RESUMEN
Prion diseases are characterized by the accumulation of amyloid fibrils. The causative agent is an infectious amyloid that comprises solely misfolded prion protein (PrPSc). Prions can convert normal cellular prion protein (PrPC) to protease K-resistance prion protein fragment (PrP-res) in vitro; however, the intermediate steps involved in this spontaneous conversion still remain unknown. We investigated whether recombinant prion protein (rPrP) can directly convert into PrP-res via liquid-liquid phase separation (LLPS) in the absence of PrPSc. We found that rPrP underwent LLPS at the interface of the aqueous two-phase system of polyethylene glycol and dextran, whereas single-phase conditions were not inducible. Fluorescence recovery assay after photobleaching revealed that the liquid-solid phase transition occurred within a short time. The aged rPrP-gel acquired a proteinase-resistant amyloid accompanied by ß-sheet conversion, as confirmed by Western blotting, Fourier transform infrared spectroscopy, and Congo red staining. The reactions required both the N-terminal region of rPrP (amino acids 23-89) and kosmotropic salts, suggesting that the kosmotropic anions may interact with the N-terminal region of rPrP to promote LLPS. Thus, structural conversion via LLPS and liquid-solid phase transition could be the intermediate steps in the conversion of prions.
Asunto(s)
Extracción Líquido-Líquido/métodos , Proteínas PrPSc/química , Priones/química , Amiloide/química , Humanos , Placa Amiloide/metabolismo , Proteínas PrPSc/metabolismo , Enfermedades por Prión , Proteínas Priónicas , Priones/metabolismo , Conformación Proteica , Proteínas Recombinantes/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier/métodosRESUMEN
Prion diseases are fatal neurodegenerative diseases characterized by the deposition of abnormal prion protein aggregates (PrPSc) in the brain. In this study, we developed hydroxyethylamino-substituted styrylchromone (SC) and 2-(2-(pyridin-3-yl)vinyl)-4H-chromen-4-one (VPC) derivatives for single-photon emission computed tomography (SPECT) imaging of PrPSc deposits in the brain. The binding affinity of these compounds was evaluated using recombinant mouse prion protein (rMoPrP) aggregates, which resulted in the inhibition constant (Ki) value of 61.5 and 88.0 nM for hydroxyethyl derivative, (E)-2-(4-((2-hydroxyethyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NHEtOH) and (E)-2-(4-((2-hydroxyethyl)(methyl)amino)styryl)-6-iodo-4H-chromen-4-one (SC-NMeEtOH), respectively. However, none of the VPC derivatives showed binding affinity for the rMoPrP aggregates. Fluorescent imaging demonstrated that the accumulation pattern of SC-NHEtOH matched with the presence of PrPSc in the brain slices from mouse-adapted bovine spongiform encephalopathy-infected mice. A biodistribution study of normal mice indicated low initial brain uptake of [125I]SC-NHEtOH (0.88% injected dose/g (% ID/g) at 2 min) despite favorable washout from the brain (0.26% ID/g, at 180 min) was displayed. [125I]SC-NHEtOH exhibited binding affinities to both artificial prion aggregates as well as prion deposits in the brain. However, significant improvement in the binding affinity for PrPSc and blood-brain barrier permeability is necessary for the development of successful in vivo imaging probes for the detection of cerebral PrPSc in the brain.
Asunto(s)
Encefalopatía Espongiforme Bovina , Enfermedades por Prión , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Bovinos , Cromonas/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Ratones , Enfermedades por Prión/diagnóstico por imagen , Enfermedades por Prión/metabolismo , Proteínas Priónicas/metabolismo , Distribución TisularRESUMEN
BACKGROUND: The neuropathology of sporadic Creutzfeldt-Jakob disease (sCJD) is usually investigated using formalin-fixed and formic acid-treated brain tissue. However, formalin and formic acid treatment can interfere with immunostaining of abnormal prion protein. Therefore, there is a need for biochemical methods other than immunostaining to investigate abnormal prion protein in postmortem tissue. We developed RT-QuIC to quantitate the seeding activity (SD50) of sCJD brain tissue treated with formalin and formic acid. METHODS: We used endpoint RT-QuIC assays to analyze SD50 in formalin-fixed brain tissue from 19 sCJD patients (14 MM1 cases, 3 MM2-thalamic form [MM2T] cases and 2 MM2-cortical form [MM2C] cases) diagnosed according to Parchi's classification. We assessed SD50 in brains after incubation in formalin solution for over 1 month, and after treating formalin-fixed brain tissue with formic acid. We also examined how the SD50 values from formalin-fixed brain samples compared with neuropathological and immunohistochemical findings. RESULTS: The SD50 values of formalin-fixed brain samples from 14 MM1 cases, 2 MM2C cases, and 2 MM2T cases were 107.77±0.57/g tissue, 107.44±0.24/g tissue and 106.00±0.77/g tissue, respectively. The average SD50 value in MM1 unfixed brains decreased by 102.04 after formalin fixation for 1 month. In MM1 cases, after combined formalin and formic acid treatment, the SD50 value was reduced by approximately 105.16 compared with that of unfixed tissue. The SD50 values of formalin-fixed tissue showed a consistent pattern with the neuropathological findings in most brain regions examined. CONCLUSION: RT-QuIC enables the study of formalin-fixed brain tissue from sCJD patients that has not previously been amenable to analysis.
Asunto(s)
Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas Priónicas/metabolismo , Anciano , Anciano de 80 o más Años , Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/patología , Femenino , Fijadores , Formaldehído , Formiatos , Humanos , Masculino , Persona de Mediana Edad , Manejo de EspecímenesAsunto(s)
Anatomía , Cadáver , Priones , Anatomía/métodos , Disección/métodos , Humanos , Priones/aislamiento & purificaciónRESUMEN
Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca2+ signaling. In this study, we evaluated the binding of PrP to metabotropic glutamate receptor 1 (mGluR1) and found that wild-type PrP (PrP-wt) and mGluR1 co-immunoprecipitated in dual-transfected Neuro-2a (N2a) cells. Fluorescence resonance energy transfer analysis revealed an energy transfer between mGluR1-Cerulean and PrP-Venus. In order to determine whether PrP can modulate mGluR1 signaling, we performed Ca2+ imaging analyses following repetitive exposure to an mGluR1 agonist. Agonist stimulation induced synchronized Ca2+ oscillations in cells coexpressing PrP-wt and mGluR1. In contrast, N2a cells expressing PrP-ΔN failed to show ligand-dependent regulation of mGluR1-Ca2+ signaling, indicating that PrP can bind to mGluR1 and modulate its function to prevent irregular Ca2+ signaling and that its N-terminal region functions as a molecular switch during Ca2+ signaling.
Asunto(s)
Señalización del Calcio , Proteínas Priónicas/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Ratones , Neuronas/metabolismo , Mapas de Interacción de ProteínasRESUMEN
Infectious prions comprising abnormal prion protein, which is produced by structural conversion of normal prion protein, are responsible for transmissible spongiform encephalopathies including Creutzfeldt-Jakob disease in humans. Prions are infectious agents that do not possess a genome and the pathogenic protein was not thought to evoke any immune response. Although we previously reported that interferon regulatory factor 3 (IRF3) was likely to be involved in the pathogenesis of prion diseases, suggesting the protective role of host innate immune responses mediated by IRF3 signalling, this remained to be clarified. Here, we investigated the reciprocal interactions of type I interferon evoked by IRF3 activation and prion infection and found that infecting prions cause the suppression of endogenous interferon expression. Conversely, treatment with recombinant interferons in an ex vivo model was able to inhibit prion infection. In addition, cells and mice deficient in type I interferon receptor (subunit interferon alpha/beta receptor 1), exhibited higher susceptibility to 22L-prion infection. Moreover, in in vivo and ex vivo prion-infected models, treatment with RO8191, a selective type I interferon receptor agonist, inhibited prion invasion and prolonged the survival period of infected mice. Taken together, these data indicated that the interferon signalling interferes with prion propagation and some interferon-stimulated genes might play protective roles in the brain. These findings may allow for the development of new strategies to combat fatal diseases.
Asunto(s)
Interferón Tipo I/fisiología , Enfermedades por Prión/patología , Priones/metabolismo , Animales , Encéfalo/patología , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Enfermedades por Prión/inmunología , Enfermedades por Prión/metabolismo , Proteínas Priónicas/metabolismo , Priones/patogenicidad , Receptor de Interferón alfa y beta/metabolismo , Transducción de SeñalRESUMEN
Human prion diseases are neurodegenerative disorders caused by prion protein. Although infectivity was historically detected only in the central nervous system and lymphoreticular tissues of patients with sporadic Creutzfeldt-Jakob disease, recent reports suggest that the seeding activity of Creutzfeldt-Jakob disease prions accumulates in various non-neuronal organs including the liver, kidney, and skin. Therefore, we reanalyzed autopsy samples collected from patients with sporadic and genetic human prion diseases and found that seeding activity exists in almost all digestive organs. Unexpectedly, activity in the esophagus reached a level of prion seeding activity close to that in the central nervous system in some CJD patients, indicating that the safety of endoscopic examinations should be reconsidered.
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Sistema Digestivo/metabolismo , Sistema Digestivo/patología , Enfermedades por Prión/metabolismo , Enfermedades por Prión/patología , Priones/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Síndrome de Creutzfeldt-Jakob/metabolismo , Síndrome de Creutzfeldt-Jakob/patología , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Cambios Post MortemRESUMEN
Prion diseases are caused by deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to develop in vivo imaging probes that can detect cerebral PrPSc deposits. We synthesized several quinacrine-based acridine (AC) derivatives with 2,9-substitution and radioiodinated them. The AC derivatives were evaluated as prion-imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. The distribution of these compounds in mice was also evaluated. The 2-methoxy derivative [125I]2 exhibited the highest binding affinity for rMoPrP aggregates with an equilibrium dissociation constant (Kd) value of 43.4nM. Fluorescence imaging with 2 showed clear signals at the thioflavin T (ThT)-positive amyloid deposits in the mBSE-infected mouse brain. Although a discrepancy was observed between the in vitro binding of AC derivatives to the aggregates and in vivo distribution of these compounds in the brain and we failed to identify prospective prion-imaging probes in this study, the AC derivatives may be considered a useful scaffold for the development of in vivo imaging probes. Further chemical modification of these AC derivatives may discover clinically applicable prion imaging probes.
Asunto(s)
Acridinas/química , Encéfalo/diagnóstico por imagen , Radioisótopos de Yodo/química , Imagen Molecular , Enfermedades por Prión/diagnóstico por imagen , Acridinas/administración & dosificación , Acridinas/síntesis química , Administración Intravenosa , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Radioisótopos de Yodo/administración & dosificación , Ratones , Estructura Molecular , Relación Estructura-Actividad , Distribución TisularRESUMEN
The phenomenon of prion strains with distinct biological characteristics has been hypothesized to be involved in the structural diversity of abnormal prion protein (PrP(Sc)). However, the molecular basis of the transmission of strain properties remains poorly understood. Real-time quaking-induced conversion (RT-QUIC) is a cell-free system that uses Escherichia coli-derived recombinant PrP (rPrP) for the sensitive detection of PrP(Sc). To investigate whether the properties of various prion strains can be transmitted to amyloid fibrils consisting of rPrP (rPrP fibrils) using RT-QUIC, we examined the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L strain. In the first round of the reaction, there were differences in the secondary structures, especially in bands attributed to ß-sheets, as determined by infrared spectroscopy, and conformational stability between Chandler-seeded (1st-rPrP-fib(Ch)) and 22L-seeded (1st-rPrP-fib(22L)) rPrP fibrils. Of note, specific identifying characteristics of the two rPrP fibril types seen in the ß-sheets resembled those of the original PrP(Sc). Furthermore, the conformational stability of 1st-rPrP-fib(Ch) was significantly higher than that of 1st-rPrP-fib(22L), as with Chandler and 22L PrP(Sc). The survival periods of mice inoculated with 1st-rPrP-fib(Ch) or 1st-rPrP-fib(22L) were significantly shorter than those of mice inoculated with mixtures from the mock 1st-round RT-QUIC procedure. In contrast, these biochemical characteristics were no longer evident in subsequent rounds, suggesting that nonspecific uninfected rPrP fibrils became predominant probably because of their high growth rate. Together, these findings show that at least some strain-specific conformational properties can be transmitted to rPrP fibrils and unknown cofactors or environmental conditions may be required for further conservation. Importance: The phenomenon of prion strains with distinct biological characteristics is assumed to result from the conformational variations in the abnormal prion protein (PrP(Sc)). However, important questions remain about the mechanistic relationship between the conformational differences and the strain diversity, including how strain-specific conformations are transmitted. In this study, we investigated whether the properties of diverse prion strains can be transmitted to amyloid fibrils consisting of E. coli-derived recombinant PrP (rPrP) generated by real-time quaking-induced conversion (RT-QUIC), a recently developed in vitro PrP(Sc) formation method. We demonstrate that at least some of the strain-specific conformational properties can be transmitted to rPrP fibrils in the first round of RT-QUIC by examining the secondary structure, conformational stability, and infectivity of rPrP fibrils seeded with PrP(Sc) derived from either the Chandler or the 22L prion strain. We believe that these findings will advance our understanding of the conformational basis underlying prion strain diversity.
Asunto(s)
Proteínas PrPSc/química , Priones/química , Animales , Ratones , Microscopía Electrónica de Transmisión , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Abnormal prion protein (PrP(Sc)) generated from the cellular isoform of PrP (PrP(C)) is assumed to be the main or sole component of the pathogen, called prion, of transmissible spongiform encephalopathies (TSE). Because PrP is a host-encoded protein, acquired immune responses are not induced in TSE. Meanwhile, activation of the innate immune system has been suggested to partially block the progression of TSE; however, the mechanism is not well understood. To further elucidate the role of the innate immune system in prion infection, we investigated the function of interferon regulatory factor 3 (IRF3), a key transcription factor of the MyD88-independent type I interferon (IFN) production pathway. We found that IRF3-deficient mice exhibited significantly earlier onset with three murine TSE strains, namely, 22L, FK-1, and murine bovine spongiform encephalopathy (mBSE), following intraperitoneal transmission, than with wild-type controls. Moreover, overexpression of IRF3 attenuated prion infection in the cell culture system, while PrP(Sc) was increased in prion-infected cells treated with small interfering RNAs (siRNAs) against IRF3, suggesting that IRF3 negatively regulates PrP(Sc) formation. Our findings provide new insight into the role of the host innate immune system in the pathogenesis of prion diseases.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Enfermedades por Prión/inmunología , Transducción de Señal , Animales , Encéfalo/metabolismo , Encéfalo/patología , Línea Celular , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas PrPSc/metabolismo , Enfermedades por Prión/genética , Enfermedades por Prión/metabolismo , Bazo/metabolismo , Bazo/patologíaRESUMEN
Prion diseases are fatal neurodegenerative disorders caused by the deposition of scrapie prion protein aggregates (PrPSc) in the brain. We previously reported that styrylchromone (SC) and benzofuran (BF) derivatives have potential as imaging probes for PrPSc. To further improve their properties, we designed and synthesized 2-(benzofuran-2-yl)-chromone (BFC) derivatives hybridized with SC and BF backbones as novel single-photon emission computed tomography probes for the detection of cerebral PrPSc deposits. Recombinant mouse prion protein (rMoPrP) aggregates and mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice were used to evaluate the binding properties of BFC derivatives to PrPSc. The BFC derivatives exhibited high binding affinities (equilibrium dissociation constant [Kd] = 22.6-47.7 nM) for rMoPrP aggregates. All BFC derivatives showed remarkable selectivity against amyloid beta aggregates. Fluorescence microscopy confirmed that the fluorescence signals of the BFC derivatives corresponded to the antibody-positive deposits of PrPSc in mBSE-infected mouse brains. Among the BFC derivatives, [125I]BFC-OMe and [125I]BFC-NH2 exhibited high brain uptake and favorable washout from the mouse brain. In vitro autoradiography demonstrated that the distribution of [125I]BFC-OMe in the brain tissues of mBSE-infected mice was colocalized with PrPSc deposits. Taken together, BFC derivatives appear to be promising prion imaging probes.
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Benzofuranos , Encefalopatía Espongiforme Bovina , Priones , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Bovinos , Cromonas/metabolismo , Encefalopatía Espongiforme Bovina/metabolismo , Ratones , Priones/metabolismoRESUMEN
Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy are characterized by aggregation of abnormal α-synuclein (α-syn) and collectively referred to as α-synucleinopathy. Because these diseases have different prognoses and treatments, it is desirable to diagnose them early and accurately. However, it is difficult to accurately diagnose these diseases by clinical symptoms because symptoms such as muscle rigidity, postural dysreflexia, and dementia sometimes overlap among these diseases. The process of conformational conversion and aggregation of α-syn has been thought similar to that of abnormal prion proteins that cause prion diseases. In recent years, in vitro conversion methods, such as real-time quaking-induced conversion (RT-QuIC), have been developed. This method has succeeded in amplifying and detecting trace amounts of abnormal prion proteins in tissues and central spinal fluid of patients by inducing conversion of recombinant prion proteins via shaking. Additionally, it has been used for antemortem diagnosis of prion diseases. Recently, aggregated α-syn has also been amplified and detected in patients by applying this method and many clinical studies have examined diagnosis using tissues or cerebral spinal fluid from patients. In this review, we discuss the utility and problems of α-syn RT-QuIC for antemortem diagnosis of α-synucleinopathies.
RESUMEN
The defining characteristic of prion diseases is conversion of a cellular prion protein (PrPC) to an abnormal prion protein (PrPSc). The exogenous attachment of PrPSc to the surface of a target cell is critical for infection. However, the initial interaction of PrPSc with the cell surface is poorly characterized. In the current study, we specifically focused on the association of PrPSc with cells during the early phase of infection, using an acute infection model. First, we treated mouse neuroblastoma N2a-58 cells with prion strain 22â¯L-infected brain homogenates and revealed that PrPSc was associated with membrane fractions within three hours, a short exposure time. These results were also observed in PrPC-deficient hippocampus cell lines. We also demonstrate here that PrPSc from 22â¯L-infected brain homogenates was associated with lipid rafts during the early phase of infection. Furthermore, we revealed that DS500, a glycosaminoglycan mimetic, inhibited both the attachment of PrPSc to membrane fractions and subsequent prion transmission, suggesting that the early association of prions with cell surface is important for prion infection.
Asunto(s)
Enfermedades por Prión , Priones , Animales , Membrana Celular , Sulfato de Dextran , Ratones , Proteínas PrPScRESUMEN
The accumulation of abnormal prion protein (PrPSc) produced by the structure conversion of PrP (PrPC) in the brain induces prion disease. Although the conversion process of the protein is still not fully elucidated, it has been known that the intramolecular chemical bridging in the most fragile pocket of PrP, known as the "hot spot," stabilizes the structure of PrPC and inhibits the conversion process. Using our original structure-based drug discovery algorithm, we identified the low molecular weight compounds that predicted binding to the hot spot. NPR-130 and NPR-162 strongly bound to recombinant PrP in vitro, and fragment molecular orbital (FMO) analysis indicated that the high affinity of those candidates to the PrP is largely dependent on nonpolar interactions, such as van der Waals interactions. Those NPRs showed not only significant reduction of the PrPSc levels but also remarkable decrease of the number of aggresomes in persistently prion-infected cells. Intriguingly, treatment with those candidate compounds significantly prolonged the survival period of prion-infected mice and suppressed prion disease-specific pathological damage, such as vacuole degeneration, PrPSc accumulation, microgliosis, and astrogliosis in the brain, suggesting their possible clinical use. Our results indicate that in silico drug discovery using NUDE/DEGIMA may be widely useful to identify candidate compounds that effectively stabilize the protein.
Asunto(s)
Simulación por Computador , Progresión de la Enfermedad , Descubrimiento de Drogas/métodos , Enfermedades por Prión/diagnóstico , Enfermedades por Prión/tratamiento farmacológico , Proteínas Priónicas/efectos de los fármacos , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Humanos , Masculino , Ratones , Ratones Transgénicos , Enfermedades por Prión/genética , Proteínas Priónicas/genética , Proteínas Priónicas/metabolismo , Unión Proteica/fisiología , Relación Estructura-ActividadRESUMEN
Human prion diseases are etiologically categorized into three forms: sporadic, genetic, and infectious. Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common type of human prion disease that manifests as subacute progressive dementia. No effective therapy for sCJD is currently available. Potential therapeutic compounds are frequently tested in rodents infected with mouse-adapted prions that differ from human prions. However, therapeutic effect varies depending on the prion strain, which is one of the reasons why candidate compounds have shown little effect in sCJD patients. We previously reported that intraperitoneal administration of FK506 was able to prolong the survival of mice infected with a mouse-adapted prion by suppressing the accumulation of abnormal prion protein (PrP) and inhibiting the activation of microglia. In this study, we tested oral administration of FK506 in knock-in mice expressing chimeric human prion protein (KiChM) that were infected with sCJD to determine if this compound is also effective against a clinically relevant human prion, i.e., one that has not been adapted to mice. Treatment with FK506, started either just before or just after disease onset, suppressed typical sCJD pathology (gliosis) and slightly but significantly prolonged the survival of sCJD-inoculated mice. It would be worthwhile to conduct a clinical trial using FK506, which has been safety-approved and is widely used as a mild immunosuppressant.
Asunto(s)
Síndrome de Creutzfeldt-Jakob/tratamiento farmacológico , Síndrome de Creutzfeldt-Jakob/mortalidad , Progresión de la Enfermedad , Inmunosupresores/administración & dosificación , Proteínas Priónicas , Tacrolimus/administración & dosificación , Anciano , Animales , Encéfalo/efectos de los fármacos , Encéfalo/patología , Síndrome de Creutzfeldt-Jakob/genética , Femenino , Humanos , Ratones , Proteínas Priónicas/genética , Tasa de Supervivencia/tendenciasRESUMEN
BACKGROUND: The prognosis of intrahepatic cholangiocarcinoma (ICC) is based on tumor localization; however, the mechanism remains unknown. Therefore, we investigated the biological characteristics of perihilar and peripheral ICC in a mouse model. METHODS: The model was established by the administration of three oncogenic plasmids harboring myristoylated AKT, mutated human YAP, and pCMV-Sleeping Beauty into the mice. The perihilar and peripheral ICC tumors that developed in the same mouse were assessed for the expression of cell adhesion factors and driver genes with immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The perihilar ICC tumors were irregularly shaped, whereas the peripheral tumors were mostly circular, similar to the differences found in patients. Alpha-smooth muscle actin was strongly expressed in the perihilar tumors at 10 weeks, and vimentin expression was significantly up-regulated in the perihilar ICC at 14 weeks. Fgfr2 level significantly increased in peripheral ICC at 10 weeks, whereas Idh2 expression was up-regulated in perihilar ICC. CONCLUSIONS: Despite diffuse injection of oncogenic plasmid, expression of driver genes and oncogenes in ICC tumor cells differs depending on the tumor localization, resulting in changes in epithelial-mesenchymal transition, which may explain the different outcomes of patients with peripheral and perihilar ICC.
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Colangiocarcinoma/genética , Tecnología de Genética Dirigida , Expresión Génica , Genes Relacionados con las Neoplasias , Neoplasias Hepáticas/genética , Animales , Moléculas de Adhesión Celular/genética , Colangiocarcinoma/patología , Modelos Animales de Enfermedad , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , PronósticoRESUMEN
INTRODUCTION: Prion diseases are fatal neurodegenerative disorders caused by the deposition of abnormal prion protein aggregates (PrPSc) in the central nervous system. This study aimed to evaluate the use of iodinated pyridyl benzofuran (IPBF) derivatives as single-photon emission computed tomography (SPECT) probes for the detection of cerebral PrPSc deposits. METHODS: In vitro binding assays of IPBF derivatives were carried out in the recombinant mouse prion protein (rMoPrP) and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. SPECT imaging of 5-(5-[123I]iodobenzofuran-2-yl)-N-methylpyridin-2-amine ([123I]IPBF-NHMe) was performed on mBSE-infected and mock-infected mice. RESULTS: Fluorescence microscopy results showed that fluorescence signals of IPBF derivatives corresponded to the thioflavin-T positive amyloid deposits of PrPSc in the brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. Among the IPBF derivatives, 5-(5-iodobenzofuran-2-yl)-N-methylpyridin-2-amine (IPBF-NHMe) exhibited the highest binding affinity to the recombinant mouse prion protein (rMoPrP) aggregates with a Ki of 14.3 nM. SPECT/computed tomography (CT) imaging and ex vivo autoradiography demonstrated that the [123I]IPBF-NHMe distribution in brain tissues of mBSE-infected mice co-localized with PrPSc deposits. CONCLUSION: [123I]IPBF-NHMe appears to be a prospective SPECT tracer for monitoring prion deposits in living brain tissues.
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Benzofuranos/química , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Proteínas Priónicas/metabolismo , Piridinas/química , Tomografía Computarizada de Emisión de Fotón Único/métodos , Animales , Estudios de Factibilidad , Ratones , Microscopía FluorescenteRESUMEN
Prion diseases are transmissible neurodegenerative disorders of humans and animals, which are characterized by the aggregation of abnormal prion protein (PrPSc) in the central nervous system. Although several small compounds that bind to normal PrP (PrPC) have been shown to inhibit structural conversion of the protein, an effective therapy for human prion disease remains to be established. In this study, we screened 1200 existing drugs approved by the US Food and Drug Administration (FDA) for anti-prion activity using surface plasmon resonance imaging (SPRi). Of these drugs, 31 showed strong binding activity to recombinant human PrP, and three of these reduced the accumulation of PrPSc in prion-infected cells. One of the active compounds, alprenolol hydrochloride, which is used clinically as a ß-adrenergic blocker for hypertension, also reduced the accumulation of PrPSc in the brains of prion-infected mice at the middle stage of the disease when the drug was administered orally with their daily water from the day after infection. Docking simulation analysis suggested that alprenolol hydrochloride fitted into the hotspot within mouse PrPC, which is known as the most fragile structure within the protein. These findings provide evidence that SPRi is useful in identifying effective drug candidates for neurodegenerative diseases caused by abnormal protein aggregation, such as prion diseases.
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Alprenolol/farmacología , Imagenología Tridimensional , Priones/antagonistas & inhibidores , Alprenolol/química , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Espectroscopía de Resonancia Magnética , Ratones , Simulación del Acoplamiento Molecular , Oxprenolol/química , Oxprenolol/farmacología , Proteínas PrPSc/metabolismo , Priones/química , Priones/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/farmacología , Resonancia por Plasmón de Superficie , Análisis de SupervivenciaRESUMEN
Prion diseases are fatal neurodegenerative disorders associated with the deposition of abnormal prion protein aggregates (PrPSc) in the brain tissue. Here, we report the development of 125I-labeled iodobenzofuran (IBF) derivatives as single photon emission computed tomography (SPECT) imaging probes to detect cerebral PrPSc deposits. We synthesized and radioiodinated several 5-IBF and 6-IBF derivatives. The IBF derivatives were evaluated as prion imaging probes using recombinant mouse prion protein (rMoPrP) aggregates and brain sections of mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice. Although all the IBF derivatives were strongly adsorbed on the rMoPrP aggregates, [125I]5-IBF-NHMe displayed the highest adsorption rate and potent binding affinity with an equilibrium dissociation constant (Kd) of 12.3 nM. Fluorescence imaging using IBF-NHMe showed clear signals of the PrPSc-positive amyloid deposits in the mBSE-infected mouse brains. Biodistribution studies in normal mice demonstrated slow uptake and clearance from the brain of 125I-IBF derivatives. Among the derivatives, [125I]6-IBF-NH2 showed the highest peak brain uptake [2.59% injected dose (ID)/g at 10 min] and good clearance (0.51% ID/g at 180 min). Although the brain distribution of IBF derivatives should still be optimized for in vivo imaging, these compounds showed prospective binding properties to PrPSc. Further chemical modification of these IBF derivatives may contribute to the discovery of clinically applicable prion imaging probes.
Asunto(s)
Benzofuranos/síntesis química , Encéfalo/metabolismo , Radioisótopos de Yodo/química , Proteínas PrPC/metabolismo , Enfermedades por Prión/diagnóstico por imagen , Animales , Benzofuranos/administración & dosificación , Benzofuranos/química , Benzofuranos/farmacocinética , Encéfalo/diagnóstico por imagen , Bovinos , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Estructura Molecular , Enfermedades por Prión/metabolismo , Distribución Tisular , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Abnormal protease-resistant prion protein (PrP-res) is the only surrogate biochemical marker for prion diseases, and a sensitive technique to detect PrP-res in blood or tissues is urgently needed. Primary cultured bone marrow stromal cells (MSCs) expressed PrP and were capable of supporting stable human prion infection. Using a mouse-adapted BSE strain, we demonstrated that PrP-res can be detected in expanded MSCs. We then analyzed the bone marrow cells collected at autopsy from two individuals with sporadic Creutzfeldt-Jakob disease (CJD), and, in both cases, cultured MSCs were positive for PrP-res. These data would suggest that ex vivo MSC expansion accompanied by PrP-res analysis could be a helpful tool in the definitive diagnosis of prion disease at an earlier stage in the disease process than is currently possible, and with considerably less distress to the patient.