RESUMEN
Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a ß-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100-110 to 227-237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung ß-solenoid fold.
Asunto(s)
Encefalopatía Espongiforme Bovina , Modelos Moleculares , Proteínas PrPSc/química , Animales , Bovinos , Ratones , Ratones TransgénicosRESUMEN
Ablation of the cellular prion protein PrP(C) leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrP(C) prevents the disease, suggesting that PrP(C) acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrP(C)-deficient mice is reduced, suggesting that PrP(C) acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrP(C) triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC-derived peptide (FT(23-50)) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT(23-50) and the equivalent type-IV collagen peptide. We conclude that PrP(C) promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrP(C), these observations are relevant to the pathogenesis of demyelinating polyneuropathies--common debilitating diseases for which there are limited therapeutic options.
Asunto(s)
Priones/metabolismo , Priones/farmacología , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacología , AMP Cíclico/metabolismo , Enfermedades Desmielinizantes/metabolismo , Femenino , Células HEK293 , Homeostasis/efectos de los fármacos , Humanos , Ligandos , Ratones , Datos de Secuencia Molecular , Vaina de Mielina/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Docilidad , Proteínas Priónicas , Priones/química , Priones/genética , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/genética , Células de Schwann/efectos de los fármacos , Células de Schwann/metabolismo , Nervio Ciático/efectos de los fármacos , Nervio Ciático/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/genéticaRESUMEN
Prions are transmissible agents causing lethal neurodegenerative diseases that are composed of aggregates of misfolded cellular prion protein (PrPSc). Despite non-fibrillar oligomers having been proposed as the most infectious prion particles, prions purified from diseased brains usually consist of large and fibrillar PrPSc aggregates, whose protease-resistant core (PrPres) encompasses the whole C-terminus of PrP. In contrast, PrPSc from Gerstmann-Sträussler-Scheinker disease associated with alanine to valine substitution at position 117 (GSS-A117V) is characterized by a small protease-resistant core, which is devoid of the C-terminus. We thus aimed to investigate the role of this unusual PrPSc in terms of infectivity, strain characteristics, and structural features. We found, by titration in bank voles, that the infectivity of GSS-A117V is extremely high (109.3 ID50 U/g) and is resistant to treatment with proteinase K (109.0 ID50 U/g). We then purified the proteinase K-resistant GSS-A117V prions and determined the amount of infectivity and PrPres in the different fractions, alongside the morphological characteristics of purified PrPres aggregates by electron microscopy. Purified pellet fractions from GSS-A117V contained the expected N- and C-terminally cleaved 7 kDa PrPres, although the yield of PrPres was low. We found that this low yield depended on the low density/small size of GSS-A117V PrPres, as it was mainly retained in the last supernatant fraction. All fractions were highly infectious, thus confirming the infectious nature of the 7 kDa PrPres, with infectivity levels that directly correlated with the PrPres amount detected. Finally, electron microscopy analysis of these fractions showed no presence of amyloid fibrils, but only very small and indistinct, non-fibrillar PrPresparticles were detected and confirmed to contain PrP via immunogold labelling. Our study demonstrates that purified aggregates of 7 kDa PrPres, spanning residues â¼90-150, are highly infectious oligomers that encode the biochemical and biological strain features of the original sample. Overall, the autocatalytic behaviour of the prion oligomers reveals their role in the propagation of neurodegeneration in patients with Gerstmann-Sträussler-Scheinker disease and implies that the C-terminus of PrPSc is dispensable for infectivity and strain features for this prion strain, uncovering the central PrP domain as the minimal molecular component able to encode infectious prions. These findings are consistent with the hypothesis that non-fibrillar prion particles are highly efficient propagators of disease and provide new molecular and morphological constraints on the structure of infectious prions.
Asunto(s)
Enfermedad de Gerstmann-Straussler-Scheinker/transmisión , Proteínas PrPSc/química , Proteínas PrPSc/aislamiento & purificación , Proteínas PrPSc/patogenicidad , Animales , Arvicolinae , HumanosRESUMEN
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/metabolismoRESUMEN
The misfolding of the cellular prion protein (PrPC) causes fatal neurodegenerative diseases. Yet PrPC is highly conserved in mammals, suggesting that it exerts beneficial functions preventing its evolutionary elimination. Ablation of PrPC in mice results in well-defined structural and functional alterations in the peripheral nervous system. Many additional phenotypes were ascribed to the lack of PrPC, but some of these were found to arise from genetic artifacts of the underlying mouse models. Here, we revisit the proposed physiological roles of PrPC in the central and peripheral nervous systems and highlight the need for their critical reassessment using new, rigorously controlled animal models.
Asunto(s)
Sistema Nervioso Central/patología , Sistema Nervioso Periférico/patología , Enfermedades por Prión/metabolismo , Proteínas Priónicas/metabolismo , Animales , Modelos Animales de Enfermedad , Ratones , Enfermedades por Prión/etiología , Proteínas Priónicas/toxicidadRESUMEN
It has been shown recently that PrP (prion protein) and the calcium channel auxiliary α2δ subunits interact in neurons and expression systems [Senatore, Colleoni, Verderio, Restelli, Morini, Condliffe, Bertani, Mantovani, Canovi, Micotti, Forloni, Dolphin, Matteoli, Gobbi and Chiesa (2012) Neuron 74, 300-313]. In the present study we examined whether there was an effect of PrP on calcium currents. We have shown that when PrP is co-expressed with calcium channels formed from CaV2.1/ß and α2δ-1 or α2δ-2, there is a consistent decrease in calcium current density. This reduction was absent when a PrP construct was used lacking its GPI (glycosylphosphatidylinositol) anchor. We have reported previously that α2δ subunits are able to form GPI-anchored proteins [Davies, Kadurin, Alvarez-Laviada, Douglas, Nieto-Rostro, Bauer, Pratt and Dolphin (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 1654-1659] and show further evidence in the present paper. We have characterized recently a C-terminally truncated α2δ-1 construct, α2δ-1ΔC, and found that, despite loss of its membrane anchor, it still shows a partial ability to increase calcium currents [Kadurin, Alvarez-Laviada, Ng, Walker-Gray, D'Arco, Fadel, Pratt and Dolphin (2012) J. Biol. Chem. 1287, 33554-33566]. We now find that PrP does not inhibit CaV2.1/ß currents formed with α2δ-1ΔC, rather than α2δ-1. It is possible that PrP and α2δ-1 compete for GPI-anchor intermediates or trafficking pathways, or that interaction between PrP and α2δ-1 requires association in cholesterol-rich membrane microdomains. Our additional finding that CaV2.1/ß1b/α2δ-1 currents were inhibited by GPI-GFP, but not cytosolic GFP, indicates that competition for limited GPI-anchor intermediates or trafficking pathways may be involved in PrP suppression of α2δ subunit function.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Canales de Calcio/metabolismo , Regulación de la Expresión Génica , Glicosilfosfatidilinositoles/metabolismo , Priones/biosíntesis , Animales , Unión Competitiva/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica/fisiología , Transporte de Proteínas/genética , Ratas , Transducción de Señal/genética , XenopusRESUMEN
Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, ß-rich oligomers that bind to PrP(C).
Asunto(s)
Agonistas de Aminoácidos Excitadores/toxicidad , Ácido Glutámico/toxicidad , Ratones Endogámicos C57BL/fisiología , Mutación/fisiología , Neuronas/fisiología , Proteínas PrPC/biosíntesis , Animales , Células Cultivadas , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Proteínas PrPC/genéticaRESUMEN
INTRODUCTION: Fontan surgery is a palliative procedure performed in children with a functionally univentricular heart. Improvements in surgical technique over the past 30 years have increased life expectancy in this rare population. However, the epidemiology of persons living with Fontan is poorly understood. This study aimed to estimate the 2020 and 2030 prevalence of persons living with a Fontan circulation in 11 countries across the US, Europe, Australia and New Zealand, by procedure type: [atriopulmonary connection (AP), lateral tunnel total cavopulmonary connection (LT-TCPC) or extracardiac total cavopulmonary connection (EC-TCPC)]; and age group: [children (< 12 years), adolescents (12-17 years), and adults (≥ 18 years old)] by building an epidemiologic model. METHODS: The annual number of Fontan surgeries by country in 2010-2020 were extracted from hospital or claims databases, via procedure codes. The epidemiology of persons living with Fontan was modelled by applying these surgery frequencies to mid-year populations from 1972 to 2020 and overlaying an uptake curve. A literature search identified: 30-day mortality rates, long-term survival, and median age at surgery. Averages of these estimates were inputted into the model to project prevalence in 2030. RESULTS: The number of persons living with Fontan in 2020 across the 11 countries was estimated to be 47,881 [66 people per million (ppm)], rising to 59,777 (79 ppm) by 2030. In 2020, this population was 55% adults, 17% adolescents and 28% children shifting to 64%, 13% and 23%, respectively, in 2030. Among all persons living with Fontan, 74%/18%/9% are estimated to have EC-TCPC/LT-TCPC/AP, respectively, in 2020, and 83%/14%/4% in 2030. CONCLUSIONS: According to this epidemiology model, the Fontan population is growing, partly driven by increased survival rates with the more recent LT-TCPC and EC-TCPC procedures (compared with AP). The 2020/2030 prevalence of persons living with Fontan is 66/79 ppm.
Asunto(s)
Procedimiento de Fontan , Cardiopatías Congénitas , Adolescente , Adulto , Niño , Procedimiento de Fontan/efectos adversos , Procedimiento de Fontan/métodos , Cardiopatías Congénitas/epidemiología , Cardiopatías Congénitas/cirugía , Humanos , Arteria Pulmonar/cirugía , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
Mammalian models are essential for brain aging research. However, the long lifespan and poor amenability to genetic and pharmacological perturbations have hindered the use of mammals for dissecting aging-regulatory molecular networks and discovering new anti-aging interventions. To circumvent these limitations, we developed an ex vivo model system that faithfully mimics the aging process of the mammalian brain using cultured mouse brain slices. Genome-wide gene expression analyses showed that cultured brain slices spontaneously upregulated senescence-associated genes over time and reproduced many of the transcriptional characteristics of aged brains. Treatment with rapamycin, a classical anti-aging compound, largely abolished the time-dependent transcriptional changes in naturally aged brain slice cultures. Using this model system, we discovered that prions drastically accelerated the development of age-related molecular signatures and the pace of brain aging. We confirmed this finding in mouse models and human victims of Creutzfeldt-Jakob disease. These data establish an innovative, eminently tractable mammalian model of brain aging, and uncover a surprising acceleration of brain aging in prion diseases.
Asunto(s)
Envejecimiento , Encéfalo , Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Priones , Animales , Encéfalo/metabolismo , Encéfalo/patología , Ratones , Enfermedades por Prión/genética , Enfermedades por Prión/patología , Priones/genéticaRESUMEN
Although prion infections cause cognitive impairment and neuronal death, transcriptional and translational profiling shows progressive derangement within glia but surprisingly little changes within neurons. Here we expressed PrPC selectively in neurons and astrocytes of mice. After prion infection, both astrocyte and neuron-restricted PrPC expression led to copious brain accumulation of PrPSc . As expected, neuron-restricted expression was associated with typical prion disease. However, mice with astrocyte-restricted PrPC expression experienced a normal life span, did not develop clinical disease, and did not show astro- or microgliosis. Besides confirming that PrPSc is innocuous to PrPC -deficient neurons, these results show that astrocyte-born PrPSc does not activate the extreme neuroinflammation that accompanies the onset of prion disease and precedes any molecular changes of neurons. This points to a nonautonomous mechanism by which prion-infected neurons instruct astrocytes and microglia to acquire a specific cellular state that, in turn, drives neural dysfunction.
Asunto(s)
Enfermedades por Prión , Priones , Animales , Astrocitos/metabolismo , Ratones , Neuroglía/metabolismo , Neuronas/metabolismo , Enfermedades por Prión/metabolismo , Priones/metabolismoRESUMEN
Prion infections cause conformational changes of the cellular prion protein (PrPC) and lead to progressive neurological impairment. Here we show that toxic, prion-mimetic ligands induce an intramolecular R208-H140 hydrogen bond ('H-latch'), altering the flexibility of the α2-α3 and ß2-α2 loops of PrPC. Expression of a PrP2Cys mutant mimicking the H-latch was constitutively toxic, whereas a PrPR207A mutant unable to form the H-latch conferred resistance to prion infection. High-affinity ligands that prevented H-latch induction repressed prion-related neurodegeneration in organotypic cerebellar cultures. We then selected phage-displayed ligands binding wild-type PrPC, but not PrP2Cys. These binders depopulated H-latched conformers and conferred protection against prion toxicity. Finally, brain-specific expression of an antibody rationally designed to prevent H-latch formation prolonged the life of prion-infected mice despite unhampered prion propagation, confirming that the H-latch is an important reporter of prion neurotoxicity.
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Proteínas PrPC , Priones , Animales , Anticuerpos/metabolismo , Cerebelo/metabolismo , Ligandos , Ratones , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas Priónicas/química , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Priones/metabolismo , Priones/toxicidadRESUMEN
Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies.
Asunto(s)
Isquemia Encefálica , Proteínas de Microfilamentos , Mutación Missense , Accidente Cerebrovascular , Anciano , Sustitución de Aminoácidos , Animales , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Persona de Mediana Edad , Accidente Cerebrovascular/genética , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
The adhesion G-protein coupled receptor Adgrg6 (formerly Gpr126) is instrumental in the development, maintenance and repair of peripheral nervous system myelin. The prion protein (PrP) is a potent activator of Adgrg6 and could be used as a potential therapeutic agent in treating peripheral demyelinating and dysmyelinating diseases. We designed a dimeric Fc-fusion protein comprising the myelinotrophic domain of PrP (FT2Fc), which activated Adgrg6 in vitro and exhibited favorable pharmacokinetic properties for in vivo treatment of peripheral neuropathies. While chronic FT2Fc treatment elicited specific transcriptomic changes in the sciatic nerves of PrP knockout mice, no amelioration of the early molecular signs demyelination was detected. Instead, RNA sequencing of sciatic nerves revealed downregulation of cytoskeletal and sarcomere genes, akin to the gene expression changes seen in myopathic skeletal muscle of PrP overexpressing mice. These results call for caution when devising myelinotrophic therapies based on PrP-derived Adgrg6 ligands. While our treatment approach was not successful, Adgrg6 remains an attractive therapeutic target to be addressed in other disease models or by using different biologically active Adgrg6 ligands.
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Enfermedades Desmielinizantes/tratamiento farmacológico , Fragmentos de Péptidos/uso terapéutico , Proteínas Priónicas/química , Receptores Acoplados a Proteínas G/agonistas , Animales , Línea Celular , Enfermedades Desmielinizantes/genética , Femenino , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Priónicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Nervio Ciático/metabolismo , TranscriptomaRESUMEN
Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here, we identified > 6,000 PrP-binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage-derived antibodies. When expressed recombinantly, these antibodies exhibited anti-PrP reactivity. Furthermore, we surveyed 48,718 samples from 37,894 hospital patients for the presence of anti-PrP IgGs and found 21 high-titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti-PrP autoimmunity is innocuous. The existence of anti-prion antibodies in unbiased human immunological repertoires suggests that they might clear nascent prions early in life. Combined with the reported lack of such antibodies in carriers of disease-associated PRNP mutations, this suggests a link to the low incidence of spontaneous prion diseases in human populations.
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Enfermedades por Prión , Priones , Anticuerpos , Linfocitos B , Humanos , InmunoterapiaRESUMEN
The key event in the pathogenesis of prion diseases is the conformational conversion of the normal prion protein (PrP) (PrP(C)) into an infectious, aggregated isoform (PrP(Sc)) that has a high content of beta-sheet. Historically, a great deal of effort has been devoted to developing antibodies that specifically recognize PrP(Sc) but not PrP(C), as such antibodies would have enormous diagnostic and experimental value. A mouse monoclonal IgM antibody (designated 15B3) and three PrP motif-grafted monoclonal antibodies (referred to as IgG 19-33, 89-112, and 136-158) have been previously reported to react specifically with infectious PrP(Sc) but not PrP(C). In this study, we extend the characterization of these four antibodies by testing their ability to immunoprecipitate and immunostain infectious and non-infectious aggregates of wild-type, mutant, and recombinant PrP. We find that 15B3 as well as the motif-grafted antibodies recognize multiple types of aggregated PrP, both infectious and non-infectious, including forms found in brain, in transfected cells, and induced in vitro from purified recombinant protein. These antibodies are exquisitely selective for aggregated PrP, and do not react with soluble PrP even when present in vast excess. Our results suggest that 15B3 and the motif-grafted antibodies recognize structural features common to both infectious and non-infectious aggregates of PrP. Our study extends the utility of these antibodies for diagnostic and experimental purposes, and it provides new insight into the structural changes that accompany PrP oligomerization and prion propagation.
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Anticuerpos Monoclonales/metabolismo , Proteínas PrPSc/inmunología , Proteínas PrPSc/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Humanos , Ratones , Ratones Transgénicos , Proteínas PrPSc/patogenicidad , Priones/química , Priones/inmunología , Priones/metabolismo , Unión Proteica/fisiología , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismoRESUMEN
Prions are the infectious agents causing transmissible spongiform encephalopathies (TSE), progressive, inexorably lethal neurological diseases. Antibodies targeting the globular domain (GD) of the cellular prion protein PrPC trigger a neurotoxic syndrome morphologically and molecularly similar to prion disease. This phenomenon raises the question whether such antibodies induce infectious prions de novo. Here we exposed cerebellar organotypic cultured slices (COCS) to the neurotoxic antibody, POM1. We then inoculated COCS homogenates into tga20 mice, which overexpress PrPC and are commonly utilized as sensitive indicators of prion infectivity. None of the mice inoculated with COCS-derived lysates developed any signs of disease, and all mice survived for at least 200 days post-inoculation. In contrast, all mice inoculated with bona fide prions succumbed to TSE after 55-95 days. Post-mortem analyses did not reveal any signs of prion pathology in mice inoculated with POM1-COCS lysates. Also, lysates from POM1-exposed COCS were unable to convert PrP by quaking. Hence, anti-GD antibodies do not catalyze the generation of prion infectivity. These data indicate that prion replication can be separated from prion toxicity, and suggest that anti-GD antibodies exert toxicity by acting downstream of prion replication.
RESUMEN
Synaptic dysfunction is an important cause of neurological symptoms in prion diseases, a class of clinically heterogeneous neurodegenerative disorders caused by misfolding of the cellular prion protein (PrP(C)). Experimental data suggest that accumulation of misfolded PrP(C) in the endoplasmic reticulum (ER) may be crucial in synaptic failure, possibly because of the activation of the translational repression pathway of the unfolded protein response. Here, we report that this pathway is not operative in mouse models of genetic prion disease, consistent with our previous observation that ER stress is not involved. Building on our recent finding that ER retention of mutant PrP(C) impairs the secretory trafficking of calcium channels essential for synaptic function, we propose a model of pathogenicity in which intracellular retention of misfolded PrP(C) results in loss of function or gain of toxicity of PrP(C)-interacting proteins. This neurotoxic modality may also explain the phenotypic heterogeneity of prion diseases.
RESUMEN
How mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α(2)δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α(2)δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.
Asunto(s)
Canales de Calcio/metabolismo , Cerebelo/citología , Mutación/genética , Neuronas/metabolismo , Priones/genética , Transmisión Sináptica/genética , Factores de Edad , Animales , Animales Recién Nacidos , Biofisica , Calcio/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Estimulación Eléctrica , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imagen por Resonancia Magnética , Potenciales de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/genética , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Enfermedades por Prión/fisiopatología , Proteínas Priónicas , Transporte de Proteínas/genética , Tiempo de Reacción/genética , Prueba de Desempeño de Rotación con Aceleración Constante , Sinaptosomas/metabolismoRESUMEN
A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.