RESUMEN
Prions are proteinaceous infectious particles that replicate by structural conversion of the host-encoded cellular prion protein (PrPC), causing fatal neurodegenerative diseases in mammals. Species-specific amino acid substitutions (AAS) arising from single nucleotide polymorphisms within the prion protein gene (Prnp) modulate prion disease pathogenesis, and, in several instances, reduce susceptibility of homo- or heterozygous AAS carriers to prion infection. However, a mechanistic understanding of their protective effects against clinical disease is missing. We generated gene-targeted mouse infection models of chronic wasting disease (CWD), a highly contagious prion disease of cervids. These mice express wild-type deer or PrPC harboring the S138N substitution homo- or heterozygously, a polymorphism found exclusively in reindeer (Rangifer tarandus spp.) and fallow deer (Dama dama). The wild-type deer PrP-expressing model recapitulated CWD pathogenesis including fecal shedding. Encoding at least one 138N allele prevented clinical CWD, accumulation of protease-resistant PrP (PrPres) and abnormal PrP deposits in the brain tissue. However, prion seeding activity was detected in spleens, brains, and feces of these mice, suggesting subclinical infection accompanied by prion shedding. 138N-PrPC was less efficiently converted to PrPres in vitro than wild-type deer (138SS) PrPC. Heterozygous coexpression of wild-type deer and 138N-PrPC resulted in dominant-negative inhibition and progressively diminished prion conversion over serial rounds of protein misfolding cyclic amplification. Our study indicates that heterozygosity at a polymorphic Prnp codon can confer the highest protection against clinical CWD and highlights the potential role of subclinical carriers in CWD transmission.
Asunto(s)
Ciervos , Enfermedades por Prión , Priones , Reno , Enfermedad Debilitante Crónica , Ratones , Animales , Priones/metabolismo , Proteínas Priónicas/genética , Ciervos/genética , Enfermedad Debilitante Crónica/genética , Ratones Transgénicos , Enfermedades por Prión/genéticaRESUMEN
Cerebral deposition of abnormally aggregated α-synuclein (αSyn) is a neuropathological hallmark of Parkinson's disease (PD). PD-associated αSyn (αSynPD) aggregates can act as proteinaceous nuclei ("seeds") able of self-templated propagation. Since this is strikingly reminiscent to properties of proteinaceous infectious particles (prions), lessons learned from prion diseases suggest to test whether transferred αSynPD can propagate and induce neurological impairments or disease in a new host. Two studies that addressed this question provided divergent results. Intracerebral (i.c.) injection of Lewy body extracts from PD patients caused cerebral αSyn pathology, as well as nigrostriatal neurodegeneration, of wild-type mice and macaques, with the mice also showing motor impairments (Recasens et al. 2014, Ann Neurol 75:351-362). In contrast, i.c. transmission of homogenates from PD brains did not stimulate, after "> 360" days post-injection (dpi), pathological αSyn conversion or clinical symptoms in transgenic TgM83+/- mice hemizygously expressing mutated (A53T) human αSyn (Prusiner et al. 2015, PNAS 112:E5308-E5317). To advance the assessment of possible αSynPD hazards by providing further data, we examined neuropathological and clinical effects upon i.c. transmission of brain, stomach wall and muscle tissue as well as blood from PD patients in TgM83+/- mice up to 612 dpi. This revealed a subtle, yet distinctive stimulation of localized αSyn aggregation in the somatodendritic compartment and dystrophic neurites of individual or focally clustered cerebral neurons after challenge with brain and stomach wall homogenates. No such effect was observed with transmitted blood or homogenized muscle tissue. The detected stimulation of αSyn aggregation was not accompanied by apparent motor impairments or overt neurological disease in TgM83+/- mice. Our study substantiated that transmitted αSynPD seeds, including those from the stomach wall, are able to propagate in new mammalian hosts. The consequences of such propagation and potential safeguards need to be further investigated.
Asunto(s)
Encéfalo/patología , Sistema Nervioso Entérico/patología , Cuerpos de Lewy/patología , Neuronas/patología , Enfermedad de Parkinson , Estómago/patología , alfa-Sinucleína , Animales , Humanos , Ratones , Músculo Esquelético/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Priones , alfa-Sinucleína/administración & dosificación , alfa-Sinucleína/sangre , alfa-Sinucleína/aislamiento & purificación , alfa-Sinucleína/metabolismoRESUMEN
The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.
Asunto(s)
Bacillus licheniformis/química , Encefalopatía Espongiforme Bovina/etiología , Calor , Péptido Hidrolasas/metabolismo , Proteínas Priónicas/química , Proteolisis , Animales , Bacillus licheniformis/enzimología , Bovinos , Ratones TransgénicosAsunto(s)
Péptidos beta-Amiloides , Hormona del Crecimiento , Encéfalo , Cadáver , Hormona de Crecimiento Humana , HumanosRESUMEN
Prions or PrPSc are proteinaceous infectious agents that consist of misfolded, self-replicating states of a sialoglycoprotein called the prion protein or PrPC The current work tests a new hypothesis that sialylation determines the fate of prions in an organism. To begin, we produced control PrPSc from PrPC using protein misfolding cyclic amplification with beads (PMCAb), and also generated PrPSc with reduced sialylation levels using the same method but with partially desialylated PrPC as a substrate (dsPMCAb). Syrian hamsters were inoculated intraperitoneally with brain-derived PrPSc or PrPSc produced in PMCAb or dsPMCAb and then monitored for disease. Animals inoculated with brain- or PMCAb-derived PrPSc developed prion disease, whereas administration of dsPMCAb-derived PrPSc with reduced sialylation did not cause prion disease. Animals inoculated with dsPMCAb-derived material were not subclinical carriers of scrapie, as no PrPSc was detected in brains or spleen of these animals by either Western blotting or after amplification by serial PMCAb. In subsequent experiments, trafficking of brain-, PMCAb-, and dsPMCAb-derived PrPSc to secondary lymphoid organs was monitored in wild type mice. PrPSc sialylation was found to be critical for effective trafficking of PrPSc to secondary lymphoid organs. By 6 hours after inoculation, brain- and PMCAb-derived PrPSc were found in spleen and lymph nodes, whereas dsPMCAb-derived PrPSc was found predominantly in liver. This study demonstrates that the outcome of prion transmission to a wild type host is determined by the sialylation status of the inoculated PrPSc Furthermore, this work suggests that the sialylation status of PrPSc plays an important role in prion lymphotropism.
Asunto(s)
Ácido N-Acetilneuramínico/metabolismo , Priones/metabolismo , Animales , Western Blotting , Cricetinae , Mesocricetus , Proteínas PrPSc/metabolismo , Espectrofotometría InfrarrojaRESUMEN
The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP(C)) into Proteinase K-resistant, infectious PrP particles (PrP(TSE)), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP(C) substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP(C) substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.
Asunto(s)
Proteínas PrPSc/química , Animales , Química Encefálica , Cricetinae , Endopeptidasa K , Mesocricetus , Microscopía de Fuerza Atómica , Proteína PrP 27-30/química , Proteína PrP 27-30/metabolismo , Proteína PrP 27-30/ultraestructura , Proteínas PrPSc/metabolismo , Proteínas PrPSc/ultraestructura , Conformación Proteica , Pliegue de Proteína , Estabilidad Proteica , Scrapie/metabolismo , Scrapie/transmisión , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
The misfolding and aggregation of endogenous proteins in the central nervous system is a neuropathological hallmark of Alzheimer's disease (AD), Parkinson's disease (PD), as well as prion diseases. A molecular mechanism referred to as "nucleation-dependent aggregation" is thought to underlie this neuropathological phenomenon. According to this concept, disease-associated protein particles act as nuclei, or seeds, that recruit cellular proteins and incorporate them, in a misfolded form, into their growing aggregate structure. Experimental studies have shown that the aggregation of the AD-associated proteins amyloid-ß (Aß) and tau, and of the PD-associated protein α-synuclein, can be stimulated in laboratory animal models by intracerebral (i.c.) injection of inocula containing aggregated species of the respective proteins. This has raised the question of whether AD or PD can be transmitted, like certain human prion diseases, between individuals by self-propagating protein particles potentially present on medical instruments or in blood or blood products. While the i.c. injection of inocula containing AD- or PD-associated protein aggregates was found to cause neuronal damage and clinical abnormalities (e.g., motor impairments) in some animal models, none of the studies published so far provided evidence for a transmission of severe or even fatal disease. In addition, available epidemiological data do not indicate a transmissibility of AD or PD between humans. The findings published so far on the effects of experimentally transmitted AD- or PD-associated protein seeds do not suggest specific precautionary measures in the context of hemotherapy, but call for vigilance in transfusion medicine and other medical areas.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Sistema Nervioso Central/metabolismo , Enfermedad de Parkinson/metabolismo , Priones/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Sistema Nervioso Central/patología , Humanos , Modelos Moleculares , Priones/genética , Pliegue de Proteína , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
Prion amyloidosis occurred in the heart of 1 of 3 macaques intraperitoneally inoculated with bovine spongiform encephalopathy prions. This macaque had a remarkably long duration of disease and signs of cardiac distress. Variant Creutzfeldt-Jakob disease, caused by transmission of bovine spongiform encephalopathy to humans, may manifest with cardiac symptoms from prion-amyloid cardiomyopathy.
Asunto(s)
Amiloidosis/patología , Cardiomiopatías/etiología , Cardiomiopatías/patología , Síndrome de Creutzfeldt-Jakob/patología , Síndrome de Creutzfeldt-Jakob/transmisión , Encefalopatía Espongiforme Bovina/transmisión , Animales , Encéfalo/patología , Bovinos , Encefalopatía Espongiforme Bovina/patología , Macaca mulatta , Músculo Esquelético/patología , Miocardio/patologíaRESUMEN
Risk for human exposure to bovine spongiform encephalopathy (BSE)-inducing agent was estimated in a nonhuman primate model. To determine attack rates, incubation times, and molecular signatures, we orally exposed 18 macaques to 1 high dose of brain material from cattle with BSE. Several macaques were euthanized at regular intervals starting at 1 year postinoculation, and others were observed until clinical signs developed. Among those who received ≥5 g BSE-inducing agent, attack rates were 100% and prions could be detected in peripheral tissues from 1 year postinoculation onward. The overall median incubation time was 4.6 years (3.7-5.3). However, for 3 macaques orally exposed on multiple occasions, incubation periods were at least 7-10 years. Before clinical signs were noted, we detected a non-type 2B signature, indicating the existence of atypical prion protein during the incubation period. This finding could affect diagnosis of variant Creutzfeldt-Jakob disease in humans and might be relevant for retrospective studies of positive tonsillectomy or appendectomy specimens because time of infection is unknown.
Asunto(s)
Encefalopatía Espongiforme Bovina/fisiopatología , Encefalopatía Espongiforme Bovina/transmisión , Enfermedades Transmitidas por los Alimentos/fisiopatología , Macaca fascicularis , Proteínas PrPSc/química , Secuencia de Aminoácidos , Animales , Encéfalo/patología , Bovinos , Síndrome de Creutzfeldt-Jakob/diagnóstico , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/fisiopatología , Síndrome de Creutzfeldt-Jakob/transmisión , Modelos Animales de Enfermedad , Encefalopatía Espongiforme Bovina/diagnóstico , Encefalopatía Espongiforme Bovina/metabolismo , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/metabolismo , Humanos , Periodo de Incubación de Enfermedades Infecciosas , Carne/envenenamiento , Datos de Secuencia Molecular , Proteínas PrPSc/genética , Proteínas PrPSc/aislamiento & purificación , Alineación de SecuenciaRESUMEN
Since the beginning prion research has been largely dependent on animal models for deciphering the disease, drug development or prion detection and quantification. Thereby, ethical as well as cost and labour-saving aspects call for alternatives in vitro. Cell models can replace or at least complement animal studies, but their number is still limited and the application usually restricted to certain strains and host species due to often strong transmission barriers. Bank voles promise to be an exception as they or materials prepared from them are uniquely susceptible to prions from various species in vivo, in vitro and in cell-free applications. Here we present a mainly astrocyte-based primary glia cell assay from bank vole, which is infectible with scrapie strains from bank vole, mouse and hamster. Stable propagation of bank vole-adapted RML, murine 22L and RML, and hamster 263K scrapie is detectable from 20 or 30 days post exposure onwards. Thereby, the infected bank vole glia cells show similar or even faster prion propagation than likewise infected glia cells of the corresponding murine or hamster hosts. We propose that our bank vole glia cell assay could be a versatile tool for studying and comparing multiple prion strains with different species backgrounds combined in one cell assay.
Asunto(s)
Arvicolinae , Bioensayo/métodos , Neuroglía , Priones/metabolismo , Scrapie/diagnóstico , Animales , Técnicas de Cultivo de Célula/métodos , Cricetinae , Ratones , Proteínas PrPSc/metabolismo , RoedoresRESUMEN
BACKGROUND AND OBJECTIVES: For early diagnosis and disease monitoring of neurodegenerative diseases (NDs), reliable blood biomarkers are needed. Elevated levels of neurofilament light chain protein (NfL), an axonal damage marker, have been described across different NDs, with highest values in prion diseases and amyotrophic lateral sclerosis (ALS). Synaptic degeneration is a common early feature in most NDs and seems to precede neuronal degeneration in prion disease. However, synaptic markers in blood are still missing. Here, we investigated whether the brain-specific protein ß-synuclein might be a suitable blood biomarker for early diagnosis and evaluation of synaptic integrity in prion disease. METHODS: We analyzed blood ß-synuclein with a newly established digital ELISA and NfL with a single-molecule array in samples obtained from human participants and prion and ALS animal models. Furthermore, ß-synuclein was investigated in brain tissue of individuals with Creutzfeldt-Jakob disease (CJD) and controls. RESULTS: We investigated 308 patients, including 129 cases with prion disease, 8 presymptomatic PRNP variation carriers, 60 with ALS, 68 with other ND, and 43 control patients. In CJD symptomatic cases, ß-synuclein and NfL were markedly increased compared to all other diagnostic groups (p < 0.001). In the large majority of presymptomatic PRNP variation carriers, ß-synuclein and NfL levels were within normal ranges. In prion disease animal models, ß-synuclein and NfL displayed normal levels in the presymptomatic phase with a sudden elevation at disease onset and a plateau in the symptomatic phase. In contrast to NfL, ß-synuclein was not elevated in either symptomatic patients with ALS or an ALS animal model. In the discrimination between prion disease and all other groups, ß-synuclein (area under the curve 0.97, 95% CI 0.94-0.99, p < 0.001) was superior to NfL (area under the curve 0.91, 95% CI 0.88-0.94, p < 0.001). In addition, brain tissue ß-synuclein showed significantly reduced levels in patients with CJD compared to control patients (p < 0.001). DISCUSSION: Blood ß-synuclein was significantly elevated in patients with CJD, reflecting ongoing synaptic damage, and showed good discriminative characteristics. We therefore propose it as a candidate blood marker for early diagnosis and monitoring of synaptic integrity in prion disease. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that serum ß-synuclein concentration accurately distinguishes patients with symptomatic CJD from controls.
Asunto(s)
Síndrome de Creutzfeldt-Jakob , Enfermedades por Prión , Sinucleína beta/biosíntesis , Biomarcadores , Síndrome de Creutzfeldt-Jakob/diagnóstico , Humanos , Filamentos Intermedios , Proteínas de Neurofilamentos , Enfermedades por Prión/diagnósticoRESUMEN
A neuropathological hallmark of Parkinson's disease (PD) is the cerebral deposition of abnormally aggregated α-synuclein (αSyn). PD-associated αSyn (αSynPD) aggregates are assumed to act, in a prion-like manner, as proteinaceous nuclei ("seeds") capable of self-templated propagation. Braak and colleagues put forward the idea of a neural gut-brain axis mediating the centripetal spread of αSynPD pathology from the enteric nervous system (ENS) to the brain in PD. This has sparked great interest and initiated passionate discussions both in support of and opposing the suggested hypothesis. A precedent for the spread of protein seeds or seeding from the gastro-intestinal (GI) tract to the central nervous system (CNS) had been previously revealed for pathological prion protein in peroral prion infections. This article scrutinizes the similarities and dissimilarities between the pathophysiological spread of disease-associated protein aggregation along the neural gut-brain axis in peroral prion infections and PD. On this basis, evidence supporting the proposed neural gut-brain axis in PD is concluded to be not as robust as that established for peroral prion infections. New tools for the ultrasensitive detection of αSynPD-associated seeding activity in archived or fresh human tissue samples such as real-time quaking induced conversion (RT-QuIC) or protein misfolding cyclic amplification (PMCA) assays can possibly help to address this deficit in the future.
Asunto(s)
Encéfalo/patología , Sistema Nervioso Entérico , Tracto Gastrointestinal/patología , Enfermedad de Parkinson/patología , Agregación Patológica de Proteínas/patología , Animales , Humanos , Ratones , Proteínas Priónicas/metabolismo , Priones/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt-Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrPSc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.
RESUMEN
The cellular prion protein (PrP(c)) is a multifunctional, highly conserved and ubiquitously expressed protein. It undergoes a number of modifications during its post-translational processing, resulting in different PrP(c) glycoforms and truncated PrP(c) fragments. Limited data are available in humans on the expression and cleavage of PrP(c). In this study we investigated the PrP(c) isoform composition in the cerebrospinal fluid from patients with different human prion diseases. The first group of patients was affected by sporadic Creutzfeldt-Jakob disease exhibiting different PrP codon 129 genotypes. The second group contained patients with a genetic form of Creutzfeldt-Jakob disease (E200K). The third group consisted of patients with fatal familial insomnia and the last group comprised cases with the Gerstmann-Sträussler-Scheinker syndrome. We examined whether the PrP codon 129 polymorphism in sporadic Creutzfeldt-Jakob disease as well as the type of prion disease in human patients has an impact on the glycosylation and processing of PrP(c). Immunoblotting analyses using different monoclonal PrP(c) antibodies directed against various epitopes of PrP(c) revealed, for all examined groups of patients, a consistent predominance of the glycosylated PrP(c) isoforms as compared with the unglycosylated form. In addition, the antibody SAF70 recognized a variety of PrP(c) fragments with sizes of 21, 18, 13 and 12 kDa. Our findings indicate that the polymorphisms at PrP codon 129, the E200K mutation at codon 200 or the examined types of human transmissible spongiform encephalopathies do not exert a measurable effect on the glycosylation and processing of PrP(c) in human prion diseases.
Asunto(s)
Codón , Síndrome de Creutzfeldt-Jakob , Mutación , Polimorfismo Genético , Priones , Isoformas de Proteínas , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/metabolismo , Síndrome de Creutzfeldt-Jakob/líquido cefalorraquídeo , Síndrome de Creutzfeldt-Jakob/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Priones/líquido cefalorraquídeo , Priones/genética , Isoformas de Proteínas/líquido cefalorraquídeo , Isoformas de Proteínas/genética , Procesamiento Proteico-Postraduccional , Adulto JovenRESUMEN
Effective disinfectants are of key importance for the safe handling and reprocessing of surgical instruments. This study tested whether new formulations containing SDS, NaOH and 1-propanol (n-propanol) are simultaneously active against a broad range of pathogens including bacteria, fungi, non-enveloped viruses and prions. Inactivation and disinfection were examined in suspension and on carriers, using coagulated blood or brain homogenate as an organic contaminant. Coomassie blue staining was used to assess whether the formulations undesirably fixed proteins to rough surfaces. A mixture of 0.2 % SDS and 0.3 % NaOH in 20 % n-propanol achieved potent decontamination of steel carriers contaminated with PrP(TSE), the biochemical marker for prion infectivity, from 263K scrapie hamsters or from patients with sporadic or variant Creutzfeldt-Jakob disease. 263K scrapie infectivity on carriers was decreased by > or =5.5 logs. Furthermore, the formulation effectively inactivated poliovirus, hepatitis A virus and caliciviruses (including murine norovirus) in suspension tests. It also yielded significant titre reductions of bacteria (Enterococcus faecium, Mycobacterium avium; >6 logs), fungi (spores of Aspergillus niger; > or =5 logs) and poliovirus (>4 logs) embedded in coagulated blood on carriers. The formulation was not found to fix proteins more than was observed with water as the cleaning reagent. In conclusion, SDS, NaOH and n-propanol can synergistically achieve fast, broad-range disinfection.
Asunto(s)
Bacterias/efectos de los fármacos , Desinfectantes/metabolismo , Desinfectantes/farmacología , Desinfección/métodos , Hongos/efectos de los fármacos , Priones/efectos de los fármacos , Virus/efectos de los fármacos , Animales , Humanos , Viabilidad Microbiana/efectos de los fármacosRESUMEN
Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrP(C)) is rich in alpha-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrP(Sc)) has an anomalously high beta-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (pre-clinical, 100 and 130 days post-infection (dpi); first clinical signs ( approximately 145 dpi); and terminal ( approximately 170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the beta-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in beta-sheet content. As the disease progressed, the beta-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly - but not solely - due to the formation of PrP(Sc) in the tissue and indicated that other proteins high in beta-sheet were produced, either by overexpression or misfolding. Elevated beta-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for a further understanding of the disease.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Scrapie/metabolismo , Animales , Cricetinae , Ganglios Espinales/química , Ganglios Espinales/patología , Mesocricetus , Proteínas del Tejido Nervioso/análisis , Proteínas PrPSc/análisis , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Estructura Secundaria de Proteína , Scrapie/etiología , Scrapie/patología , Espectroscopía Infrarroja por Transformada de Fourier , Factores de TiempoRESUMEN
The involvement of muscles in the pathogenesis of transmissible spongiform encephalopathies (TSEs) is irregular and unpredictable. We show that the TSE-specific protein (PrP(TSE)) is present in muscles of mice fed with a mouse-adapted strain of bovine spongiform encephalopathy as early as 100 days post-infection, corresponding to about one-third of the incubation period. The proportion of mice with PrP(TSE)-positive muscles and the number of muscles involved increased as infection progressed, but never attained more than a limited distribution, even at the clinical stage of disease. The appearance of PrP(TSE) in muscles during the preclinical stage of disease was probably due to the haematogenous/lymphatic spread of infectivity from the gastrointestinal tract to lymphatic tissues associated with muscles, whereas in symptomatic animals, the presence of PrP(TSE) in the nervous system, in neuromuscular junctions and in muscle fibres suggests a centrifugal spread from the central nervous system, as already observed in other TSE models.
Asunto(s)
Encefalopatía Espongiforme Bovina/metabolismo , Tejido Linfoide/química , Priones/aislamiento & purificación , Animales , Bovinos , Encefalopatía Espongiforme Bovina/patología , Ratones , Músculo EsqueléticoRESUMEN
Prion infectivity and its molecular marker, the pathological prion protein PrP(Sc), accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrP(Sc) was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrP(Sc) deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrP(Sc) was detected before the onset of symptoms, but the bulk of skin-associated PrP(Sc) accumulated in the clinical phase. PrP(Sc) was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrP(Sc) in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrP(Sc) by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.