RESUMEN
Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126. Exposure to its aggregated form induced a massive neuronal death within 24 h. Apoptosis was characterized by nuclear fragmentation, neuritic retraction and fragmentation and activation of caspase-3. During the early decision phase, reactive oxygen species were detected after 3 h. Using immunocytochemistry, we showed a peak of phosphorylated c-Jun-N-terminal kinase (JNK) translocation into the nucleus after 8 h, along with the activation of the nuclear c-Jun transcription factor. Both pharmacological inhibition of JNK by SP600125 and overexpression of a dominant negative form of c-Jun significantly reduced neuronal death, while the MAPK p38 inhibitor SB203580 had no effect. Apoptosis was also studied after exposure of tg338 cortical neurons in primary culture to sheep scrapie agent. In this model, prion-induced neuronal apoptosis gradually increased with time and induced a 40% cell death after 2 weeks exposure. Immunocytochemical analysis showed early c-Jun activation after 7 days. In summary, the JNK-c-Jun pathway plays an important role in neuronal apoptosis induced by PrP106-126. This pathway is also activated during scrapie infection and may be involved in prion-induced neuronal death. Pharmacological blockade of early pathways opens new therapeutic prospects for scrapie PrP-based pathologies.
Asunto(s)
Apoptosis/fisiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/farmacología , Priones/farmacología , Scrapie/metabolismo , Scrapie/patología , Animales , Núcleo Celular/metabolismo , Corteza Cerebral/patología , Femenino , Ratones , Ratones Mutantes , Neuronas/patología , Fosforilación , Embarazo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cell-binding and internalization studies on neuronal and non-neuronal cells have demonstrated that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor for the cellular prion protein (PrP). Here we identify direct and heparan sulfate proteoglycan (HSPG)-dependent interaction sites mediating the binding of the cellular PrP to its receptor, which we demonstrated in vitro on recombinant proteins. Mapping analyses in the yeast two-hybrid system and cell-binding assays identified PrPLRPbd1 [amino acids (aa) 144-179] as a direct and PrPLRPbd2 (aa 53-93) as an indirect HSPG-dependent laminin receptor precursor (LRP)-binding site on PrP. The yeast two-hybrid system localized the direct PrP-binding domain on LRP between aa 161 and 179. Expression of an LRP mutant lacking the direct PrP-binding domain in wild-type and mutant HSPG-deficient Chinese hamster ovary cells by the Semliki Forest virus system demonstrates a second HSPG-dependent PrP-binding site on LRP. Considering the absence of LRP homodimerization and the direct and indirect LRP-PrP interaction sites, we propose a comprehensive model for the LRP-PrP-HSPG complex.
Asunto(s)
Proteoglicanos de Heparán Sulfato/metabolismo , Priones/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Laminina/metabolismo , Técnicas del Sistema de Dos Híbridos , Animales , Sitios de Unión/fisiología , Células CHO , Línea Celular , Cromatografía en Gel , Cricetinae , Galactósidos/metabolismo , Glutatión Transferasa/genética , Proteoglicanos de Heparán Sulfato/genética , Humanos , Ratones , Oligopéptidos , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/genética , Priones/genética , Unión Proteica/fisiología , Precursores de Proteínas/genética , Estructura Terciaria de Proteína/fisiología , Receptores de Laminina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus de los Bosques Semliki/genéticaRESUMEN
Recently, we identified the 37-kDa laminin receptor precursor (LRP) as an interactor for the prion protein (PrP). Here, we show the presence of the 37-kDa LRP and its mature 67-kDa form termed high-affinity laminin receptor (LR) in plasma membrane fractions of N2a cells, whereas only the 37-kDa LRP was detected in baby hamster kidney (BHK) cells. PrP co-localizes with LRP/LR on the surface of N2a cells and Semliki Forest virus (SFV) RNA transfected BHK cells. Cell-binding assays reveal the LRP/LR-dependent binding of cellular PrP by neuronal and non-neuronal cells. Hyperexpression of LRP on the surface of BHK cells results in the binding of exogenous PrP. Cell binding is similar in PrP(+/+) and PrP(0/0) primary neurons, demonstrating that PrP does not act as a co-receptor of LRP/LR. LRP/LR-dependent internalization of PrP is blocked at 4 degrees C. Secretion of an LRP mutant lacking the transmembrane domain (aa 86-101) from BHK cells abolishes PrP binding and internalization. Our results show that LRP/LR acts as the receptor for cellular PrP on the surface of mammalian cells.
Asunto(s)
Priones/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Laminina/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Citometría de Flujo , Humanos , Riñón/citología , Riñón/metabolismo , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida , Neuroblastoma/metabolismo , Neuronas/citología , Neuronas/metabolismo , Oligopéptidos , Péptidos/genética , Enfermedades por Prión/etiología , Priones/genética , Unión Proteica/fisiología , Precursores de Proteínas/genética , Estructura Terciaria de Proteína/fisiología , Receptores de Laminina/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Virus de los Bosques Semliki/genética , TransfecciónRESUMEN
It has been shown recently that the generation of an abnormal transmembrane form of the prion protein ((Ctm)PrP) is involved in the neurodegeneration process during inherited and infectious prion diseases but a causative relationship has never been established. We wanted to know if and how the proposed transmembrane domain of PrP could induce neuronal dysfunction. Thus, we investigated the neurotoxic properties of two peptides whose sequences are encompassed within this domain. We show that PrP peptides 118-135 and 105-132 as well as an amidated more soluble peptide 105-132 induce the death of pure cortical neurons originating from normal and PrP knockout mice. This can be correlated with the high propensity of these peptides to insert stably into and to destabilize cell membranes. Through this study, we have identified a novel mechanism of neurotoxicity for PrP, which directly involves membrane perturbation; this mechanism is independent of fibril formation and probably corresponds to the effect of the transmembrane insertion of (Ctm)PrP.
Asunto(s)
Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Enfermedades por Prión/metabolismo , Priones/toxicidad , Péptidos beta-Amiloides/toxicidad , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Aminoácidos Excitadores/farmacología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Modelos Moleculares , Método de Montecarlo , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/ultraestructura , Enfermedades por Prión/etiología , Priones/biosíntesis , Priones/síntesis química , Priones/química , Priones/ultraestructura , Estructura Terciaria de ProteínaRESUMEN
During brain development, neuronal and glial cells are generated from neural precursors on a precise schedule involving steps of proliferation, fate commitment and differentiation. We report that telomerase activity is highly expressed during embryonic murine cortical neurogenesis and early steps of gliogenesis and progressively decreases thereafter during cortex maturation to be undetectable in the normal adult brain. We evidenced neural precursor cells (NPC) as the principal telomerase-expressing cells in primary cultures from E15 mouse embryo cortices. Their differentiation either in neurons or in glial cells lead to a down regulation of telomerase activity that was directly correlated to the decrease of telomerase core protein (mTERT) mRNA synthesis. Furthermore, we show that FGF2 (fibroblast growth factor 2), one of the main regulators of CNS development, induces a dose-dependant increase of both the proliferation of NPC and telomerase activity in primary cortical cultures without affecting the mTERT mRNA synthesis compared to that of glyceraldehyde-3-phosphate dehydrogenase (mGAPDH). Finally, we evidenced that AZT (3'-azido-2', 3'-dideoxythymidine), known to inhibit telomerase activity, blocks in a dose dependant manner the FGF2-induced proliferation of NPC. Altogether, our results are in favor of an important role of telomerase activity during brain organogenesis. Oncogene (2000).
Asunto(s)
Encéfalo/enzimología , Factor 2 de Crecimiento de Fibroblastos/fisiología , ARN , Telomerasa/genética , Regulación hacia Arriba/fisiología , Animales , Secuencia de Bases , Encéfalo/citología , Encéfalo/embriología , Diferenciación Celular , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Telomerasa/metabolismo , Zidovudina/farmacologíaRESUMEN
Transmissible subacute spongiform encephalopathies (TSE) are animal and human neurodegenerative diseases. The nature of the transmissible agent remains unknown. The specific molecular marker of these diseases is the abnormal isoform of the prion protein (PrP). This protein is encoded by a cellular gene and accumulates in a pathological isoform (PrPres) which is partially resistant to proteolysis. The tridimensional structure of this protein remains theoretical. F. Cohen proposed one of the most realistic models. According to this model and from molecular mechanics calculation, we suggest a PrP oligomeric ionic channel model that may be involved in TSE-induced neuronal apoptosis.
Asunto(s)
Canales Iónicos/fisiología , Modelos Biológicos , Neuronas/patología , Enfermedades por Prión/fisiopatología , Priones/patogenicidad , Animales , Apoptosis/fisiología , Humanos , Enfermedades por Prión/patología , Priones/fisiología , Conformación ProteicaRESUMEN
The scrapie isoform of the prion protein (PrPres) induces neurodegeneration and gliosis in the central nervous system. These features may be reproduced in vitro on exposure of neuronal and glial cultures to PrPres and the peptide HuPr P106-126. In the present study, we investigated the role of microglial cells and astrocytes in the pathological process by studying their molecular response to PrP 106-126 exposure. PrP 106-126 elicited a specific overproduction of pro-inflammatory cytokines IL1beta and IL6 in microglial cells (but not increased expression of TNFalpha, IL10, and TGFbeta1) and over-expression of GFAP in astrocytes. These effects were strictly dependent on the ability of the peptide to form amyloid fibrils. These data strongly suggest that microglial cells contribute to prion-related neurodegenerative processes by producing proinflammatory cytokines in the brain areas of amyloid PrP deposition.
Asunto(s)
Citocinas/biosíntesis , Microglía/metabolismo , Fragmentos de Péptidos/farmacología , Priones/farmacología , Animales , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Humanos , Inflamación/metabolismo , Interleucina-1/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Degeneración Nerviosa/patología , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
Spongiform transmissible encephalopathies are neurodegenerative diseases characterized by the accumulation, in infected brains, of a pathological form of a normal host-encoded protein called PrP. Previous data have shown that PrP could interact with cytosolic factors, including nuclear molecules, emphasizing the possible function of such interactions. Moreover, in infected cells, PrP is observed not only at the plasma membrane but also in the nuclear compartment. The N-terminal extremity of the mature PrP has been thought to harbor a nuclear localization signal reminiscent of the nuclear localization signal of the simian virus 40 large T antigen. By designing a fusion protein between the putative nuclear localization signal of PrP and the green fluorescent protein, we have shown that the N-terminal sequence of PrP is not efficient in targeting the protein in the nuclear compartment. This implies new insights regarding the way by which PrP could, however, reach the nuclear compartment.
Asunto(s)
Núcleo Celular/química , Núcleo Celular/metabolismo , Señales de Localización Nuclear , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico/fisiología , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Inmunohistoquímica , Indicadores y Reactivos , Proteínas Luminiscentes , Microscopía Confocal , Datos de Secuencia Molecular , Plásmidos , Proteínas PrPC/genética , Análisis de Secuencia de ADN , TransfecciónRESUMEN
The agent responsible for transmissible spongiform encephalopathies (TSEs) is thought to be a malfolded, protease-resistant version (PrPres) of the normal cellular prion protein (PrP). The interspecies transmission of bovine spongiform encephalopathy (BSE) to mice was studied. Although all of the mice injected with homogenate from BSE-infected cattle brain exhibited neurological symptoms and neuronal death, more than 55 percent had no detectable PrPres. During serial passage, PrPres appeared after the agent became adapted to the new host. Thus, PrPres may be involved in species adaptation, but a further unidentified agent may actually transmit BSE.