RESUMO
The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP(c)). This pathological isoform (PrP(Sc)) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP(Sc) in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP(Sc)-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP(c) expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP(Sc) in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.
Assuntos
Fragmentos de Peptídeos/farmacocinética , Príons/farmacocinética , Animais , Contagem de Células , Células Cultivadas , Cerebelo/citologia , Cerebelo/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/química , Microglia/ultraestrutura , Microscopia Confocal , Microscopia Eletrônica , Neurofibrilas/metabolismo , Neurofibrilas/ultraestrutura , Neurônios/química , Neurônios/ultraestrutura , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/metabolismo , Príons/análise , Príons/metabolismoRESUMO
Clusterin mRNA, detected in increased quantities in the cervical spinal cord of cattle with bovine spongiform encephalopathy (BSE), was localized mainly in the neuroglia (including astrocytes) of the lateral and ventral areas of white matter. Axonal degeneration was also observed in these areas. The dorsal horns of the spinal cord in which BSE prion protein (PrP(BSE)) was deposited did not exhibit strong clusterin "up-regulation" but showed increased clusterin immunolabelling with a punctate distribution in the neuropil. Labelling of adjacent sections of the grey matter in BSE-affected spinal cord and thalamus demonstrated that the clusterin was deposited in association with extracellular PrP(BSE).
Assuntos
Sistema Nervoso Central/metabolismo , Encefalopatia Espongiforme Bovina/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares , Animais , Northern Blotting , Bovinos , Contagem de Células , Sistema Nervoso Central/patologia , Clusterina , Encefalopatia Espongiforme Bovina/patologia , Imuno-Histoquímica , Hibridização In Situ , Proteínas do Tecido Nervoso/metabolismo , Neurópilo/metabolismo , Príons/metabolismo , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Tálamo/metabolismoRESUMO
The prion/amyloid neuropeptide 106-126 spontaneously aggregates to form fibrillar structures in vitro. The aggregation in vitro could be prevented in a dose-related manner by clusterin, and the specificity of this action was confirmed by reversal with antibody to clusterin. The relevance of these observations is discussed in relation to previous observations that clusterin and PrPBSE colocalise in naturally occurring cases of BSE.