RESUMEN
The transmissible spongiform encephalopathies (TSE) or prion diseases are fatal neurodegenerative disorders in which the central event is the conversion of a normal host-encoded protein (PrP(c)) into an abnormal isoform (PrP(sc)) which accumulates as amyloid in TSE brain. The two PrP(c) and PrP(sc) prion protein isoforms are membrane sialoglycoproteins synthesized in the central nervous system and various peripheral organ tissues. In this review, we describe the ultrastructural localization of prion proteins in human and animal cerebral and non-cerebral tissues whether or not infected by TSE agents. In addition to the plasma membrane of several cells, PrP(c) was found in association with cytoplasmic organelles of central and nerve-muscle synapses, and secretory granules of epithelial cells. Fibrils of amyloid plaques, synaptic structures, and lysosome-like organelles constitute the subcellular sites harboring PrP(sc). These findings have led to discussions on the physiological role of PrP(c) and the pathological mechanisms underlying prion spongiform encephalopathies.
Asunto(s)
Encéfalo/metabolismo , Enfermedades por Prión/fisiopatología , Priones/análisis , Animales , Encéfalo/ultraestructura , Cricetinae , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Microscopía Inmunoelectrónica , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Tejido Nervioso/metabolismo , Tejido Nervioso/ultraestructura , Unión Neuromuscular/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/ultraestructura , Enfermedades por Prión/metabolismo , Priones/fisiología , Isoformas de Proteínas/análisis , Isoformas de Proteínas/ultraestructura , Ovinos , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Cerebral cortex biopsy from a patient with new variant Creutzfeldt-Jakob disease (nvCJD) has been examined at the electron microscope level. Spongiform changes corresponded mostly to distended neurites scattered in the neuropil or surrounding amyloid plaques. These latter exhibited heterogeneous submicroscopic morphology including variable amount of loosely interwoven amyloid fibrils admixed in a cellular-rich environment constituted essentially by abnormal neuronal processes. By immunoelectron microscopy, fibrils and some membrane structures reacted with anti-prion protein (PrP) antibodies. One striking aspect was the presence of many small dystrophic neurites without paired helical filaments. Moreover, amyloid fibrils showed unexpected intimate association with abnormal membranes, suggesting a relationship between PrP fibrillogenesis and membrane alteration. These ultrastructural findings provide an additional criterion to distinguish nvCJD-from sporadic CJD-type plaques and reinforce the hypothesis that nvCJD brain is infected by a distinctive strain of the transmissible agent encephalopathy.
Asunto(s)
Encéfalo/patología , Encéfalo/ultraestructura , Síndrome de Creutzfeldt-Jakob/patología , Placa Amiloide/patología , Placa Amiloide/ultraestructura , Adulto , Humanos , Masculino , Microscopía ElectrónicaRESUMEN
We analyzed the distribution and organization of the pathological prion protein isoform (PrPsc) in the brain of new variant Creutzfeldt-Jakob disease using a sensitive post-embedding immunogold electron microscopy method. On methacrylate semithin sections, silver-PrP staining showed florid plaques, containing microvacuoles. It also revealed scattered granular and perivacuolar deposits. At the electron microscope level, plaque PrP-gold labeling was associated with filaments and flocculent amorphous material sometimes observed inside microvacuoles, considered as degenerative neurites. Outside the plaques, PrP-gold labeling was predominantly found over flocculent amorphous material and the presynaptic domain of synapses. Some lysosome-like organelles seen in the neuron perikaryon, were also found to be PrP-immunoreactive. These results are consistent with the hypothesis that the synapse is a privileged target in prion disease.
Asunto(s)
Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Proteínas PrPSc/metabolismo , Adulto , Encéfalo/ultraestructura , Síndrome de Creutzfeldt-Jakob/patología , Humanos , Inmunohistoquímica , Microscopía Inmunoelectrónica , Orgánulos/metabolismo , Orgánulos/ultraestructura , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Distribución Tisular/fisiología , Vacuolas/metabolismo , Vacuolas/ultraestructuraRESUMEN
We examined the localization of the normal cellular isoform of prion protein (PrPc) in mammalian skeletal muscle. Using two anti-PrP antibodies, the neuromuscular junction (NMJ) was preferentially stained after immunohistofluorescence. The mouse, hamster, and human NMJ displayed a fluorescent signal specific for PrPc. Postembedding immunoelectron microscopy analysis performed in the mouse muscle showed that the PrPc-specific colloidal gold immunolabelling was concentrated over the sarcoplasmic cytoplasm. The membrane of the postsynaptic domain was devoid of gold particles, while a weak signal was occasionally observed close to the presynaptic vesicles of the terminal axons. These results indicate that the PrP gene is expressed in mammalian muscle at the NMJ. The subsynaptic sarcoplasm of the NMJ appears to be the privileged site where PrPc presumably associated with endosome membrane may play a role in either physiological activity or maintenance of the morphological integrity of the synapse.
Asunto(s)
Unión Neuromuscular/metabolismo , Unión Neuromuscular/ultraestructura , Priones/metabolismo , Animales , Western Blotting , Cricetinae , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Mesocricetus , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Microscopía Electrónica , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestructura , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructuraRESUMEN
In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.
Asunto(s)
Proteínas PrPC/metabolismo , Animales , Química Encefálica , Cricetinae , Humanos , Mesocricetus , Microscopía Inmunoelectrónica/métodos , Especificidad de Órganos , Fracciones Subcelulares/química , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/ultraestructura , Adhesión del TejidoRESUMEN
The cellular prion protein (PrPc) is a membrane sialoglycoprotein synthesized in the central nervous system and extraneural tissues. Its post-translational modification produces an accumulation of abnormal isoform PrPsc found in brains of transmissible neurodegenerative disorders in animals (scrapie and bovine spongiform encephalopathy) and humans (Kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome). One major unanswered question relative to PrPc concerns its physiological role in brain neurons, depending largely on the limited knowledge of its subcellular localization. Using a highly-sensitive immunogold electron microscopy technique, we reported that in the hamster dentate gyrus, the synaptic boutons constituted the submicroscopic site where PrPc was observed. This detection was obtained with 2 highly-specific polyclonal antibodies for prion protein. PrPc localization was assigned, both on structural basis and on its co-localization with synaptophysin. The presence of PrPc in synaptic terminals should provide additional informations on its possible role in neuronal transmission and on the implication of synapses in the pathogenesis of spongiform encephalopathies.
Asunto(s)
Hipocampo/ultraestructura , Terminales Presinápticos/química , Priones/análisis , Animales , Anticuerpos , Cricetinae , Inmunohistoquímica , Sinaptofisina/inmunologíaRESUMEN
The analysis of human immunodeficiency virus type 1 (HIV-1) RNA sequences in CEM and Jurkat lymphoid cells infected with the virus has been performed at the subcellular level. Using a biotinylated DNA probe specific for HIV-1, virus RNA sequences were detected on Lowicryl thin sections after immunogold cytochemistry. The labelling observed on the cytoplasm was localized near the plasma membrane connected with extracellular cluster of virions. On free immature and nascent form of the virus the detection of HIV-1 RNA was associated with the peripheral electron-dense structure, whereas on mature form the labelling was concentrated on the central nucleoid known to be the site of the HIV-1 genomic RNA. The identification of virus RNA was also performed simultaneously with the detection of HIV-1 core protein p24 or p17 using a double immunogold labelling. Whereas the HIV-1 RNA showed again a cytoplasmic and virions localization, the structural protein was only observed on viral formations. The cytoplasmic localization of virus RNA, at the time of virus production, suggests that they are of genomic origin destined to be packaged in virions once the assembly of virus structural proteins has taken place in the plasma membrane at the viral budding site. The present molecular investigation conducted at the subcellular level provides insight into the cell periphery distribution of HIV-1 RNA observed at the light microscope as corresponding to the detection of HIV-1 infected lymphoid cells actually releasing virions.
Asunto(s)
VIH-1/química , ARN Viral/análisis , Proteínas del Núcleo Viral/análisis , Biotina , Línea Celular/microbiología , Membrana Celular/microbiología , Sondas de ADN , VIH-1/ultraestructura , Inmunohistoquímica , Hibridación in SituRESUMEN
Using several HIV-1-specific antibodies and the immunogold labelling technique, we have detected and localized distinct viral proteins on ultrathin sections of HIV-1 infected cells embedded in the Lowicryl K4M resin. Monoclonal antibodies (MAbs) against p24, p17 and gp160/gp41 showed a preferential labelling on viral formations still attached to the cell membrane (budding process) or free in the extracellular space. The anti-p24 and the anti-p17 MAb yielded a gold labelling not only on mature but also on immature virions where the gold particles were associated with the ring-like electron-dense material. The three HIV-1-specific antibodies against p24, p17 and p55 yielded a cross-reaction with HIV-2 in agreement with the conservation of the internal antigenic determinants of both viruses. In all instances, there was no specific immunogold labelling over the cells, suggesting that once the virus structural proteins were synthesized, they were promptly utilized for virus assembly at the plasma membrane level.
Asunto(s)
Infecciones por VIH/microbiología , VIH-1/química , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica/métodos , Proteínas Estructurales Virales/aislamiento & purificación , Resinas Acrílicas , Anticuerpos Monoclonales , Oro , Anticuerpos Anti-VIH , VIH-1/inmunología , VIH-1/ultraestructura , Humanos , Microtomía , Células Tumorales CultivadasRESUMEN
Human mitochondrial transcripts have been examined at the ultrastructural level. After contact with ultrathin sections of a human lymphoid cell line (CEM) embedded in Lowicryl K4M, biotinylated mitochondrial probes yield specific hybrids identified by a colloidal gold immunocytochemistry marker that visualizes rRNA and mRNA coding for respiratory chain polypeptides CO II, CO III and ATPase-6. The mitochondrial transcripts are preferentially located close to the inner membrane, particularly the cristae, suggesting that intra-organelle protein synthesis is intimately associated with the mitochondrial membrane system. Quantitative analysis indicates that the mitochondria concentrate the labeling with intensities that vary with the type of RNA and that the nucleus induces a light hybridization signal with each mitochondrial probe. The visualization of human mitochondrial DNA expression in correlation with the fine anatomy of the mitochondria constitutes a new approach for fundamental research on the organelle and for analyzing its behaviour in human mitochondrial diseases.
Asunto(s)
Mitocondrias/fisiología , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , ARN Ribosómico/análisis , Adenosina Trifosfatasas/genética , Complejo IV de Transporte de Electrones/genética , Humanos , Inmunohistoquímica , Membranas/ultraestructura , Microscopía Inmunoelectrónica , Mitocondrias/ultraestructura , ARN Mensajero/aislamiento & purificación , ARN Ribosómico/aislamiento & purificación , Células Tumorales CultivadasRESUMEN
We describe a procedure for detecting and localizing cellular rRNA and HIV virus RNA, based on in situ hybridization at the ultrastructural level. After contact with ultrathin sections of cells embedded in Lowicry K4M, biotinylated DNA probes yield specific hybrids identified by a colloidal gold immunocytochemistry marker. The cellular transcripts are preferentially located in the ribosome structures of the cytoplasme and in the dense fibrillar component of the nucleolus. With the HIV probe, viral RNA molecules, were observed over infected cell and some extracellular viral particles. In the cytoplasm the hybridization signal is detected frequently at the periphery close to the plasma membrane, whereas the gold signal associated with virions is localized mainly over the center (core) of the particle.
Asunto(s)
Sondas de ADN , VIH/genética , ARN Ribosómico/análisis , ARN Viral/análisis , Biotina , Humanos , ARN Ribosómico/ultraestructura , ARN Viral/ultraestructuraRESUMEN
A genomic probe containing ribosomal sequences and labelled with biotin was used to hybridize rRNA molecules in ultrathin sections of animal cells embedded in Lowicryl K4M. After detection with streptavidin conjugated with 10 nm gold particles, ribosomal target sequences were localized preferentially in the dense fibrillar component of the nucleolus and in the polyribosome structures of the cytoplasm. The method presently described offers the possibility to detect rapidly and precisely ribosomal gene expression at the ultrastructural level, particularly under different physiological and pathological conditions.