RESUMEN
Background Fragile X syndrome (FXS) is the most common inherited cause of intellectual disability and autism. Gene therapy may offer an efficient method to ameliorate the symptoms of this disorder. Methods An AAVphp.eb-hSyn-mFMR1IOS7 vector and an empty control were injected into the tail vein of adult Fmr1 knockout (KO) mice and wildtype (WT) controls. The KO mice were injected with 2 × 1013 vg/kg of the construct. The control KO and WT mice were injected with an empty vector. Four weeks following treatment, the animals underwent a battery of tests: open field, marble burying, rotarod, and fear conditioning. The mouse brains were studied for levels of the Fmr1 product FMRP. Results: No significant levels of FMRP were found outside the CNS in the treated animals. The gene delivery was highly efficient, and it exceeded the control FMRP levels in all tested brain regions. There was also improved performance in the rotarod test and partial improvements in the other tests in the treated KO animals. Conclusion: These experiments demonstrate efficient, brain-specific delivery of Fmr1 via peripheral administration in adult mice. The gene delivery led to partial alleviation of the Fmr1 KO phenotypical behaviors. FMRP oversupply may explain why not all behaviors were significantly affected. Since AAV.php vectors are less efficient in humans than in the mice used in the current experiment, studies to determine the optimal dose using human-suitable vectors will be necessary to further demonstrate feasibility.
Asunto(s)
Barrera Hematoencefálica , Síndrome del Cromosoma X Frágil , Humanos , Animales , Ratones , Ratones Noqueados , Barrera Hematoencefálica/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Estudios de Factibilidad , Terapia GenéticaRESUMEN
Alpha-synuclein (SNCA), a presynaptic protein, is significantly reduced in individuals with Down syndrome (DS) and Ts65Dn mice, a mouse model of DS. Methylation analyses of promoter proximal CpG sites indicate similar reduction in Ts65Dn mice compared to control mice. Epigallocatechin-3-gallate (EGCG), a polyphenolic catechin present in green tea extract, increases methylation of SNCA promoter proximal CpG sites and expression in Ts65Dn mice. These results suggest a positive link between CpG methylation and SNCA expression in Down syndrome.
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Síndrome de Down/metabolismo , Epigénesis Genética/efectos de los fármacos , alfa-Sinucleína/metabolismo , Animales , Catequina/análogos & derivados , Catequina/farmacología , Metilación de ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Síndrome de Down/tratamiento farmacológico , Epigénesis Genética/fisiología , Ratones , Regiones Promotoras GenéticasRESUMEN
Two members of the senescence-accelerated mouse group, SAMP8 and SAMP10, are characterized by learning and memory deficits, while the SAMR1 strain is not. In this study, we used two behavioral tests, social approach and object recognition and compared the results observed for the SAMP strains with those seen in the control strain, SAMR1. In social approach experiments, the 2 SAMP strains showed decreased sociability compared to SAMR1 as shown by their reluctance to spend time near a stranger mouse and increased immobility. In object recognition experiments, SAMP strains spent more time in the thigmotaxis zone and less time in the more exposed central zone than SAMR1 mice. From a behavioral standpoint, SAMP mice were less interactive and showed increased anxiety-like behavior compared to SAMR1.
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Envejecimiento Prematuro/fisiopatología , Envejecimiento Prematuro/psicología , Envejecimiento/fisiología , Ansiedad/psicología , Relaciones Interpersonales , Animales , Trastornos de la Memoria , Ratones , Actividad Motora/fisiología , Reconocimiento en Psicología/fisiología , Especificidad de la EspecieRESUMEN
The triplication of genes located on chromosome 21 is known to cause a wide spectrum of pathology seen in Down syndrome (DS), including leukemia, seizures, stroke, and mental retardation. Studies on RNA and protein expression of genes in DS brain have demonstrated the role of triplicated genes in several DS phenotypes. Significant changes in the expression of nontriplicated genes have also been observed. However, little information is available regarding the role of nonchromosome 21 genes in DS pathology. We have found that α-synuclein (SNCA), a presynaptic protein whose gene is located on chromosome 6 in the Ts65Dn mouse model for DS, is significantly reduced in the cortex and other brain regions. We hypothesize that this alteration may play a critical role in the reduced synaptic function observed in DS. We have found an increase in the level of neurosin, a key negative regulator of SNCA in Ts65Dn cortex. We have also found increased levels of protein phosphatase 2A, a negative regulator of the activation of tyrosine hydroxylase and a key enzyme in the biosynthetic pathway for dopamine in Ts65Dn cortex. These findings reveal potential target sites for intervention in the treatment of DS pathology.
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Encéfalo/metabolismo , Síndrome de Down/metabolismo , Calicreínas/metabolismo , Transmisión Sináptica/fisiología , alfa-Sinucleína/biosíntesis , Adolescente , Adulto , Anciano , Animales , Western Blotting , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Síndrome de Down/fisiopatología , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Proteína Fosfatasa 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
The pathogenesis of Alzheimer's disease (AD) is thought to be related to the accumulation of amyloid beta (Abeta) in amyloid deposits and toxic oligomeric species. Immunomodulation is emerging as an effective means of shifting the equilibrium from Abeta accumulation to clearance; however, excessive cell mediated inflammation and cerebral microhemorrhages are two forms of toxicity which can occur with this approach. Vaccination studies have so far mainly targeted the adaptive immune system. In the present study, we have stimulated the innate immune system via the Toll-like receptor 9 (TLR9) with cytosine-guanosine-containing DNA oligodeoxynucleotides in Tg2576 AD model transgenic mice. This treatment produced a 66% and 80% reduction in the cortical (p = 0.0001) and vascular (p = 0.0039) amyloid burden, respectively, compared with nontreated AD mice. This was in association with significant reductions in Abeta42, Abeta40, and Abeta oligomer levels. We also show that treated Tg mice performed similarly to wild-type mice on a radial arm maze. Our data suggest that stimulation of innate immunity via TLR9 is highly effective at reducing the parenchymal and vascular amyloid burden, along with Abeta oligomers, without apparent toxicity.
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Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Transducción de Señal/fisiología , Receptor Toll-Like 9/biosíntesis , Enfermedad de Alzheimer/prevención & control , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Actividad Motora/fisiología , Receptor Toll-Like 9/fisiologíaRESUMEN
Previous studies have reported that various inbred SAM mouse strains differ markedly with regard to a variety of parameters, such as capacity for learning and memory, life spans and brain histopathology. A potential cause of differences seen in these strains may be based on the fact that some strains have a high concentration of infectious murine leukemia virus (MuLV) in the brain, whereas other strains have little or no virus. To elucidate the effect of a higher titer of endogenous retrovirus in astroglial cells of the brain, we established astroglial cell lines from SAMR1 and SAMP8 mice, which are, respectively, resistant and prone to deficit in learning and memory and shortened life span. MuLV-negative astroglial cell lines established from ICR mice served as controls. Comparison of these cell lines showed differences in: 1) levels of the capsid antigen CAgag in both cell lysates and culture media, 2) expression of genomic retroelements, 3) the number of virus particles, 4) titer of infectious virus, 5) morphology, 6) replication rate of cells in culture and final cell concentrations, 7) expression pattern of proinflammatory cytokine genes. The results show that the expression of MuLV is much higher in SAMP8 than SAMR1 astrocyte cultures and that there are physiological differences in astroglia from the 2 strains. These results raise the possibility that the distinct physiological differences between SAMP8 and SAMR1 are a function of activation of endogenous retrovirus.
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Astrocitos/fisiología , Astrocitos/virología , Retrovirus Endógenos/aislamiento & purificación , Retrovirus Endógenos/fisiología , Animales , Proteínas de la Cápside/biosíntesis , Línea Celular , Citocinas/biosíntesis , Perfilación de la Expresión Génica , Ratones , Microscopía Electrónica de Transmisión , ARN Mensajero/biosíntesis , ARN Viral/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayo de Placa Viral , Virión/aislamiento & purificaciónRESUMEN
Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.
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Enfermedades por Prión/inmunología , Receptor Toll-Like 4/inmunología , Animales , Western Blotting , Encéfalo/patología , Femenino , Interleucina-6/metabolismo , Macrófagos/inmunología , Ratones , Ratones Endogámicos C3H , Enfermedades por Prión/fisiopatología , Factores de Tiempo , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Prion diseases are characterized by conversion of the cellular prion protein (PrP(C)) to a protease-resistant conformer, the srapie form of PrP (PrP(Sc)). Humoral immune responses to nondenatured forms of PrP(Sc) have never been fully characterized. We investigated whether production of antibodies to PrP(Sc) could occur in PrP null (Prnp(-/-)) mice and further, whether innate immune stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN) 1826 could enhance this process. Whether such stimulation could raise anti-PrP(Sc) antibody levels in wild-type (Prnp(+/+)) mice was also investigated. Prnp(-/-) and Prnp(+/+) mice were immunized with nondenatured 139A scrapie-associated fibrils (SAF), with or without ODN 1826, and were tested for titers of PrP-specific antibodies. In Prnp(-/-) mice, inclusion of ODN 1826 in the immunization regime increased anti-PrP titers more than 13-fold after two immunizations and induced, among others, antibodies to an N-terminal epitope, which were only present in the immune repertoire of mice receiving ODN 1826. mAb 6D11, derived from such a mouse, reacts with the N-terminal epitope QWNK in native and denatured forms of PrP(Sc) and recombinant PrP and exhibits a K(d) in the 10(-)(11) M range. In Prnp(+/+) mice, ODN 1826 increased anti-PrP levels as much as 84% after a single immunization. Thus, ODN 1826 potentiates adaptive immune responses to PrP(Sc) in 139A SAF-immunized mice. These results represent the first characterization of humoral immune responses to nondenatured, infectious PrP(Sc) and suggest methods for optimizing the generation of mAbs to PrP(Sc), many of which could be used for diagnosis and treatment of prion diseases.
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Anticuerpos Monoclonales/biosíntesis , ADN/inmunología , Proteína PrP 27-30/inmunología , Proteínas PrPSc/inmunología , Animales , Formación de Anticuerpos , Epítopos , Inmunidad Innata , Inmunización , Cambio de Clase de Inmunoglobulina , Ratones , Ratones Noqueados , Oligodesoxirribonucleótidos , Proteínas PrPSc/biosíntesis , Proteínas PrPSc/genética , Células TH1/inmunología , Células Th2/inmunología , Receptor Toll-Like 9/inmunologíaRESUMEN
Chronic wasting disease (CWD) of elk (Cervus elaphus nelsoni) and mule deer (Odocoileus hemionus) is one of three naturally occurring forms of prion disease, the others being Creutzfeldt-Jakob disease in humans and scrapie in sheep. In the last few decades, CWD has spread among captive and free-ranging cervids in 13 US states, two Canadian provinces and recently in Korea. The origin of the CWD agent(s) in cervids is not known. This study describes the development of a transgenic mouse line (TgElk) homozygous for a transgene array encoding the elk prion protein (PrP(C)) and its use in propagating and simulating CWD in mice. Intracerebral injection of one mule deer and three elk CWD isolates into TgElk mice led to disease with incubation periods of 127 and 95 days, respectively. Upon secondary passage, the incubation time was reduced to 108 and 90 days, respectively. Upon passage into TgElk mice, CWD prions (PrP(Sc)) maintained the characteristic Western blot profiles seen in CWD-affected mule deer and elk and produced histopathological modifications consistent with those observed in the natural disease. The short incubation time observed on passage from cervid to mouse with both mule deer and elk CWD brain homogenates and the demonstrated capacity of the animals to propagate (mouse to mouse) CWD agents make the TgElk line a valuable model to study CWD agents in cervid populations. In addition, these results with this new transgenic line suggest the intriguing hypothesis that there could be more than one strain of CWD agent in cervids.
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Ciervos , Modelos Animales de Enfermedad , Proteínas PrPC/genética , Enfermedad Debilitante Crónica , Animales , Western Blotting , Encéfalo/patología , Electroforesis en Gel de Poliacrilamida , Histocitoquímica , Inmunohistoquímica , Ratones , Ratones Transgénicos , Proteínas PrPC/aislamiento & purificación , Enfermedad Debilitante Crónica/patología , Enfermedad Debilitante Crónica/transmisiónRESUMEN
Quantitative immunogold procedure was used to study the distribution of metallothionein I/II (MT-I/II) at the ultrastructural level in the perivascular areas, including microvascular endothelial cells (ECs) and astrocytes with their perivascular end-feet, in brains of scrapie-infected hyperglycemic (diabetic) and normoglycemic (non-diabetic) mice. Samples of the fronto-parietal cortex obtained from diabetic and non-diabetic scrapie-infected, as well as from non-infected (control) SJL/J mice, were processed for immunocytochemical examination. In control mice, the labelling of the ECs was of low intensity, restricted to few immunogold particles in the cytoplasm. More intense labelling was present in the cytoplasm of astrocytic perivascular processes and perikarya, where it was associated with endoplasmic reticulum and fibrils. A few immunosignals were also present inside the nuclei of astrocytes. In diabetic mice the labelling of the EC cytoplasm was slightly increased, whereas in the cytoplasm of perivascular processes and pericarya of astrocytes, including their nuclei, there was significant enhancement of labelling. In these cells the density of immunosignals was highest in the areas of cytoplasm containing bundles of fibrils. In non-diabetic, scrapie-infected mice the intensity of immunolabelling was higher than in control mice but slightly lower than in diabetic mice. These results are similar to those in Alzheimer's disease reported by other authors, and suggest that neurodegenerative diseases as well as metabolic stress enhance the metallothionein expression in perivascular regions of brain cerebral cortex, predominantly in astrocytes.
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Química Encefálica , Diabetes Mellitus/metabolismo , Metalotioneína/análisis , Scrapie/metabolismo , Regulación hacia Arriba/genética , Animales , Astrocitos/química , Astrocitos/ultraestructura , Encéfalo/irrigación sanguínea , Encéfalo/patología , Corteza Cerebral/química , Corteza Cerebral/patología , Diabetes Mellitus/patología , Células Endoteliales/química , Células Endoteliales/ultraestructura , Endotelio Vascular/patología , Endotelio Vascular/ultraestructura , Inmunohistoquímica , Ratones , Scrapie/patologíaRESUMEN
Senescence-accelerated mice (SAMP8) have a short life span, whereas SAMR1 mice are resistant to accelerated senescence. Previously it has been reported that the Akv strain of ecotropic murine leukemia virus (E-MuLV) was detected in brains of SAMP8 mice but not in brains of SAMR1 mice. In order to determine the change of MuLV levels following scrapie infection, we analyzed the E-MuLV titer and the RNA expression levels of E-MuLV, xenotropic MuLV, and polytropic MuLV in brains and spinal cords of scrapie-infected SAM mice. The expression levels of the 3 types of MuLV were increased in scrapie-infected mice compared to control mice; E-MuLV expression was detected in infected SAMR1 mice, but only in the terminal stage of scrapie disease. We also examined incubation periods and the levels of PrPSc in scrapie-infected SAMR1 (sR1) and SAMP8 (sP8) mice. We confirmed that the incubation period was shorter in sP8 (210+/-5 days) compared to sR1 (235+/-10 days) after intraperitoneal injection. The levels of PrPSc in sP8 were significantly greater than sR1 at 210+/-5 days, but levels of PrPSc at the terminal stage of scrapie in both SAM strains were virtually identical. These results show the activation of MuLV expression by scrapie infection and suggest acceleration of the progression of scrapie pathogenesis by MuLV.
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Encéfalo/virología , Virus de la Leucemia Murina/metabolismo , Scrapie/metabolismo , Scrapie/virología , Médula Espinal/virología , Animales , Encéfalo/metabolismo , Progresión de la Enfermedad , Inmunohistoquímica , Ratones , Proteínas PrPSc/metabolismo , Priones/metabolismo , Médula Espinal/metabolismo , Factores de Tiempo , Replicación ViralRESUMEN
Prion diseases are transmissible and invariably fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K (PK)-resistant conformer, scrapie PrP (PrP(Sc)). Humoral immunity may significantly prolong the incubation period and even prevent disease in murine models of prionoses. However, the mechanism(s) of action of anti-PrP monoclonal antibodies (Mabs) remain(s) obscure. The murine neuroblastoma N2a cell line, infected with the 22L mouse-adapted scrapie strain, was used to screen a large library of Mabs with similar binding affinities to PrP, to identify those antibodies which could clear established infection and/or prevent infection de novo. Three Mabs were found capable of complete and persistent clearing of already-infected N2a cells of PrP(Sc). These antibodies were 6D11 (generated to PK-resistant PrP(Sc) and detecting PrP residues 93-109), and 7H6 and 7A12, which were raised against recombinant PrP and react with neighbouring epitopes of PrP residues 130-140 and 143-155, respectively. Mabs were found to interact with PrP(Sc) formation both on the cell surface and after internalization in the cytosol. Treatment with Mabs was not associated with toxicity nor did it result in decreased expression of PrP(C). Both preincubation of N2a cells with Mabs prior to exposure to 22L inoculum and preincubation of the inoculum with Mabs prior to infecting N2a cells resulted in a significant reduction in PrP(Sc) levels. Information provided in these studies is important for the rational design of humoral immune therapy for prion infection in animals and eventually in humans.
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Anticuerpos Monoclonales/farmacología , Proteínas PrPC/efectos de los fármacos , Proteínas PrPSc/efectos de los fármacos , Enfermedades por Prión/inmunología , Enfermedades por Prión/prevención & control , Animales , Afinidad de Anticuerpos , Células Cultivadas , Mapeo Epitopo , Epítopos Inmunodominantes , Ratones , Proteínas PrPC/inmunología , Proteínas PrPSc/inmunología , Proteínas PrPSc/patogenicidadRESUMEN
This article describes a time-tested method for avoiding common problems encountered while pouring crown and bridge impressions.
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Técnica de Impresión Dental , Coronas , Materiales de Impresión Dental , Dentadura Parcial Fija , Humanos , Modelos DentalesRESUMEN
Quantitative immunogold procedure was used to study the distribution of molecular components of interendothelial junctions in blood-brain barrier (BBB) microvessels of scrapie infected SJL/J hyperglycemic mice showing obesity and reduced glucose tolerance. Samples of brain (fronto-parietal cerebral cortex and thalamo-hypothalamic region) obtained from hyperglycemic (diabetic) mice and from non- infected, normoglycemic (non-diabetic) SJL/J mice, were processed for immunocytochemical examination. The localization of the following tight junction (TJ)-associated proteins was studied: occludin as an integral membrane (transmembrane) protein, and zonula occludens one (ZO-1) as a peripheral protein. The localization of beta-catenin as a representative of the cadherin/catenin complex that is typical for adherens junctions (AJs) also was studied. Morphometric analysis revealed that the density of immunosignals for occludin, represented by colloidal gold particles (GPs), was significantly lower in the brain microvessels of diabetic than in non-diabetic mice. No significant differences in the density of immunosignals for ZO-1 and beta-catenin between both experimental mouse groups were observed. It indicates that abnormal glucose metabolism affects mostly occludin which is believed to play a fundamental role in the maintenance of the tightness of endothelial lining in brain microvascular network and thereby in the preservation of its barrier function. These results also support the previously expressed opinion that occludin, detected with the applied morphological method, can be considered a sensitive indicator of altered molecular architecture of the interendothelial junctions due to the action of some metabolic or pathological insults.
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Barrera Hematoencefálica/metabolismo , Diabetes Mellitus/metabolismo , Endotelio Vascular/metabolismo , Hiperglucemia/metabolismo , Proteínas de la Membrana/metabolismo , Scrapie/metabolismo , Uniones Adherentes/metabolismo , Uniones Adherentes/ultraestructura , Animales , Biomarcadores/metabolismo , Barrera Hematoencefálica/patología , Diabetes Mellitus/etiología , Diabetes Mellitus/patología , Modelos Animales de Enfermedad , Endotelio Vascular/ultraestructura , Femenino , Hiperglucemia/etiología , Hiperglucemia/patología , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos , Obesidad/metabolismo , Obesidad/patología , Ocludina , Fosfoproteínas/metabolismo , Scrapie/complicaciones , Scrapie/patología , Uniones Estrechas/metabolismo , Uniones Estrechas/ultraestructura , Proteína de la Zonula Occludens-1 , beta Catenina/metabolismoRESUMEN
The mouse is a popular and versatile model for the study of scrapie and other transmissible spongiform encephalopathies. In this chapter, information is given for preparation of infectious material for inoculation and a method of clinical scoring that yields accurate and reproducible quantification of the scrapie incubation period. With the help of histopathological and immunopathological techniques, we can detect brain pathological changes in scrapie-infected animals at the cellular and molecular level. We will also describe the histological and immunocytochemistry methods we use for scrapie research, outline step-by-step procedures, discuss tissue preparation, fixation, and processing of specimens, and provide special hints to achieve successful staining. We also include our results of PrPSc and GFAP immunostaining in scrapie research. In conclusion, immunopathological staining is an important and useful tool in the research of scrapie pathology.
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Modelos Animales de Enfermedad , Scrapie , Animales , Gliosis/metabolismo , Inmunohistoquímica/métodos , Ratones , Scrapie/diagnóstico , Scrapie/metabolismo , Scrapie/fisiopatologíaRESUMEN
The diagnosis of prion diseases in humans is challenging due to a lack of specific and sensitive non-invasive tests. Many forms of human prion disease including variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, and 10% of sporadic CJD cases are associated with amyloid deposition. Several positron emission tomography (PET) ligands have recently been developed to directly image beta-amyloid associated with Alzheimer disease. One of them, methoxy-X04, is a fluorescent derivative of Congo red with high binding affinity toward amyloid fibrils and good blood-brain barrier permeability. Using methoxy-X04, we investigated whether amyloid-targeting ligands can be also employed for direct imaging of amyloid deposits associated with some prion diseases. Such a method could potentially become a novel diagnostic approach for these conditions. Studies were performed on MB mice infected with the 87V mouse-adapted scrapie strain. Labeling of PrP amyloid plaques in brains of presymptomatic and symptomatic mice was demonstrated using in vivo transcranial two-photon microscopy after systemic administration of methoxy-X04. During real-time imaging, PrP amyloid deposits could be clearly distinguished 15 min after intravenous administration of methoxy-X04. The ligand showed rapid clearance from brain areas that did not contain amyloid deposits. PrP amyloid deposits could also be detected by direct application of methoxy-X04 on cerebellar sections from GSS patients. These results suggest that methoxy-X04 or similar derivatives could be used as PET imaging agents to improve the diagnosis of human prion diseases associated with amyloid deposition.
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Alquenos , Péptidos beta-Amiloides/análisis , Derivados del Benceno , Placa Amiloide/patología , Enfermedades por Prión/diagnóstico por imagen , Enfermedades por Prión/patología , Tomografía Computarizada de Emisión/métodos , Alquenos/metabolismo , Alquenos/farmacocinética , Animales , Derivados del Benceno/metabolismo , Derivados del Benceno/farmacocinética , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Ratones , Placa Amiloide/metabolismo , Valor Predictivo de las Pruebas , Enfermedades por Prión/metabolismo , Reproducibilidad de los Resultados , Scrapie/diagnóstico por imagen , Scrapie/metabolismo , Scrapie/patología , EstilbenosRESUMEN
Scrapie is a fatal, but slow, infectious disease. C57BL/6J, SAMP8 (a strain that develops early senescence), SAMR1 (a strain that is resistant to senescence) and AKR/J (a progenitor of the SAM strains) mice were infected with 22A, 139A, 22L and ME7 scrapie strains. Histopathological stains included haematoxylin and eosin (HE), and periodic acid-Schiff (PAS). Vacuolation was found in the brains of all scrapie-infected mice. The 22A strain caused more extensive vacuolation in the brains of SAMP8 and SAMR1 mice than in C57BL mice. PAS-positive plaques (PP) were found in 22A-infected mice in cortex, corpus callosum, hippocampus, subependymal zone area and thalamus. PP were significantly increased in 22A-infected SAMR1 mice compared to mice from other scrapie-infected strains. Clusters of small, round, homogeneous PAS-positive granular structures (PGS) were found in all mouse strains, especially in aging control and 22A-infected C57BL mice, predominantly in the stratum radiatum of the CA1, CA2 and CA3 areas of the hippocampus. Some of these structures were also observed in stratum oriens and piriform cortex, and in cerebellar Purkinje cell areas. Some of the PGS were associated with astrocytes and blood vessels. Each granule was 1-5 microm in diameter and there were clusters consisting of several to 40 PGS; the sizes of the clusters ranged from 10 to 80 microm in diameter. There were more PGS clusters in uninfected C57BL and AKR mice than in uninfected SAMP8 and SAMR1 mice. PGS were not increased in scrapie-infected mice. These findings suggest that PGS accumulation was more dependent on the genetic information of the mouse strain, whereas PP and vacuolation patterns depended on the scrapie strain-mouse strain combination.
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Envejecimiento/patología , Encéfalo/patología , Scrapie/genética , Scrapie/patología , Animales , Astrocitos/metabolismo , Astrocitos/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos , Reacción del Ácido Peryódico de Schiff , Placa Amiloide/patologíaRESUMEN
The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy.
Asunto(s)
Quelantes/farmacología , Quelantes/uso terapéutico , Cobre/metabolismo , Enfermedades por Prión/prevención & control , Animales , Química Encefálica/efectos de los fármacos , Terapia por Quelación/métodos , Cobre/análisis , Cobre/sangre , Relación Dosis-Respuesta a Droga , Endopeptidasa K/efectos de los fármacos , Endopeptidasa K/metabolismo , Ratones , Ratones Endogámicos , Penicilamina/farmacología , Penicilamina/uso terapéutico , Proteínas PrPSc/química , Proteínas PrPSc/efectos de los fármacos , Proteínas PrPSc/metabolismo , Enfermedades por Prión/tratamiento farmacológico , Factores de TiempoRESUMEN
Signal abnormalities on magnetic resonance imaging (MRI) T2-weighted images (T2WI) have been described in patients with Creutzfeldt-Jakob disease; however, the pathology underlying these findings remains to be fully described. We investigated the time-course of signal alterations in a murine model of prion disease using in vivo 9.4 Tesla micro magnetic resonance imaging (muMRI). The topography of muMRI signal changes was correlated with the accumulation of proteinase resistant PrP(Sc) in corresponding brain sections. Increased signal intensity on T2WI was observed in the septum and in the hippocampus of presymptomatic mice 120 days post infection (dpi). Mildly symptomatic animals (150 dpi) and animals with apparent neurological deficit (180 dpi) had a greater increase of signal intensity on T2WI in the septum and the hippocampus; in addition, abnormalities in the cortex and in the thalamus were found. Neuropathological evaluation demonstrated accumulation of PrP(Sc) and astrogliosis but only minimal or no spongiform changes in structures where abnormal signal was detected. These observations suggest that early pathological changes related to the accumulation of PrP(Sc) may be detectable in presymptomatic subjects using MRI systems with higher magnetic field strength.
Asunto(s)
Scrapie/patología , Animales , Imagen por Resonancia Magnética , Masculino , Ratones , Proteínas PrPSc/metabolismo , Scrapie/metabolismoRESUMEN
Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation.