the intestine. In the combined absence of the GALTs and FDCs (lymphotoxin (LT)alpha(-/-) mice and LTbeta(-/-) mice), scrapie agent transmission was blocked. When FDC maturation was induced in remaining lymphoid tissues, mice that lacked both Peyer's patches (PPs) and mesenteric lymph nodes (wild-type (WT)-->LTalpha(-/-) mice) or PPs alone (WT-->LTbeta(-/-) mice) remained refractory to disease, demonstrating an important role for the PPs. Although early scrapie agent accumulation also occurs within the mesenteric lymph nodes, their presence in WT-->LTbeta(-/-) mice did not restore disease susceptibility. We have also shown that isolated lymphoid follicles (ILFs) are important novel sites of TSE agent accumulation in the intestine. Mice that lacked PPs but contained numerous FDC-containing mature ILFs succumbed to scrapie at similar times to control mice. Because the formation and maturation status of ILFs is inducible and influenced by the gut flora, our data suggest that such factors could dramatically affect susceptibility to orally acquired TSE agents. In conclusion, these data demonstrate that following oral exposure TSE agent accumulation upon FDCs within lymphoid tissue within the intestine itself is critically required for efficient neuroinvasion.
Asunto(s)
Células Dendríticas Foliculares/inmunología , Intestinos/inmunología , Linfotoxina beta/inmunología , Ganglios Linfáticos Agregados/inmunología , Proteínas PrPSc/farmacología , Scrapie/inmunología , Administración Oral , Animales , Células Dendríticas Foliculares/patología , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/genética , Intestinos/patología , Linfotoxina-alfa/deficiencia , Linfotoxina-alfa/inmunología , Linfotoxina beta/deficiencia , Ratones , Ratones Noqueados , Ganglios Linfáticos Agregados/patología , Proteínas PrPSc/inmunología , Scrapie/genética , Scrapie/transmisiónRESUMEN
Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrPSc using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.
Asunto(s)
Endopeptidasa K/farmacología , Proteínas PrPSc/aislamiento & purificación , Proteínas PrPSc/metabolismo , Animales , Química Encefálica , Centrifugación por Gradiente de Densidad , Fraccionamiento Químico , Cricetinae , Endopeptidasa K/metabolismo , Hidrólisis , Mesocricetus , Proteínas PrPSc/química , Proteínas PrPSc/farmacología , Priones/metabolismo , Desnaturalización Proteica , Scrapie/metabolismo , Tripsina/metabolismoRESUMEN
Prions represent a unique class of infectious agents in which the normal cellular prion protein (PrPC) is converted to an abnormal isoform (PrPSc), which accumulates in the brain and constitutes the major, if not the only, component of the infectious particle. Factors that still remain to be identified may facilitate the conversion of PrPC to PrPSc. In the present study, we first demonstrated that a growth factor of the neurotrophin family, brain-derived neurotrophic factor (BDNF), stimulates the formation of PrPSc in a gonadotropin-releasing hormone-secreting neuronal cell line (GT1-1 cells) infected with the Rocky Mountain Laboratory (RML) strain of scrapie as determined by Western blot analysis. We then observed that the prion-infected cells can be cleared from PrPSc by treatment with three inhibitors of mitogen-activated protein kinase kinase 1/2 (MEK1/2) [1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene and 2-(2-amino-3-methyoxyphenyl)-4H-1-benzopyran-4-one, as well as alpha-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile, which passes the blood-brain barrier], a component of one of the intracellular signaling pathways activated by BDNF. The MEK1/2 inhibitors were also efficient in clearing PrPSc from prion-infected GT1-1 cells stimulated to accumulate high levels of PrPSc by enhanced serum concentrations in the medium or by the use of a serum-free neuron-specific neurobasal medium. PrPSc did not reappear in the cultures within 5 weeks after completion of treatment. We conclude that inhibitors of the MEK1/2 pathway can efficiently and probably irreversibly clear PrP(Sc) from prion-infected cells. The MEK pathway may therefore be a suitable target for therapeutic intervention in prion diseases.
Asunto(s)
Hipotálamo/fisiología , MAP Quinasa Quinasa 1/fisiología , MAP Quinasa Quinasa 2/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Neuronas/fisiología , Proteínas PrPSc/farmacología , Animales , Factor Neurotrófico Derivado del Encéfalo/farmacología , Células Cultivadas , Medios de Cultivo , Hormona Liberadora de Gonadotropina/metabolismo , Hipotálamo/efectos de los fármacos , MAP Quinasa Quinasa 1/efectos de los fármacos , MAP Quinasa Quinasa 2/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Neuronas/efectos de los fármacosRESUMEN
Complement activation products C1q and C3d, serum amyloid P component (SAP) and activated glial cells accumulate in amyloid deposits of conformationally changed prion protein (PrP(Sc)) in Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker disease and scrapie-infected mouse brain. Biological properties, including the potential to activate microglia, relate to prion (PrP) peptide fibrillogenic abilities. We investigated if SAP and C1q influence the fibrillogenic properties of human and mouse PrP peptide and concomitantly their stimulatory effects on human microglia in vitro. PrP-peptide induced microglial IL-6 and TNF-alpha release significantly increased in the presence of SAP and C1q. Also, SAP and C1q enhanced PrP-peptide fibril formation as revealed by electron microscopy and thioflavin S-based quantitative assays. This suggests that SAP and C1q contribute to fibrillar state-dependent cellular effects of PrP. Combined, ultrastructural and thioflavin assays, together with microglial cytokine release measurements, provide a test system to screen potential, fibrillarity impeding therapeutics for prion disease.
Asunto(s)
Complemento C1q/farmacología , Microglía/metabolismo , Microglía/patología , Fragmentos de Péptidos/farmacología , Proteínas PrPSc/farmacología , Componente Amiloide P Sérico/farmacología , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Persona de Mediana Edad , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Proteínas PrPSc/genéticaRESUMEN
The essential component of infectious prions is a misfolded protein termed PrPSc, which is produced by conformational change of a normal host protein, PrPC. It is currently unknown whether PrPSc molecules exist in a unique conformation or whether they are able to undergo additional conformational changes. Under commonly used experimental conditions, PrPSc molecules are characteristically protease-resistant and capable of inducing the conversion of PrPC molecules into new PrPSc molecules. We describe the effects of ionic strength, copper, and zinc on the conformation-dependent protease resistance and conversion-inducing activity of PrPSc molecules in scrapie-infected hamster brains. In the absence of divalent cations, PrPSc molecules were > 20-fold more sensitive to proteinase K digestion in low ionic strength buffers than in high ionic strength buffers. Addition of micromolar concentrations of copper or zinc ions restored the protease resistance of PrPSc molecules under conditions of low ionic strength. These transition metals also controlled the conformation of purified truncated PrP-(27-30) molecules at low ionic strength, confirming that the N-terminal octapeptide repeat region of PrPSc is not required for binding to copper or zinc ions. The protease-sensitive and protease-resistant conformations of PrPSc were reversibly interchangeable, and only the protease-resistant conformation of PrPSc induced by high ionic strength was able to induce the formation of new protease-resistant PrP (PrPres) molecules in vitro. These findings show that PrPSc molecules are structurally interconvertible and that only a subset of PrPSc conformations are able to induce the conversion of other PrP molecules.
Asunto(s)
Iones , Proteínas PrPSc/farmacología , Animales , Encéfalo/metabolismo , Cationes , Membrana Celular/metabolismo , Cobre/química , Cricetinae , Relación Dosis-Respuesta a Droga , Endopeptidasa K/farmacología , Endopeptidasas/química , Immunoblotting , Mesocricetus , Metales , Ratones , Proteínas PrPSc/química , Conformación Proteica , Scrapie/metabolismo , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Zinc/químicaRESUMEN
The inflammatory response in prion diseases is dominated by microglial activation. As macrophages of the central nervous system, the phagocytic capacity of microglia is well recognized, and it is possible that microglia are involved in the removal and processing of amyloid fibrils, thus preventing their harmful effect. We have analyzed the effects of a synthetic peptide of the human prion protein, PrP(106-126), which can form fibrils, and the pathogenic form of prion protein, PrPsc, on phagocytosis in microglia isolated from neonatal rat brain cultures. To some extent, fibrillar PrP(106-126) is internalized and processed. However, both synthetic prion peptide PrP(106-126) in a fibrillar form and pathogenic prion protein PrPsc severely hamper the phagocytic activity as measured by the uptake of beads by microglia. At a concentration that does not induce microglial death, PrP(106-126) reduced the number of beads internalized and altered their cytoplasmic distribution. This effect was not due to decreased binding of beads to the cell surface, nor restricted to specific classes of receptors. Although the PrP(106-126) did not prevent F-actin and Rac1 accumulation at sites of particle engulfment, it appeared to interfere with a later step of the internalization process.
Asunto(s)
Microglía/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fagocitosis/efectos de los fármacos , Proteínas PrPSc/farmacología , Priones/farmacología , Actinas/metabolismo , Animales , Anticuerpos , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Microglía/inmunología , Microglía/ultraestructura , Microscopía Electrónica , Fragmentos de Péptidos/inmunología , Fagocitosis/inmunología , Priones/inmunología , Ratas , Ratas Wistar , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The accumulation and activation of microglial cells at sites of amyloid prion deposits or plaques have been documented extensively. Here, we investigate the in vivo recruitment of microglial cells soon after intraocular injection of scrapie-infected cell homogenate (hgtsc+) using immunohistochemistry on retinal sections. A population of CD11b/CD45-positive microglia was specifically detected within the ganglion and internal plexiform retinal cell layers by 2 d after intravitreal injection of hgtsc+. Whereas no chemotactism properties were ascribed to hgtsc+ alone, a massive migration of microglial cells was observed by incubating primary cultured neurons and astrocytes with hgtsc+ in a time- and concentration-dependent manner. hgtsc+ triggered the recruitment of microglial cells by interacting with both neurons and astrocytes by upregulation of the expression levels of a broad spectrum of neuronal and glial chemokines. We show that, in vitro and in vivo, the microglia migration is at least partly under the control of chemokine receptor-5 (CCR-5) activation, because highly specific CCR-5 antagonist TAK-779 significantly reduced the migration rate of microglia. Activated microglia recruited in the vicinity of prion may, in turn, cause neuronal cell damage by inducing apoptosis. These findings provide insight into the understanding of the cell-cell communication that takes place during the development of prion diseases.
Asunto(s)
Astrocitos/metabolismo , Microglía/fisiología , Neuronas/metabolismo , Proteínas PrPSc/farmacología , Enfermedades por Prión/fisiopatología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Recuento de Células , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Endopeptidasa K/química , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/patología , Neuroblastoma , Neuronas/efectos de los fármacos , Neuronas/patología , Nitritos/metabolismo , Nervio Óptico/patología , Proteínas PrPSc/patogenicidad , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Receptores CCR5/metabolismo , Retina/efectos de los fármacos , Retina/patología , Fracciones Subcelulares/químicaRESUMEN
Previous studies in Creutzfeldt-Jakob disease (CJD) have shown that myeloid cells in the periphery as well as derivative microglial cells in the brain are infectious. Microglia can show an activated phenotype before prion protein (PrP) pathology is detectable in brain, and isolated infectious microglia contain very little PrP. To find whether a set of inflammatory genes are significantly induced or suppressed with infection, we analyzed RNA from isolated microglia with relevant cDNA arrays, and identified approximately 30 transcripts not previously examined in any transmissible spongiform encephalopathy. This CJD expression profile contrasted with that of uninfected microglia exposed to prototypic inflammatory stimuli such as lipopolysaccharide and IFN-gamma, as well as PrP amyloid. These findings underscore inflammatory pathways evoked by the infectious agent in brain. Transcript profiles unique for CJD microglia and other myeloid cells provide opportunities for more sensitive preclinical diagnoses of infectious and noninfectious neurodegenerative diseases.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/metabolismo , Perfilación de la Expresión Génica , Inflamación/metabolismo , Microglía/metabolismo , Animales , Células Cultivadas , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas PrPSc/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Feline spongiform encephalopathy (FSE) is thought to have resulted from consumption of food contaminated with bovine spongiform encephalopathy and the latter is believed to result from the consumption of food contaminated with scrapie. However, no direct experimental documentation exists to indicate that the scrapie agent is capable of amplifying in cats, and, therefore, crossing the species barrier. During 1979, 6 cats ranging in age from 3.5 to 18 months were intracerebrally inoculated with sheep scrapie (inoculum G-639-PP) and were observed for an extended period. Inoculated cats did not develop neurologic disease, and microscopic lesions of spongiform encephalopathy were not evident. Immunohistochemistry and Western blot techniques failed to detect the abnormal form of prion protein (PrP(res)). These results indicate that the sheep scrapie agent (G-639-PP) used in this study was not capable of amplifying in cats and therefore was unable to cross the species barrier to produce FSE.
Asunto(s)
Enfermedades de los Gatos/inducido químicamente , Enfermedades de los Gatos/transmisión , Proteínas PrPSc/administración & dosificación , Proteínas PrPSc/farmacología , Enfermedades por Prión/transmisión , Enfermedades por Prión/veterinaria , Enfermedades de las Ovejas/transmisión , Animales , Western Blotting , Gatos , Femenino , Inmunohistoquímica , Inyecciones Intraventriculares , Masculino , Enfermedades por Prión/inducido químicamente , Oveja Doméstica , Especificidad de la Especie , Estados UnidosRESUMEN
To identify molecular interaction partners of the cellular prion protein (PrP(C)), we sought to apply an in situ crosslinking method that maintains the microenvironment of PrP(C). Mild formaldehyde crosslinking of mouse neuroblastoma cells (N2a) that are susceptible to prion infection revealed the presence of PrP(C) in high molecular mass (HMM) protein complexes of 200 to 225 kDa. LC/MS/MS analysis identified three murine splice-variants of the neural cell adhesion molecule (N-CAM) in the complexes, which isolate with caveolae-like domains (CLDs). Enzymatic removal of N-linked sugar moieties did not disrupt the complexes, arguing that the interaction of PrP with N-CAM occurs through amino acid side-chains. Additionally, similar levels of PrP/N-CAM complexes were found in N2a and prion-infected N2a (ScN2a) cells. With the use of an N-CAM-specific peptide library, the PrP-binding site was determined to comprise beta-strands C and C' within the two consecutive fibronectin type III (FNIII) modules found in proximity of the membrane-attachment site of N-CAM. As revealed by in situ crosslinking of PrP deletion mutants, the PrP face of the binding site is formed by the N terminus, helix A (residues 144-154) and the adjacent loop region of PrP. N-CAM-deficient (N-CAM(-/-)) mice that were intracerebrally challenged with scrapie prions succumbed to disease with a mean incubation period of 122 (+/-4.1, SEM) days, arguing that N-CAM is not involved in PrP(Sc) replication. Our findings raise the possibility that N-CAM may join with PrP(C) in carrying out some as yet unidentified physiologic cellular function.
Asunto(s)
Moléculas de Adhesión de Célula Nerviosa/química , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Empalme Alternativo/genética , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Caveolas/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Endopeptidasa K/metabolismo , Formaldehído/metabolismo , Sustancias Macromoleculares , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Peso Molecular , Mutación/genética , Moléculas de Adhesión de Célula Nerviosa/genética , Neuroblastoma/metabolismo , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Fosfatidilinositol Diacilglicerol-Liasa , Proteínas PrPC/genética , Proteínas PrPSc/farmacología , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Sitios de Empalme de ARN/genética , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismoRESUMEN
Behavioural testing can reveal effects in scrapie-infected mice long before overt clinical signs appear (Betmouni et al., 1999, Psychobiology, 27, 63-71). These effects may be partly attributable to an early, atypical inflammatory response in the brain (Betmouni et al., 1996, Neuroscience, 74, 1-5). The present study replicated and extended these findings, and examined the effect of chronic treatment with dapsone. This anti-inflammatory compound has been reported to delay disease onset in a rat model of Creutzfeldt-Jakob disease (Manuelidis et al., 1998, Lancet, 352, 456). Although the doses used in the present study were higher than those of Manuelidis et al. (1998), no attenuation of the disease was seen in either behavioural or subsequent histological tests. Burrowing, i.e. displacing food pellets from a tube in the home cage, decreased from around week 12 in scrapie-infected mice, as did consumption of palatable glucose solution. Concurrently, ambulation in an open field increased, as did rearing at around week 17. Spontaneous alternation was impaired around this time. Around 18 weeks, motor performance on an inverted screen, horizontal bar, rotating rod and static rods decreased. Nest construction was impaired at 20 weeks. Overt clinical signs (reduction in mobility, hunched posture, poor coat condition, bladder enlargement) only occurred after week 20, when the mice were prepared for histology. The ME7 scrapie-infected mice thus showed a characteristic complex of neurological and behavioural changes during the course of the disease that were not ameliorated by dapsone. These changes appeared well before clinical signs were prominent.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Dapsona/farmacología , Encefalitis/tratamiento farmacológico , Ratones Endogámicos C57BL/fisiología , Scrapie/tratamiento farmacológico , Animales , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Conducta Animal/fisiología , Peso Corporal/efectos de los fármacos , Peso Corporal/fisiología , Encéfalo/patología , Encéfalo/fisiopatología , Extractos Celulares/farmacología , Modelos Animales de Enfermedad , Ingestión de Alimentos/efectos de los fármacos , Ingestión de Alimentos/fisiología , Encefalitis/fisiopatología , Femenino , Gliosis/inducido químicamente , Gliosis/patología , Gliosis/fisiopatología , Inmunohistoquímica , Ratones , Microglía/efectos de los fármacos , Microglía/metabolismo , Microglía/patología , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Degeneración Nerviosa/fisiopatología , Proteínas PrPSc/farmacología , Scrapie/patología , Scrapie/fisiopatología , Factores de TiempoRESUMEN
The sequence of events involved in the neurodegeneration caused by transmissible spongiform encephalopathies (TSEs) is not yet known. Using a murine scrapie model in which neurodegeneration in the hippocampus is restricted to CA2, we show that pyramidal neuron damage and death by an apoptotic mechanism occur early in the incubation period, prior to the appearance of CA2 disease-specific accumulation of PrP and the onset of clinical disease. We suggest that the initial hippocampal pathological event in this model is dendritic dysfunction and activation of an apoptotic pathway rather than PrP accumulation.
Asunto(s)
Apoptosis/fisiología , Dendritas/patología , Priones/metabolismo , Scrapie/metabolismo , Scrapie/patología , Animales , Apoptosis/efectos de los fármacos , Dendritas/efectos de los fármacos , Modelos Animales de Enfermedad , Genes jun/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/patología , Inmunohistoquímica , Ratones , Proteínas PrPSc/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/patologíaRESUMEN
Prion diseases are characterized by the accumulation of a specific disease-associated isoform of the prion protein (PrP), termed PrPSc, which is the main, if not the only, component of the infectious agent termed prion. PrPSc is derived by an autocatalytic post-translational process involving conformational changes from the normal host-encoded isoform of the prion protein, termed PrPC. PrPC is a copper-binding glycoprotein attached to the cell membrane of neurons and other cells by means of a GPI anchor. The pattern of neurodegeneration differs between variants of prion disease and is related to the pattern of PrPSc deposition and differences in susceptibility of different cell types to the disease process. The pattern of PrPSc deposition depends on the strain of the agent and the PrP genotype of the host. Strain properties of prions appear to be related to different pathological conformations of PrPSc. Neuronal cell death is a salient feature in the pathology of prion diseases. Histological and electron microscopical studies have shown that cell death in prion disease occurs by apoptosis. Apoptosis of neuronal cells can also be induced in vitro by exposure to PrPSc or a neurotoxic peptide fragment corresponding to amino acids 106-126 of human prion protein (PrP106-126). Both in vitro and in vivo, the toxicity of PrPSc and PrP fragments appears to depend on neuronal expression of PrPC and on microglial activation. Activated microglial cells release pro-inflammatory cytokines and reactive oxygen species. Cell culture experiments suggest an important role of microglia-mediated oxidative stress in the induction of neuronal cell death. Only limited data are available on direct effects of PrPSc on neuronal cells. Potential effects include increased formation of an aberrant transmembrane form of PrP, termed CtmPrP, and changes in plasma membrane properties. In addition to direct and indirect toxic effects of PrPSc, a loss of function of PrPC may contribute to neuronal cell death. Potential mechanisms include disturbances in cerebral copper metabolism and antioxidative defense mechanisms. A better understanding of the pathogenesis of neuronal cell death in prion diseases may also have important therapeutic implications in the future.
Asunto(s)
Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/etiología , Síndrome de Creutzfeldt-Jakob/patología , Neuronas/patología , Secuencia de Aminoácidos , Animales , Apoptosis , Encéfalo/efectos de los fármacos , Células Cultivadas , Cobre/metabolismo , Síndrome de Creutzfeldt-Jakob/veterinaria , Humanos , Microglía/efectos de los fármacos , Microglía/patología , Datos de Secuencia Molecular , Neuronas/metabolismo , Estrés Oxidativo , Proteínas PrPC/patogenicidad , Proteínas PrPSc/química , Proteínas PrPSc/patogenicidad , Proteínas PrPSc/farmacología , Priones/patogenicidadRESUMEN
The scrapie isoform of the prion protein (PrPSc) induces pathological changes in the central nervous system including neurodegeneration and gliosis. A synthetic prion protein (PrP) peptide corresponding to amino acid residues 106-126 has been shown to be toxic to neurons that express PrPC, the cellular isoform of PrP. Here we show that in mixed glial cultures PrP106-126 induces astroglial proliferation that is dependent on cellular PrPc expression. In purified cultures of glial subtypes only microglia proliferated in response to PrP106-126. This effect was independent of PrP expression. Destruction of microglia in mixed glial cultures by L-leucine methyl ester (LLME) treatment abolished enhanced proliferation caused by PrP106-126. This proliferative effect can be restored by co-culturing LLME-treated astrocytes with microglia. Microglia therefore seem to mediate the proliferative effect exerted by PrP106-126 on astrocytes.