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1.
PLoS Pathog ; 13(11): e1006733, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-29176838

RESUMEN

Prion infections cause inexorable, progressive neurological dysfunction and neurodegeneration. Expression of the cellular prion protein PrPC is required for toxicity, suggesting the existence of deleterious PrPC-dependent signaling cascades. Because group-I metabotropic glutamate receptors (mGluR1 and mGluR5) can form complexes with the cellular prion protein (PrPC), we investigated the impact of mGluR1 and mGluR5 inhibition on prion toxicity ex vivo and in vivo. We found that pharmacological inhibition of mGluR1 and mGluR5 antagonized dose-dependently the neurotoxicity triggered by prion infection and by prion-mimetic anti-PrPC antibodies in organotypic brain slices. Prion-mimetic antibodies increased mGluR5 clustering around dendritic spines, mimicking the toxicity of Aß oligomers. Oral treatment with the mGluR5 inhibitor, MPEP, delayed the onset of motor deficits and moderately prolonged survival of prion-infected mice. Although group-I mGluR inhibition was not curative, these results suggest that it may alleviate the neurological dysfunctions induced by prion diseases.


Asunto(s)
Proteínas PrPC/toxicidad , Enfermedades por Prión/tratamiento farmacológico , Enfermedades por Prión/metabolismo , Piridinas/administración & dosificación , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Animales , Anticuerpos/administración & dosificación , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Enfermedades por Prión/genética , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Receptor del Glutamato Metabotropico 5/genética , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo
2.
Elife ; 62017 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-28527237

RESUMEN

PrPC, the cellular isoform of the prion protein, serves to transduce the neurotoxic effects of PrPSc, the infectious isoform, but how this occurs is mysterious. Here, using a combination of electrophysiological, cellular, and biophysical techniques, we show that the flexible, N-terminal domain of PrPC functions as a powerful toxicity-transducing effector whose activity is tightly regulated in cis by the globular C-terminal domain. Ligands binding to the N-terminal domain abolish the spontaneous ionic currents associated with neurotoxic mutants of PrP, and the isolated N-terminal domain induces currents when expressed in the absence of the C-terminal domain. Anti-PrP antibodies targeting epitopes in the C-terminal domain induce currents, and cause degeneration of dendrites on murine hippocampal neurons, effects that entirely dependent on the effector function of the N-terminus. NMR experiments demonstrate intramolecular docking between N- and C-terminal domains of PrPC, revealing a novel auto-inhibitory mechanism that regulates the functional activity of PrPC.


Asunto(s)
Homeostasis , Proteínas PrPC/toxicidad , Proteínas Priónicas/toxicidad , Animales , Dendritas/patología , Hipocampo/patología , Espectroscopía de Resonancia Magnética , Ratones , Neuronas/patología , Proteínas PrPC/química , Proteínas Priónicas/química , Conformación Proteica
3.
Neurobiol Aging ; 36(3): 1369-77, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25599875

RESUMEN

A major feature of Alzheimer's disease is the accumulation of ß-amyloid (Aß) peptide in the brain. Recent studies have indicated that Aß oligomers (Aßo) can interact with the cellular prion protein (PrPc). Therefore, this interaction might be driving some of Aß toxic effects in the synaptic region. In the present study, we report that Aßo binds to PrPc in the neuronal membrane playing a role on toxic effects induced by Aß. Phospholipase C-enzymatic cleavage of PrPc from the plasma membrane attenuated the association of Aßo to the neurons. Furthermore, an anti-PrP antibody (6D11) decreased the association of Aßo to hippocampal neurons with a concomitant reduction in Aßo and PrPc co-localization. Interestingly, this antibody blocked the increase in membrane conductance and intracellular calcium induced by Aßo. Thus, the data indicate that PrPc plays a role on the membrane perforations produced by Aßo, the increase in calcium ions and the release of synaptic vesicles that subsequently leads to synaptic failure. Future studies blocking Aßo interaction with PrPc could be important for the discovery of new therapeutic strategies for Alzheimer's disease.


Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Membrana Celular/patología , Proteínas PrPC/toxicidad , Sinapsis/patología , Péptidos beta-Amiloides/fisiología , Animales , Calcio/metabolismo , Células Cultivadas , Femenino , Hipocampo/citología , Terapia Molecular Dirigida , Neuronas/patología , Proteínas PrPC/fisiología , Embarazo , Dominios y Motivos de Interacción de Proteínas , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo
4.
Nat Commun ; 5: 4347, 2014 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-25005024

RESUMEN

Prions are lethal infectious agents thought to consist of multi-chain forms (PrP(Sc)) of misfolded cellular prion protein (PrP(C)). Prion propagation proceeds in two distinct mechanistic phases: an exponential phase 1, which rapidly reaches a fixed level of infectivity irrespective of PrP(C) expression level, and a plateau (phase 2), which continues until clinical onset with duration inversely proportional to PrP(C) expression level. We hypothesized that neurotoxicity relates to distinct neurotoxic species produced following a pathway switch when prion levels saturate. Here we show a linear increase of proteinase K-sensitive PrP isoforms distinct from classical PrP(Sc) at a rate proportional to PrP(C) concentration, commencing at the phase transition and rising until clinical onset. The unaltered level of total PrP during phase 1, when prion infectivity increases a million-fold, indicates that prions comprise a small minority of total PrP. This is consistent with PrP(C) concentration not being rate limiting to exponential prion propagation and neurotoxicity relating to critical concentrations of alternate PrP isoforms whose production is PrP(C) concentration dependent.


Asunto(s)
Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Animales , Femenino , Humanos , Cinética , Ratones , Proteínas PrPC/química , Proteínas PrPSc/química , Proteínas PrPSc/toxicidad
5.
Prion ; 8(2)2014.
Artículo en Inglés | MEDLINE | ID: mdl-24721836

RESUMEN

Knockout of the cellular prion protein (PrP(C)) in mice is tolerated, as is complete elimination of the protein's N-terminal domain. However, deletion of select short segments between the N- and C-terminal domains is lethal. How can one reconcile this apparent paradox? Research over the last few years demonstrates that PrP(C) undergoes α-cleavage in the vicinity of residue 109 (mouse sequence) to release the bioactive N1 and C1 fragments. In biophysical studies, we recently characterized the action of relevant members of the ADAM (A Disintegrin And Metalloproteinase) enzyme family (ADAM8, 10, and 17) and found that they all produce α-cleavage, but at 3 distinct cleavage sites, with proteolytic efficiency modulated by the physiologic metals copper and zinc. Remarkably, the shortest lethal deletion segment in PrP(C) fully encompasses the 3 α-cleavage sites. Analysis of all reported PrP(C) deletion mutants suggests that elimination of α-cleavage, coupled with retention of the protein's N-terminal residues, segments 23-31 and longer, confers the lethal phenotype. Interestingly, these N-terminal residues are implicated in the activation of several membrane proteins, including synaptic glutamate receptors. We propose that α-cleavage is a general mechanism essential for downregulating PrP(C)'s intrinsic activity, and that blockage of proteolysis leads to constitutively active PrP(C) and consequent dyshomeostasis.


Asunto(s)
Proteínas PrPC/fisiología , Enfermedades por Prión/metabolismo , Animales , Ratones , Ratones Noqueados , Proteínas PrPC/genética , Proteínas PrPC/toxicidad , Proteolisis , Zinc/metabolismo
6.
Biochem J ; 459(1): 103-15, 2014 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-24438129

RESUMEN

Misfolding of PrPC (cellular prion protein) to ß-strand-rich conformations constitutes a key event in prion disease pathogenesis. PrPC can undergo either of two constitutive endoproteolytic events known as α- and ß-cleavage, yielding C-terminal fragments known as C1 and C2 respectively. It is unclear whether C-terminal fragments generated through α- and ß-cleavage, especially C2, influence pathogenesis directly. Consequently, we compared the biophysical properties and neurotoxicity of recombinant human PrP fragments recapitulating α- and ß-cleavage, namely huPrP-(112-231) (equating to C1) and huPrP-(90-231) (equating to C2). Under conditions we employed, huPrP-(112-231) could not be induced to fold into a ß-stranded isoform and neurotoxicity was not a feature for monomeric or multimeric assemblies. In contrast, huPrP-(90-231) easily adopted a ß-strand conformation, demonstrated considerable thermostability and was toxic to neurons. Synthetic PrP peptides modelled on α- and ß-cleavage of the unique Y145STOP (Tyr145→stop) mutant prion protein corroborated the differential toxicity observed for recombinant huPrP-(112-231) and huPrP-(90-231) and suggested that the persistence of soluble oligomeric ß-strand-rich conformers was required for significant neurotoxicity. Our results additionally indicate that α- and ß-cleavage of PrPC generate biophysically and biologically non-equivalent C-terminal fragments and that C1 generated through α-cleavage appears to be pathogenesis-averse.


Asunto(s)
Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/toxicidad , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Secuencia de Aminoácidos , Animales , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Humanos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Proteínas PrPC/genética
7.
Brain Pathol ; 23(3): 321-32, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23587138

RESUMEN

Research on prions, the infectious agents of devastating neurological diseases in humans and animals, has been in the forefront of developing the concept of protein aggregation diseases. Prion diseases are distinguished from other neurodegenerative diseases by three peculiarities. First, prion diseases, in addition to being sporadic or genetic like all other neurodegenerative diseases, are infectious diseases. Animal models were developed early on (a long time before the advent of transgenic technology), and this has made possible the discovery of the prion protein as the infectious agent. Second, human prion diseases have true equivalents in animals, such as scrapie, which has been the subject of experimental research for many years. Variant Creutzfeldt-Jakob disease (vCJD) is a zoonosis caused by bovine spongiform encephalopathy (BSE) prions. Third, they show a wide variety of phenotypes in humans and animals, much wider than the variants of any other sporadic or genetic neurodegenerative disease. It has now become firmly established that particular PrP(Sc) isoforms are closely related to specific human prion strains. The variety of human prion diseases, still an enigma in its own right, is a focus of this article. Recently, a series of experiments has shown that the concept of aberrant protein folding and templating, first developed for prions, may apply to a variety of neurodegenerative diseases. In the wake of these discoveries, the term prion has come to be used for Aß, α-synuclein, tau and possibly others. The self-propagation of alternative conformations seems to be the common denominator of these "prions," which in future, in order to avoid confusion, may have to be specified either as "neurodegenerative prions" or "infectious prions."


Asunto(s)
Enfermedades por Prión/patología , Pliegue de Proteína , Deficiencias en la Proteostasis/patología , Animales , Barrera Hematoencefálica , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/patología , Estudio de Asociación del Genoma Completo , Humanos , Kuru/genética , Kuru/patología , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/toxicidad , Enfermedades por Prión/genética , Enfermedades por Prión/transmisión , Deficiencias en la Proteostasis/genética
8.
PLoS One ; 7(3): e33472, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22428057

RESUMEN

Insight into the normal function of PrP(C), and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants, Δ105-125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown. To investigate this question, we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice, which are marked by the presence or absence of GFP, are differentiated together to yield neurons, astrocytes, and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity, we assayed sensitivity of the cells to the cationic antibiotic, Zeocin. In a previous study, we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics, an effect that is suppressed by co-expression of wild type PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system, we find that while ΔCR-dependent toxicity is cell-autonomous, the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C), and the cellular mechanisms underlying the rescuing process.


Asunto(s)
Comunicación Celular/fisiología , Proteínas de la Membrana/metabolismo , Células-Madre Neurales/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Animales , Bleomicina/toxicidad , Western Blotting , Células Cultivadas , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Microscopía Fluorescente , Mutación/genética , Células-Madre Neurales/efectos de los fármacos , Proteínas PrPC/toxicidad , Imagen de Lapso de Tiempo
9.
Neurobiol Aging ; 33(5): 1006.e1-10, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22036844

RESUMEN

The human prion protein fragment, PrP (106-126), may contain a majority of the pathological features associated with the infectious scrapie isoform of PrP, known as PrP(Sc). Based on our previous findings that hypoxia protects neuronal cells from PrP (106-126)-induced apoptosis and increases cellular prion protein (PrP(C)) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate PrP(C) expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes. Under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by PrP (106-126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of PrP(C) mRNA and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In PrP(C) knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length Prnp gene (Ad-Prnp) were utilized for overexpression of the Prnp gene in PrP(C) knockout hippocampal neuron cells. Adenoviral transfection of PrP(C) knockout cells with Prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal PrP(C) is regulated by HIF-1α, and PrP(C) overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases.


Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/fisiología , Degeneración Nerviosa/prevención & control , Neuronas/metabolismo , Proteínas PrPC/biosíntesis , Enfermedades por Prión/prevención & control , Animales , Línea Celular , Línea Celular Tumoral , Humanos , Hipoxia Encefálica/genética , Hipoxia Encefálica/patología , Hipoxia Encefálica/prevención & control , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Neuroblastoma , Neuronas/efectos de los fármacos , Neuronas/patología , Proteínas PrPC/genética , Proteínas PrPC/toxicidad , Enfermedades por Prión/genética , Enfermedades por Prión/patología
10.
Muscle Nerve ; 44(6): 957-67, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22102467

RESUMEN

INTRODUCTION: Cobalamin (Cbl) deficiency affects the peripheral nervous system (PNS) morphologically and functionally. We investigated whether the octapeptide repeat (OR) region of prion protein (PrP(C)) (which is claimed to have myelinotrophic properties) is involved in the pathogenesis of rat Cbl-deficient (Cbl-D) polyneuropathy. METHODS: We intracerebroventricularly administered antibodies (Abs) against the OR region (OR-Abs) to Cbl-D rats to prevent myelin damage and maximum nerve conduction velocity (MNCV) abnormalities, and PrP(C)s to normal rats to reproduce PNS Cbl-D-like lesions. We measured nerve PrP(C) levels and MNCV. RESULTS: The OR-Abs normalized myelin ultrastructure, MNCV values, and tumor necrosis factor (TNF)-α levels in the sciatic and tibial nerves of Cbl-D rats. PrP(C) levels increased in Cbl-D nerves. The nerves of the PrP(C)-treated rats showed typical Cbl-D lesions, significantly decreased MNCV values, and significantly increased TNF-α levels. CONCLUSIONS: OR-Abs prevent the myelin damage caused by increased OR regions, and excess TNF-α is involved in the pathogenesis of Cbl-D polyneuropathy.


Asunto(s)
Oligopéptidos/toxicidad , Polineuropatías/metabolismo , Proteínas PrPC/toxicidad , Vitamina B 12/toxicidad , Animales , Ratones , Conducción Nerviosa/efectos de los fármacos , Conducción Nerviosa/fisiología , Polineuropatías/inducido químicamente , Polineuropatías/fisiopatología , Ratas , Ratas Sprague-Dawley , Factor de Necrosis Tumoral alfa/biosíntesis , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Vitamina B 12/metabolismo
11.
J Neurosci ; 31(39): 14005-17, 2011 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-21957261

RESUMEN

Transgenic mice expressing prion protein (PrP) molecules with several different internal deletions display spontaneous neurodegenerative phenotypes that can be dose-dependently suppressed by coexpression of wild-type PrP. Each of these deletions, including the largest one (Δ32-134), retains 9 aa immediately following the signal peptide cleavage site (residues 23-31; KKRPKPGGW). These residues have been implicated in several biological functions of PrP, including endocytic trafficking and binding of glycosaminoglycans. We report here on our experiments to test the role of this domain in the toxicity of deleted forms of PrP. We find that transgenic mice expressing Δ23-134 PrP display no clinical symptoms or neuropathology, in contrast to mice expressing Δ32-134 PrP, suggesting that residues 23-31 are essential for the toxic phenotype. Using a newly developed cell culture assay, we narrow the essential region to amino acids 23-26, and we show that mutant PrP toxicity is not related to the role of the N-terminal residues in endocytosis or binding to endogenous glycosaminoglycans. However, we find that mutant PrP toxicity is potently inhibited by application of exogenous glycosaminoglycans, suggesting that the latter molecules block an essential interaction between the N terminus of PrP and a membrane-associated target site. Our results demonstrate that a short segment containing positively charged amino acids at the N terminus of PrP plays an essential role in mediating PrP-related neurotoxicity. This finding identifies a protein domain that may serve as a drug target for amelioration of prion neurotoxicity.


Asunto(s)
Mutación/genética , Oligopéptidos/genética , Oligopéptidos/toxicidad , Proteínas PrPC/genética , Proteínas PrPC/toxicidad , Secuencia de Aminoácidos , Animales , Bovinos , Técnicas de Cultivo de Célula/métodos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , Datos de Secuencia Molecular , Oligopéptidos/química , Proteínas PrPC/química , Estructura Terciaria de Proteína/genética , Ovinos , Tortugas , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/toxicidad , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/toxicidad
12.
Neurochem Res ; 36(10): 1863-9, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21607610

RESUMEN

Prion diseases are conformational diseases, many factors are involved in altering the conformation of prion, such as RNA, DNA, pH, and copper etc. However the neurotoxic mechanism of prion diseases is not clear yet. The aim of this study is to investigate the effect of the nucleoprotein complex of RNA and recombinant ovine prion protein (OvPrP(C)) on the cultured rat cortical neurons in vitro. Our previous study revealed that the nucleoprotein complex (OvPrP(C)-RNA) is characterized with high ß sheet conformation and proteinase K resistance. Here we found that the OvPrP(C)-RNA induced marked neuronal cell death by the MTT (3-(4,5-dimethyl-thiazole -2-yl)-2,5-diphenyl -tetrazolium bromide) and TUNEL (TdT mediated biotin-dUTP nicked-end labeling) assay, and the neurotoxic effects were confirmed by testing the content of Bcl-2 Associated X protein (Bax) in the immunoprecipitation assay and Western blot assay. Compared to the control group, there is no significant difference of active Bax or total Bax after RNA alone treatment or OvPrP(C) alone treatment, but the OvPrP(C)-RNA induced significant increases of active Bax level, while the contents of total Bax had no obvious changes after OvPrP(C)-RNA treatment. The results suggested that OvPrP(C)-RNA is neurotoxic in vitro, which added further evidence to the current understanding of mechanism of cellular injury by RNA molecules for transformation of the PrP(C) to PrP(Sc).


Asunto(s)
Corteza Cerebral/citología , Neuronas/efectos de los fármacos , Proteínas PrPC/toxicidad , Enfermedades por Prión/metabolismo , ARN/toxicidad , Ovinos , Animales , Muerte Celular/efectos de los fármacos , Células Cultivadas , Humanos , Etiquetado Corte-Fin in Situ , Neuronas/citología , Neuronas/metabolismo , Síndromes de Neurotoxicidad/metabolismo , Proteínas PrPC/genética , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína X Asociada a bcl-2/metabolismo
14.
EMBO J ; 30(10): 2057-70, 2011 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-21441896

RESUMEN

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a ß-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various ß-sheet-rich (ß) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid ß-peptide, (iii) yeast prion proteins or (iv) designed ß-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with ß-conformers. Interestingly, a secreted version of N-PrP associated with ß-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various ß-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.


Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/toxicidad , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Enfermedades por Prión/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Muerte Celular , Humanos , Proteínas de la Membrana/química , Neuronas/efectos de los fármacos , Neuronas/fisiología , Proteínas PrPC/química , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/toxicidad
15.
Am J Pathol ; 176(6): 2695-706, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20472884

RESUMEN

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.


Asunto(s)
Muerte Celular/fisiología , Cerebelo/citología , Neuronas/fisiología , Proteínas PrPC/toxicidad , Animales , Apoptosis/fisiología , Autofagia/fisiología , Biomarcadores/metabolismo , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Forma de la Célula , Activación Enzimática , Ratones , Ratones Endogámicos CBA , Ratones Mutantes Neurológicos , Ratones Transgénicos , Neuronas/patología , Neuronas/ultraestructura , Proteínas PrPC/genética , Priones/genética , Priones/metabolismo
16.
J Biol Chem ; 285(10): 7752-65, 2010 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-19940127

RESUMEN

In prion diseases, the infectious isoform of the prion protein (PrP(Sc)) may subvert a normal, physiological activity of the cellular isoform (PrP(C)). A deletion mutant of the prion protein (Delta105-125) that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrP(C) and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Delta105-125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Delta105-125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Delta105-125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.


Asunto(s)
Bioensayo/métodos , Evaluación Preclínica de Medicamentos/métodos , Proteínas PrPC , Proteínas PrPSc , Enfermedades por Prión/metabolismo , Amebicidas/farmacología , Animales , Antibióticos Antineoplásicos/farmacología , Bleomicina/farmacología , Línea Celular/efectos de los fármacos , Supervivencia Celular , Cinamatos/farmacología , Proteínas Ligadas a GPI , Gentamicinas/farmacología , Humanos , Higromicina B/análogos & derivados , Higromicina B/farmacología , Ratones , Ratones Transgénicos , Fenotipo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Proteínas PrPSc/genética , Proteínas PrPSc/metabolismo , Proteínas PrPSc/toxicidad , Priones/genética , Priones/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/fisiología
17.
Biochemistry ; 48(19): 4198-208, 2009 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-19301918

RESUMEN

Prion diseases result from a post-translational modification of the physiological prion protein (PrP(C)) into a scrapie isoform (PrP(Sc)). The PrP(106-126) fragment is conserved among various abnormal variants and shows PrP(Sc) pathogenic properties. It has been proposed that the PrP(106-126) fragment may exhibit its toxic effects through membrane pore formation. Our previous studies showed that PrP(106-126) does not interact with membranes under physiological conditions. In the present study, PrP(106-126) affinity for membranes was increased by modifying PrP(106-126) with a M112W substitution, and pore formation was further evaluated. However, while the peptide exhibited an increased local concentration in the membrane, this did not lead to the induction of membrane permeabilization, as verified by fluorescence methodologies and surface plasmon resonance. These results further support the idea that PrP(106-126) toxicity is not a consequence of peptide-membrane interaction and pore formation.


Asunto(s)
Membrana Celular/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Proteínas PrPC/toxicidad , Priones/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Potenciales de la Membrana , Datos de Secuencia Molecular , Permeabilidad , Proteínas PrPC/metabolismo , Priones/toxicidad , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , Termodinámica , Triptófano/metabolismo , Agua/química
18.
Protein Pept Lett ; 16(1): 14-26, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19149668

RESUMEN

The prion protein is a cell surface glycoprotein that is converted to a protease resistant abnormal isoform during the course of prion disease. The normal isoform of this protein has been shown to be an antioxidant that aids the survival of neurones. The abnormal isoform is associated with both the transmissible agent of prion diseases and is also toxic. Recent studies have shown that there are multiple end states in terms of aggregation of the protein. Both soluble oligomers and insoluble fibrils can form from the abnormal isoform. Although fibrils are characteristic of the disease, the most infectious prions are associated with oligomers. Neurotoxicity can be associated with fibrils but mostly appears to be due to small aggregates. For many years fibrils were believed to be central to the disease process but currently evidence supports the notion that fibrils represent a "bulk" form of abnormal protein, which is largely inert, but carried along a small active component. This review will examine what is known about the mechanisms behind prion protein aggregation, and the relevance of each form for the disease.


Asunto(s)
Proteínas PrPC/química , Apoptosis/fisiología , Glicosaminoglicanos/farmacología , Humanos , Metales/farmacología , Proteínas PrPC/efectos de los fármacos , Proteínas PrPC/genética , Proteínas PrPC/toxicidad , Proteínas PrPSc/toxicidad , Enfermedades por Prión/genética , Procesamiento Proteico-Postraduccional , Estructura Cuaternaria de Proteína
19.
J Neurosci ; 28(49): 13258-67, 2008 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-19052217

RESUMEN

The neurotoxic forms of the prion protein (PrP) that cause neurodegeneration in prion diseases remain to be conclusively identified. Considerable evidence points to the importance of noninfectious oligomers of PrP in the pathogenic process. In this study, we describe lines of Tg(WT) transgenic mice that over-express wild-type PrP by either approximately 5-fold or approximately 10-fold (depending on whether the transgene array is, respectively, hemizygous or homozygous). Homozygous but not hemizygous Tg(WT) mice develop a spontaneous neurodegenerative illness characterized clinically by tremor and paresis. Both kinds of mice accumulate large numbers of punctate PrP deposits in the molecular layer of the cerebellum as well as in several other brain regions, and they display abnormally enlarged synaptic terminals accompanied by a dramatic proliferation of membranous structures. The over-expressed PrP in Tg(WT) mice assembles into an insoluble form that is mildly protease-resistant and is recognizable by aggregation-specific antibodies, but that is not infectious in transmission experiments. Together, our results demonstrate that noninfectious aggregates of wild-type PrP are neurotoxic, particularly to synapses, and they suggest common pathogenic mechanisms shared by prion diseases and nontransmissible neurodegenerative disorders associated with protein misfolding.


Asunto(s)
Encéfalo/fisiopatología , Enfermedades Neurodegenerativas/fisiopatología , Proteínas PrPC/toxicidad , Enfermedades por Prión/fisiopatología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Homocigoto , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones Transgénicos , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Pliegue de Proteína , Sinapsis/metabolismo , Sinapsis/patología
20.
Cell Mol Life Sci ; 60(1): 133-43, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12613663

RESUMEN

Recent findings strongly support the hypothesis that diverse human disorders, including the most common neurodegenerative diseases, arise from misfolding and aggregation of an underlying protein. Despite the good evidence for the involvement of protein misfolding in disease pathogenesis, the mechanism by which protein conformational changes participate in the disease is still unclear. Among the best-studied diseases of this group are the transmissible spongiform encephalopathies or prion-related disorders, in which misfolding of the normal prion protein plays a key role in the disease. In this article we review recent data on the link between prion protein misfolding and the pathogensis of spongiform encephalopathies.


Asunto(s)
Enfermedades por Prión/etiología , Pliegue de Proteína , Secuencia de Aminoácidos , Animales , Apoptosis , Sitios de Unión , Encéfalo/metabolismo , Encéfalo/patología , Humanos , Modelos Biológicos , Modelos Teóricos , Proteínas PrPC/metabolismo , Proteínas PrPC/toxicidad , Proteínas PrPSc/metabolismo , Enfermedades por Prión/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Relación Estructura-Actividad
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