Uninfected Brains Research Articles

Sites of prion protein accumulation in scrapie‐infected mouse spleen revealed by immuno‐electron microscopy

Prion protein (PrP) from the brains of animals with transmissible spongiform encephalopathies is partially protease resistant (PrPres) compared with fully sensitive PrP (PrPsen) from uninfected brains. In most experimental models, PrPres is a reliable indicator of infectivity. Light microscopic studies have suggested that both PrPsen and disease-specific accumulations of PrP are associated with follicular dendritic cells (FDCs). Using immunogold electron microscopy, this study has demonstrated disease-specific accumulation of PrP in the spleens of C57 BL mice, 70 days after intracerebral infection with the ME7 strain of scrapie and at the terminal stage of disease at 170 days. At both stages, tingible body macrophages contained PrP within lysosomes and PrP was also detected at the plasmalemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labyrinthine glomerular complexes, within which PrP was consistently seen between FDC processes in association with abundant electron dense material, interpreted as antigen–antibody complexes. Within some glomeruli, fibrillar forms of PrP consistent with amyloid were seen. At 70 days after challenge, large or hyperplastic labyrinthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC processes at this stage. The ubiquitous accumulation of extracellular PrP in complex glomerular dendrites of FDCs in spleens from terminally affected mice, contrasted with simple FDC profiles, sparse PrP and limited electron dense deposits in all but a few FDCs of 70-day post-infected mice. This suggests that FDCs continually release PrP from the cell surface, where it is associated with trapped antigen–antibody complexes and dendritic extension. It is likely that tingible body macrophages acquire PrP following phagocytosis of PrP within iccosomes or from the extracellular space around FDC dendrites. These studies would not support an intracellular phase of PrP accumulation in FDCs but show that PrP is produced in excess by scrapie-infected cells from where it is released into the extracellular space. We suggest that PrPsen is involved in dendritic extension or in the process of antibody–antigen trapping, perhaps as part of the binding mechanism for antigen–antibody complexes. © Crown copyright 2000. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.

Sites of prion protein accumulation in scrapie-infected mouse spleen revealed by immuno-electron microscopy.

Prion protein (PrP) from the brains of animals with transmissible spongiform encephalopathies is partially protease resistant (PrP(res)) compared with fully sensitive PrP (PrP(sen)) from uninfected brains. In most experimental models, PrP(res) is a reliable indicator of infectivity. Light microscopic studies have suggested that both PrP(sen) and disease-specific accumulations of PrP are associated with follicular dendritic cells (FDCs). Using immunogold electron microscopy, this study has demonstrated disease-specific accumulation of PrP in the spleens of C57 BL mice, 70 days after intracerebral infection with the ME7 strain of scrapie and at the terminal stage of disease at 170 days. At both stages, tingible body macrophages contained PrP within lysosomes and PrP was also detected at the plasmalemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labyrinthine glomerular complexes, within which PrP was consistently seen between FDC processes in association with abundant electron dense material, interpreted as antigen-antibody complexes. Within some glomeruli, fibrillar forms of PrP consistent with amyloid were seen. At 70 days after challenge, large or hyperplastic labyrinthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC processes at this stage. The ubiquitous accumulation of extracellular PrP in complex glomerular dendrites of FDCs in spleens from terminally affected mice, contrasted with simple FDC profiles, sparse PrP and limited electron dense deposits in all but a few FDCs of 70-day post-infected mice. This suggests that FDCs continually release PrP from the cell surface, where it is associated with trapped antigen-antibody complexes and dendritic extension. It is likely that tingible body macrophages acquire PrP following phagocytosis of PrP within iccosomes or from the extracellular space around FDC dendrites. These studies would not support an intracellular phase of PrP accumulation in FDCs but show that PrP is produced in excess by scrapie-infected cells from where it is released into the extracellular space. We suggest that PrP(sen) is involved in dendritic extension or in the process of antibody-antigen trapping, perhaps as part of the binding mechanism for antigen-antibody complexes. Reproduced with the permission of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.

Molecular analysis of prion protein (PrP) and glial fibrillary acidic protein (GFAP) transcripts in experimental Creutzfeldt-Jakob disease in mice.

Prion protein (PrPsc) which accumulates in the brains affected with subacute spongiform encephalopathies (SSE) is altered isoform of normal, cellular isoform (PrPc), and PrP deposition is accompanied with spongiosis and astrogliosis. To find the amounts of PrP and GFAP transcripts during progression of experimental Creutzfeldt-Jakob disease we performed comparative RT-PCR on the terminally sick mice brains, 22 weeks following inoculation with Fujisaki strain of CJD agent, and on control brains. The intensity of bands for PrP-mRNA and control beta-actin were similar for infected and uninfected brains, while amounts of transcripts for GFAP increased as for cytokines released by glial cells-TNF-alpha and IL-1 alpha. This study supports thesis that PrPc to PrPsc conversion is post-translational process not related to PrP overproduction. Increased amounts of GFAP-mRNA during the course of the disease correlated with astrocytosis estimated by immunohistochemistry with anti-GFAP antibody.

Regional mapping of prion proteins in brain.

Scrapie is characterized by the accumulation of a protease-resistant isoform of the prion protein PrPSc. Limited proteolysis and chaotropes were used to map the distribution of PrPSc in cryostat sections of scrapie-infected brain blotted onto nitrocellulose membranes, designated histoblots. Proteolysis was omitted in order to map the cellular isoform of the prion protein (PrPC) in uninfected brains. Compared with immunohistochemistry, histoblots increased the sensitivity for PrPSc detection and showed different patterns of PrPSc accumulation. In Syrian hamsters with Sc237 scrapie, the most intense PrPSc signals occurred in sites with relatively little PrPC, suggesting that aberrant localization of prion protein may be an important feature in the pathogenesis of prion diseases. Immunostaining of PrPSc in white-matter tracts suggested that prions spread along neuroanatomical pathways. PrPSc immunostaining in histoblots was quantitated by densitometry, permitting assessment of the extent of PrPSc accumulation within specific structures. Histoblots were also useful in localizing PrPCJD and beta/A4-amyloid peptide in the brains of patients with Creutzfeldt-Jakob disease and Alzheimer disease, respectively.

Immortality of cell cultures derived from brains of mice and hamsters infected with Creutzfeldt-Jakob disease agent.

Isolates from six patients with Creutzfeldt-Jakob disease (CJD) were injected into various strains of hamsters and mice, and the infective agent was propagated. Serially passaged cultures were established from these CJD agent-infected brains and from uninfected control brains. All healthy cultures (21 out of 21) from CJD agent-infected brains became immortal and/or transformed. In contrast only 3 out of 13 normal brain cultures became immortal, and the rest died out with serial propagation in vitro. The fact that permanent cell lines were readily derived from multiple rodent strains and all CJD isolates tested suggests that a transforming capability is an intrinsic property of CJD agents. This conclusion is supported by demonstrations of in vitro cell transformation by CJD infectious brain fractions. Although the molecular mechanism of transformation events associated with the CJD agent is not presently known, a provocative possibility is that the CJD agent has a direct effect on the host genome by mechanisms analogous to those known for slowly oncogenic retroviruses.

A 54-kDa normal cellular protein may be the precursor of the scrapie agent protease-resistant protein.

Scrapie is the best understood of the transmissible spongiform encephalopathies. These neurologic disorders include the human diseases kuru and Creutzfeldt-Jakob disease and are caused by pathogens with unique biological and molecular properties. One major protein, protease-resistant protein (PrP)-27-30, is present in fractions isolated from scrapie-infected hamster brain that contain highly purified scrapie agent. PrP-27-30 appears to be the major protein component of the hamster scrapie agent. An antiserum generated to electrophoretically purified hamster scrapie PrP-27-30 identified higher molecular weight proteins in immunoblots of homogenates of uninfected hamster and mouse brains. Antibodies to hamster and mouse scrapie agent proteins were obtained by immunoaffinity purification of this antiserum. These antibodies to hamster and mouse PrPs recognized a 54-kDa protein present in uninfected brain homogenates. Antibodies immunoaffinity purified from this antiserum using whole immunoblots of normal brain antigens also identified the 54-kDa protein and PrPs. Our findings demonstrate that scrapie agent proteins share epitopes with normal proteins and suggest that the 54-kDa protein is the normal protein precursor of the scrapie agent PrPs.

Separation and properties of cellular and scrapie prion proteins.

Purified preparations of scrapie prions contain a sialoglycoprotein of Mr 27,000-30,000, designated PrP 27-30, which is derived from the scrapie prion protein [Mr, 33,000-35,000 (PrP 33-35Sc)] by limited proteolysis. Under these same conditions of proteolysis, a cellular protein of the same size (PrP 33-35C) is completely degraded. Subcellular fractionation of hamster brain showed that both PrP 33-35Sc and PrP 33-35C were found only in membrane fractions. NaCl, EDTA, and osmotic shock failed to release the prion proteins from microsomal membranes. Electron microscopy of these microsomal fractions showed membrane vesicles but not prion amyloid rods. Detergent treatment of scrapie-infected membranes solubilized PrP 33-35C, while PrP 33-35Sc aggregated into amyloid rods; the concentration of PrP 33-35C was similar to that recovered from analogous fractions prepared from uninfected control brains. The apparent amphipathic character of the PrP 33-35Sc may explain the association of scrapie infectivity with both membranes and amyloid filaments.

Antibody to Scrapie-associated Fibril Protein Identifies a Cellular Antigen

Antibody to scrapie-associated fibril (SAF) protein was produced in a rabbit by a primary injection of 50 brain equivalents of native SAF protein emulsified in complete Freund's adjuvant and by a secondary inoculation of polyacrylamide gel which contained 50 brain equivalents of SAF protein emulsified with incomplete Freund's adjuvant. The antibody produced was found to decorate the SAF as demonstrated by the use of goat anti-rabbit colloidal gold second antibody. Immunoblots (dot blots and Western blots) revealed that the antibody reacted with scrapie hamster brain extracts but not with age-matched normal hamster brain extracts. However, the antibody also identified an immunoreactive host cellular protein with an apparent mol. wt. of 28 000 from unextracted brain homogenates of both uninfected and scrapie-infected hamster and mouse brains up to a dilution of 1/10(5) brain equivalent. The SAF purification procedures caused selective enrichment of SAF proteins with selective elimination of this immunoreactive cellular antigen. It may be possible that SAFs are composed of a host cellular protein which has been changed in such a way that it aggregates non-covalently to form fibrils and becomes partially resistant to protease digestion.

Characterization of nucleic acids in membrane vesicles from scrapie-infected hamster brain

This study reports the partial characterization of nucleic acids present in gradient fractions enriched for large membrane vesicles from scrapie-infected and uninfected hamster brains. Labeling of phenol-extracted nucleic acids at the 3' or 5' ends revealed abundant amounts of low-molecular-weight RNA and little or no DNA. These nucleic acids survived nuclease treatment of membrane vesicles but were sensitive to RNase after phenol extraction. Analysis of 5'-end-labeled nucleic acids by one- and two-dimensional gel electrophoresis revealed an RNA of ca. 100 bases in preparations from scrapie-infected hamster brain that could not be detected in uninfected brain. The possibility that this apparently unique small RNA may result from tissue damage or abnormal RNA processing or may be a component of the infectious complex is discussed.

Characterization of lipids in membrane vesicles from scrapie-infected hamster brain.

The lipid compositions of membrane vesicles from scrapie-infected and uninfected hamster brains were examined before and after detergent extraction. No differences were observed in polar lipids, glycolipids, gangliosides or neutral lipids examined by thin-layer chromatography. Analysis of detergent-extracted CsCl gradient fractions with high scrapie infectivity failed to reveal any glycerolphosphatides, although neutral lipids were demonstrated. The major neutral lipid associated with detergent-extracted membrane vesicles from both infected and uninfected brain was an unidentified lipid which was found to absorb u.v. radiation strongly from 250 to 300 nm wavelengths. Membrane neutral lipids that strongly absorb u.v. radiation at wavelengths normally used to inactivate viruses may protect a small nucleic acid essential for scrapie infectivity.

Antibodies to a scrapie prion protein.

Scrapie is a slow infection of the nervous system which progresses in the absence of any apparent immune response. The recent development of a large-scale purification protocol for scrapie prions made it possible to obtain substantial quantities of electrophoretically purified prion protein (PrP 27-30) and we report here on the successful production of a rabbit antiserum to PrP 27-30. The antiserum reacted with PrP 27-30 and several lower molecular weight proteins as shown by Western blots; it did not react with protein preparations from uninfected brains. Discrete structures in the subependymal region of scrapie-infected hamster brains were stained immunocytochemically. These same structures also stained with Congo red dye and showed green birefringence with polarized light, a characteristic of purified prion rods. This staining pattern suggests that they are amyloid plaques.