RESUMO
A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting cellular host-factors that can modify prion propagation. We exposed prion-infected cells in high-density microplates to 35,364 ternary pools of 52,746 siRNAs targeting 17,582 genes representing the majority of the mouse protein-coding transcriptome. We identified 1,191 modulators of prion propagation. While 1,151 modified the expression of both the pathological prion protein, PrPSc , and its cellular counterpart, PrPC , 40 genes selectively affected PrPSc . Of the latter 40 genes, 20 augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC . Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Further, the unexpected identification of a prion-controlling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.
Assuntos
Doenças Priônicas , Príons , Camundongos , Animais , Ovinos/genética , Príons/genética , Príons/metabolismo , Drosophila melanogaster/genética , Ribonucleoproteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Doenças Priônicas/genética , Doenças Priônicas/patologia , Mamíferos/genéticaRESUMO
Microbes or danger signals trigger inflammasome sensors, which induce polymerization of the adaptor ASC and the assembly of ASC specks. ASC specks recruit and activate caspase-1, which induces maturation of the cytokine interleukin 1ß (IL-1ß) and pyroptotic cell death. Here we found that after pyroptosis, ASC specks accumulated in the extracellular space, where they promoted further maturation of IL-1ß. In addition, phagocytosis of ASC specks by macrophages induced lysosomal damage and nucleation of soluble ASC, as well as activation of IL-1ß in recipient cells. ASC specks appeared in bodily fluids from inflamed tissues, and autoantibodies to ASC specks developed in patients and mice with autoimmune pathologies. Together these findings reveal extracellular functions of ASC specks and a previously unknown form of cell-to-cell communication.
Assuntos
Apoptose/imunologia , Caspase 1/imunologia , Proteínas do Citoesqueleto/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Anticorpos/imunologia , Proteínas Reguladoras de Apoptose , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Proteínas Adaptadoras de Sinalização CARD , Proteínas de Transporte/genética , Caspase 1/genética , Inibidores de Caspase/farmacologia , Comunicação Celular/imunologia , Proteínas do Citoesqueleto/genética , Humanos , Inflamassomos/imunologia , Lisossomos/patologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Fagocitose/imunologia , Príons/química , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Transdução de Sinais/imunologiaRESUMO
The misfolding and aggregation of the human prion protein (PrP) is associated with transmissible spongiform encephalopathies (TSEs). Intermediate conformations forming during the conversion of the cellular form of PrP into its pathological scrapie conformation are key drivers of the misfolding process. Here, we analyzed the properties of the C-terminal domain of the human PrP (huPrP) and its T183A variant, which is associated with familial forms of TSEs. We show that the mutation significantly enhances the aggregation propensity of huPrP, such as to uniquely induce amyloid formation under physiological conditions by the sole C-terminal domain of the protein. Using NMR spectroscopy, biophysics, and metadynamics simulations, we identified the structural characteristics of the misfolded intermediate promoting the aggregation of T183A huPrP and the nature of the interactions that prevent this species to be populated in the wild-type protein. In support of these conclusions, POM antibodies targeting the regions that promote PrP misfolding were shown to potently suppress the aggregation of this amyloidogenic mutant.
Assuntos
Mutação , Proteínas Priônicas/química , Proteínas Priônicas/genética , Dobramento de Proteína , Amiloide/química , Amiloide/metabolismo , Humanos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Doenças Priônicas/etiologia , Doenças Priônicas/metabolismo , Príons , Agregação Patológica de Proteínas/metabolismo , Conformação Proteica , Deficiências na Proteostase , Relação Estrutura-AtividadeRESUMO
The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas PrPC/biossíntese , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular , Estudo de Associação Genômica Ampla , Humanos , Interferência de RNARESUMO
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.
Assuntos
Encefalopatia Espongiforme Bovina , Modelos Moleculares , Proteínas PrPSc/química , Animais , Bovinos , Camundongos , Camundongos TransgênicosRESUMO
The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance and splicing alterations during the course of disease in prion-inoculated mice. Prion infection induced PrP-dependent transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical signs, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, could be the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed signs in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.
Assuntos
Estudo de Associação Genômica Ampla , Microglia/metabolismo , Neurônios/metabolismo , Doenças Priônicas , RNA Mensageiro , Transcriptoma , Animais , Masculino , Camundongos , Camundongos Knockout , Doenças Priônicas/genética , Doenças Priônicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) real-time quaking-induced conversion (RT-QuIC) has a high degree of sensitivity and specificity for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD) and this has led to its being included in revised European CJD Surveillance Network diagnostic criteria for sCJD. As CSF RT-QuIC becomes more widely established, it is crucial that the analytical performance of individual laboratories is consistent. The aim of this ring-trial was to ascertain the degree of concordance between European countries undertaking CSF RT-QuIC. METHODS: Ten identical CSF samples, seven from probable or neuropathologically confirmed sCJD and three from non-CJD cases, were sent to 13 laboratories from 11 countries for RT-QuIC analysis. A range of instrumentation and different recombinant prion protein substrates were used. Each laboratory analysed the CSF samples blinded to the diagnosis and reported the results as positive or negative. RESULTS: All 13 laboratories correctly identified five of the seven sCJD cases and the remaining two sCJD cases were identified by 92% of laboratories. Of the two sCJD cases that were not identified by all laboratories, one had a disease duration >26 months with a negative 14-3-3, whilst the remaining case had a 4-month disease duration and a positive 14-3-3. A single false positive CSF RT-QuIC result was observed in this study. CONCLUSIONS: This study shows that CSF RT-QuIC demonstrates an excellent concordance between centres, even when using a variety of instrumentation, recombinant prion protein substrates and CSF volumes. The adoption of CSF RT-QuIC by all CJD surveillance centres is recommended.
Assuntos
Síndrome de Creutzfeldt-Jakob , Príons , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquidiano , Síndrome de Creutzfeldt-Jakob/diagnóstico , Humanos , Proteínas Priônicas , Príons/líquido cefalorraquidiano , Proteínas Recombinantes , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Príons/metabolismo , Príons/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Colágeno Tipo IV/química , Colágeno Tipo IV/farmacologia , AMP Cíclico/metabolismo , Doenças Desmielinizantes/metabolismo , Feminino , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Bainha de Mielina/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Maleabilidade , Proteínas Priônicas , Príons/química , Príons/genética , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
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.
Assuntos
Doença de Gerstmann-Straussler-Scheinker/transmissão , Proteínas PrPSc/química , Proteínas PrPSc/isolamento & purificação , Proteínas PrPSc/patogenicidade , Animais , Arvicolinae , HumanosRESUMO
Antibodies to the prion protein, PrP, represent a promising therapeutic approach against prion diseases but the neurotoxicity of certain anti-PrP antibodies has caused concern. Here we describe scPOM-bi, a bispecific antibody designed to function as a molecular prion tweezer. scPOM-bi combines the complementarity-determining regions of the neurotoxic antibody POM1 and the neuroprotective POM2, which bind the globular domain (GD) and flexible tail (FT) respectively. We found that scPOM-bi confers protection to prion-infected organotypic cerebellar slices even when prion pathology is already conspicuous. Moreover, scPOM-bi prevents the formation of soluble oligomers that correlate with neurotoxic PrP species. Simultaneous targeting of both GD and FT was more effective than concomitant treatment with the individual molecules or targeting the tail alone, possibly by preventing the GD from entering a toxic-prone state. We conclude that simultaneous binding of the GD and flexible tail of PrP results in strong protection from prion neurotoxicity and may represent a promising strategy for anti-prion immunotherapy.
Assuntos
Anticorpos Biespecíficos/farmacologia , Cerebelo/imunologia , Imunoterapia , Doenças Priônicas/terapia , Proteínas Priônicas/imunologia , Príons/toxicidade , Animais , Anticorpos Biespecíficos/imunologia , Células Cultivadas , Regiões Determinantes de Complementaridade/imunologia , Camundongos , Camundongos Transgênicos , Doenças Priônicas/imunologia , Príons/imunologiaRESUMO
Transmissible spongiform encephalopathies (TSEs) are caused by the prion, which consists essentially of PrPSc, an aggregated, conformationally modified form of the cellular prion protein (PrPC). Although TSEs can be experimentally transmitted by intracerebral inoculation, most instances of infection in the field occur through extracerebral routes. The epidemics of kuru and variant Creutzfeldt-Jakob disease were caused by dietary exposure to prions, and parenteral administration of prion-contaminated hormones has caused hundreds of iatrogenic TSEs. In all these instances, the development of postexposure prophylaxis relies on understanding of how prions propagate from the site of entry to the brain. While much evidence points to lymphoreticular invasion followed by retrograde transfer through peripheral nerves, prions are present in the blood and may conceivably cross the blood-brain barrier directly. Here we have addressed the role of the blood-brain barrier (BBB) in prion disease propagation using Pdgfbret/ret mice which possess a highly permeable BBB. We found that Pdgfbret/ret mice have a similar prion disease incubation time as their littermate controls regardless of the route of prion transmission. These surprising results indicate that BBB permeability is irrelevant to the initiation of prion disease, even when prions are administered parenterally.
Assuntos
Barreira Hematoencefálica/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Animais , Transporte Biológico , Encéfalo/irrigação sanguínea , Encéfalo/patologia , Bovinos , Síndrome de Creutzfeldt-Jakob/patologia , Modelos Animais de Doenças , Encefalopatia Espongiforme Bovina/patologia , Humanos , Camundongos , Doenças Priônicas/transmissão , Proteínas Priônicas/metabolismo , Príons/patogenicidade , Scrapie/patologiaRESUMO
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.
Assuntos
Proteínas PrPC/toxicidade , Doenças Priônicas/tratamento farmacológico , Doenças Priônicas/metabolismo , Piridinas/administração & dosagem , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptores de Glutamato Metabotrópico/antagonistas & inibidores , Animais , Anticorpos/administração & dosagem , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Doenças Priônicas/genética , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Receptor de Glutamato Metabotrópico 5/genética , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMO
Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the α1 and α3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides.
Assuntos
Anticorpos/imunologia , Anticorpos/toxicidade , Maleabilidade , Príons/química , Príons/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/toxicidade , Sítios de Ligação de Anticorpos , Calpaína/metabolismo , Cerebelo , Síndrome de Creutzfeldt-Jakob/metabolismo , Reagentes de Ligações Cruzadas , Mapeamento de Epitopos , Feminino , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/toxicidade , Técnicas In Vitro , Ligantes , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Proteínas PrPC/química , Proteínas PrPC/genética , Proteínas PrPC/imunologia , Príons/genética , Espécies Reativas de Oxigênio/metabolismo , Deleção de Sequência/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/toxicidadeRESUMO
The cellular prion protein, PrPC, is attached by a glycosylphosphatidylinositol anchor to the outer leaflet of the plasma membrane. Its misfolded isoform PrPSc is the causative agent of prion diseases. Conversion of PrPC into PrPSc is thought to take place at the cell surface or in endolysosomal organelles. Understanding the intracellular trafficking of PrPC may, therefore, help elucidate the conversion process. Here we describe a time-resolved fluorescence energy transfer (FRET) assay reporting membrane expression and real-time internalization rates of PrPC The assay is suitable for high-throughput genetic and pharmaceutical screens for modulators of PrPC trafficking. Simultaneous administration of FRET donor and acceptor anti-PrPC antibodies to living cells yielded a measure of PrPC surface density, whereas sequential addition of each antibody visualized the internalization rate of PrPC (Z' factor >0.5). RNA interference assays showed that suppression of AP2M1 (AP-2 adaptor protein), RAB5A, VPS35 (vacuolar protein sorting 35 homolog), and M6PR (mannose 6-phosphate receptor) blocked PrPC internalization, whereas down-regulation of GIT2 and VPS28 increased PrPC internalization. PrPC cell-surface expression was reduced by down-regulation of RAB5A, VPS28, and VPS35 and enhanced by silencing EHD1. These data identify a network of proteins implicated in PrPC trafficking and demonstrate the power of this assay for identifying modulators of PrPC trafficking.
Assuntos
Endocitose , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Células A549 , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Proteínas PrPC/genética , Proteínas PrPSc/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
Antibodies against the prion protein PrPC can antagonize prion replication and neuroinvasion, and therefore hold promise as possible therapeutics against prion diseases. However, the safety profile of such antibodies is controversial. It was originally reported that the monoclonal antibody D13 exhibits strong target-related toxicity, yet a subsequent study contradicted these findings. We have reported that several antibodies against certain epitopes of PrPC, including antibody POM1, are profoundly neurotoxic, yet antibody ICSM18, with an epitope that overlaps with POM1, was reported to be innocuous when injected into mouse brains. In order to clarify this confusing situation, we assessed the neurotoxicity of antibodies D13 and ICSM18 with dose-escalation studies using diffusion-weighted magnetic resonance imaging and various histological techniques. We report that both D13 and ICSM18 induce rapid, dose-dependent, on-target neurotoxicity. We conclude that antibodies directed to this region may not be suitable as therapeutics. No such toxicity was found when antibodies against the flexible tail of PrPC were administered. Any attempt at immunotherapy or immunoprophylaxis of prion diseases should account for these potential untoward effects.
Assuntos
Anticorpos Monoclonais/toxicidade , Imunoterapia/métodos , Proteínas PrPC/imunologia , Doenças Priônicas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epitopos de Linfócito B/imunologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Priônicas/patologiaRESUMO
The self-replicating properties of proteins into amyloid fibrils is a common phenomenon and underlies a variety of neurodegenerative diseases. Because propagation-active fibrils are chemically indistinguishable from innocuous aggregates and monomeric precursors, their detection requires measurements of their replicative capacity. Here we present a digital amyloid quantitative assay (d-AQuA) with insulin as model protein for the absolute quantification of single replicative units, propagons. D-AQuA is a microfluidics-based technology that performs miniaturized simultaneous propagon-induced amplification chain reactions within hundreds to thousands of picoliter-sized droplets. At limiting dilutions, the d-AQuA reactions follow a stochastic regime indicative of the detection of single propagons. D-AQuA thus enables absolute quantification of single propagons present in a given sample at very low concentrations. The number of propagons quantified by d-AQuA was similar to that of fibrillar insulin aggregates detected by atomic-force microscopy and to an equivalent microplate-based assay, providing independent evidence for the identity of insulin propagons with a subset of morphologically defined protein aggregates. The sensitivity, precision, and accuracy of d-AQuA enable it to be suitable for multiple biotechnological and medical applications.
Assuntos
Peptídeos beta-Amiloides/análise , Técnicas Analíticas Microfluídicas , Humanos , Microscopia de Força Atômica , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Prions induce lethal neurodegeneration and consist of PrPSc, an aggregated conformer of the cellular prion protein PrPC. Antibody-derived ligands to the globular domain of PrPC (collectively termed GDL) are also neurotoxic. Here we show that GDL and prion infections activate the same pathways. Firstly, both GDL and prion infection of cerebellar organotypic cultured slices (COCS) induced the production of reactive oxygen species (ROS). Accordingly, ROS scavenging, which counteracts GDL toxicity in vitro and in vivo, prolonged the lifespan of prion-infected mice and protected prion-infected COCS from neurodegeneration. Instead, neither glutamate receptor antagonists nor inhibitors of endoplasmic reticulum calcium channels abolished neurotoxicity in either model. Secondly, antibodies against the flexible tail (FT) of PrPC reduced neurotoxicity in both GDL-exposed and prion-infected COCS, suggesting that the FT executes toxicity in both paradigms. Thirdly, the PERK pathway of the unfolded protein response was activated in both models. Finally, 80% of transcriptionally downregulated genes overlapped between prion-infected and GDL-treated COCS. We conclude that GDL mimic the interaction of PrPSc with PrPC, thereby triggering the downstream events characteristic of prion infection.
Assuntos
Anticorpos , Proteínas PrPSc/imunologia , Doenças Priônicas/induzido quimicamente , Doenças Priônicas/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Animais , Anticorpos/imunologia , Anticorpos/toxicidade , Camundongos , Camundongos Transgênicos , Proteínas PrPSc/genética , Doenças Priônicas/genética , Doenças Priônicas/patologia , Espécies Reativas de Oxigênio/imunologia , Transdução de Sinais/genética , eIF-2 Quinase/genética , eIF-2 Quinase/imunologiaRESUMO
Two lines of transgenic mice expressing mouse/elk and mouse/horse prion protein (PrP) hybrids, which both form a well-structured ß2-α2 loop in the NMR structures at 20 °C termed rigid-loop cellular prion proteins (RL-PrP(C)), presented with accumulation of the aggregated scrapie form of PrP in brain tissue, and the mouse/elk hybrid has also been shown to develop a spontaneous transmissible spongiform encephalopathy. Independently, there is in vitro evidence for correlations between the amino acid sequence in the ß2-α2 loop and the propensity for conformational transitions to disease-related forms of PrP. To further contribute to the structural basis for these observations, this paper presents a detailed characterization of RL-PrP(C) conformations in solution. A dynamic local conformational polymorphism involving the ß2-α2 loop was found to be evolutionarily preserved among all mammalian species, including those species for which the WT PrP forms an RL-PrP(C). The interconversion between two ensembles of PrP(C) conformers that contain, respectively, a 310-helix turn or a type I ß-turn structure of the ß2-α2 loop, exposes two different surface epitopes, which are analyzed for their possible roles in the still evasive function of PrP(C) in healthy organisms and/or at the onset of a transmissible spongiform encephalopathy.
Assuntos
Química Encefálica , Encéfalo , Proteínas PrPC/química , Doenças Priônicas , Animais , Cervos , Cavalos , Humanos , Camundongos , Camundongos Transgênicos , Proteínas PrPC/genética , Proteínas PrPC/metabolismo , Estrutura Secundária de ProteínaRESUMO
Zoonotic prion transmission was reported after the bovine spongiform encephalopathy (BSE) epidemic, when >200 cases of prion disease in humans were diagnosed as variant Creutzfeldt-Jakob disease. Assessing the risk of cross-species prion transmission remains challenging. We and others have studied how specific amino acid residue differences between species impact prion conversion and have found that the ß2-α2 loop region of the mouse prion protein (residues 165-175) markedly influences infection by sheep scrapie, BSE, mouse-adapted scrapie, deer chronic wasting disease, and hamster-adapted scrapie prions. The tyrosine residue at position 169 is strictly conserved among mammals and an aromatic side chain in this position is essential to maintain a 310-helical turn in the ß2-α2 loop. Here we examined the impact of the Y169G substitution together with the previously described S170N, N174T "rigid loop" substitutions on cross-species prion transmission in vivo and in vitro. We found that transgenic mice expressing mouse PrP containing the triple-amino acid substitution completely resisted infection with two strains of mouse prions and with deer chronic wasting disease prions. These studies indicate that Y169 is important for prion formation, and they provide a strong indication that variation of the ß2-α2 loop structure can modulate interspecies prion transmission.
Assuntos
Substituição de Aminoácidos/genética , Doenças Priônicas/genética , Doenças Priônicas/transmissão , Príons/genética , Animais , Bovinos , Cricetinae , Cervos , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doenças Priônicas/prevenção & controle , Proteínas Priônicas , Estrutura Secundária de Proteína , Ovinos , Especificidade da EspécieRESUMO
In the otherwise highly conserved NMR structures of cellular prion proteins (PrP(C)) from different mammals, species variations in a surface epitope that includes a loop linking a ß-strand, ß2, with a helix, α2, are associated with NMR manifestations of a dynamic equilibrium between locally different conformations. Here, it is shown that this local dynamic conformational polymorphism in mouse PrP(C) is eliminated through exchange of Tyr169 by Ala or Gly, but is preserved after exchange of Tyr 169 with Phe. NMR structure determinations of designed variants of mouse PrP(121-231) at 20 °C and of wild-type mPrP(121-231) at 37 °C together with analysis of exchange effects on NMR signals then resulted in the identification of the two limiting structures involved in this local conformational exchange in wild-type mouse PrP(C), and showed that the two exchanging structures present characteristically different solvent-exposed epitopes near the ß2-α2 loop. The structural data presented in this paper provided a platform for currently ongoing, rationally designed experiments with transgenic laboratory animals for renewed attempts to unravel the so far elusive physiological function of the cellular prion protein.