Brains Of Scrapie-infected Mice Research Articles

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice

The presence of cytokines, prostaglandins and lipocortin-1 was investigated in terminally affected mice in two models of scrapie. There was marked induction of glial interleukin-1β, tumour necrosis factor α, prostaglandin E 2, prostaglandin F 2α and lipocortin-1 immunoreactivity in those areas of the brain showing the characteristic vacuolation of scrapie. A comparison of these staining patterns with those of GFAP and F4/80 showed that their expression occurred predominantly in astrocytes. It is possible that cytokines play a significant role in the pathogenesis of neurodegeneration in scrapie.

Activation of cerebral cytokine gene expression and its correlation with onset of reactive astrocyte and acute-phase response gene expression in scrapie

The pathogenesis of scrapie, a transmissible subacute spongiform encephalopathy, is unclear. However, certain aspects of the known cellular and molecular neuropathology in scrapie led us to hypothesize that cytokines could mediate cerebral pathological changes in this neurodegenerative disease. Therefore, expression of multiple cytokine genes in the brain and peripheral organs of scrapie-infected mice was examined. Late in the course of scrapie, expression of tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and IL-1 beta mRNA was markedly increased in the brain but not the spleen, kidneys, or liver. In time course studies, scrapie-infected mice exhibited increased cerebral expression of the TNF-alpha, IL-1 alpha, and IL-1 beta mRNAs by week 15 postinoculation--a time point that coincided with the onset of clinical symptoms. Thereafter, the levels of these cytokine transcripts increased progressively to the terminal stages of of the disease (week 25). To determine the relationship of the increased cerebral expression of the cytokine mRNAs to the development of pathological changes in scrapie, we examined the expression of the glial fibrillary acidic protein gene (a marker for astrocytosis) and the murine acute-phase response gene homologous to the alpha 1-antichymotrypsin gene (designated EB22/5.3). Markedly increased expression of both the glial fibrillary acidic protein and EB22/5.3 mRNAs was observed in the brain but not the peripheral organs of scrapie-infected mice. The increased expression of both these gene products also occurred at week 15 of infection and, thereafter, increased progressively to the terminal stages of the disease. Therefore, infection of mice with scrapie resulted in significant increases in the expression of the TNF-alpha, IL-1 alpha, and IL-1 beta gene products, whose pattern correlated with the onset and development of molecular and clinical pathological changes. Since scrapie is known not to evoke an immune response, the present findings strongly suggest the existence of a localized cerebral host response to the agent during which proinflammatory cytokines could be key pathogenic mediators.

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice : A.E. Williams, A.-M. van Dam ∗, F. Berkenbosch ∗, P. Eikelenboom † and H. Fraser. AFRC/MRC Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, EH9 3JF, Scotland and Departments of ∗Pharmacology and †Psychiatry, Free University, Amsterdam, Netherlands

Cytokines, prostaglandins and lipocortin-1 are present in the brains of scrapie-infected mice : A.E. Williams, A.-M. van Dam ∗, F. Berkenbosch ∗, P. Eikelenboom † and H. Fraser. AFRC/MRC Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, EH9 3JF, Scotland and Departments of ∗Pharmacology and †Psychiatry, Free University, Amsterdam, Netherlands

Astrocytes are the primary source of tissue factor in the murine central nervous system. A role for astrocytes in cerebral hemostasis.

Hemostasis in the brain is of paramount importance because bleeding into the neural parenchyma can result in paralysis, coma, and death. Consistent with this sensitivity to hemorrhage, the brain contains large amounts of tissue factor (TF), the major cellular initiator of the coagulation protease cascades. However, to date, the cellular source for TF in the central nervous system has not been identified. In this study, analysis of murine brain sections by in situ hybridization demonstrated high levels of TF mRNA in cells that expressed glial fibrillary acidic protein, a specific marker for astrocytes. Furthermore, primary mouse astrocyte cultures and astrocyte cell lines from mouse, rat, and human constitutively expressed TF mRNA and functional protein. These data indicated that astrocytes are the primary source of TF in the central nervous system. We propose that astrocytes forming the glia limitans around the neural vasculature and deep to the meninges are intimately involved in controlling hemorrhage in the brain. Finally, we observed an increase in TF mRNA expression in the brains of scrapie-infected mice. This modulation of TF expression in the absence of hemorrhage suggested that TF may function in processes other than hemostasis by altering protease generation in normal and diseased brain.

Detection of proteinase K-resistant prion protein and infectivity in mouse spleen by 2 weeks after scrapie agent inoculation.

The sequential accumulation of the protease-resistant form of the endogenous prion protein (PrP-res) was compared to levels of scrapie infectivity in the spleen and brain of scrapie-infected mice at various times after inoculation. In mouse spleen PrP-res was detected 1 week after inoculation, and increased 65-fold between 1 and 3 weeks post-inoculation and an additional 15-fold during the next 17 weeks. Infectivity in spleen reached a maximum plateau level by 3 weeks. In contrast, in mouse brain PrP-res was not detected until 8 weeks after inoculation and then increased 200-fold during the next 12 weeks. During this same time, infectivity increased approximately 10,000-fold. Therefore, in both spleen and brain of scrapie-infected mice accumulation of PrP-res and infectivity appear to be associated. However, it was not possible to show quantitative correlations between PrP-res detection and infectivity, perhaps owing to the inaccuracy of the infectivity assay.

Variations in prion protein and glial fibrillary acidic protein mRNAs in the brain of scrapie-infected newborn mouse.

To begin to understand the molecular basis of cases of Creutzfeldt-Jakob disease recently described in young children, the expression of prion protein and glial fibrillary acidic protein (GFAP) mRNAs was investigated during the development of the brain of scrapie-infected newborn mice. Changes in the time course of expression were identified by Northern blot quantification between days 1 and 172. Although scrapie-infected and control animals showed no detectable changes in brain development (first 56 days of life), GFAP mRNAs were found to increase significantly as early as day 84. A 10-fold increase in the level of GFAP mRNA was observed in brain between day 112 and death (day 172).

Lysosomes as key organelles in the pathogenesis of prion encephalopathies.

The causation, structural origin, and mechanism of formation of spongiform lesions in transmissible encephalopathies are unknown. We have used immunogold electron microscopy to locate ubiquitin conjugates, hsp 70, and beta-glucuronidase (markers of the lysosomal compartment) and prion protein (PrP) in both control and scrapie-infected mouse brain. In scrapie-infected brain, lysosomes and lysosome-related structures (multivesicular and tubulovesicular dense bodies) are present in abnormally high numbers in neuronal cell processes. These structures contain PrP, together with the lysosomal markers ubiquitin conjugates, hsp 70, and beta-glucuronidase, which could also be identified spilling from tubulovesicular dense bodies into areas of early rarefaction in neuronal processes; we suggest that these areas of rarefaction are the precursor lesions of spongiform change. We advance the hypothesis that spongiform change is brought about by cytoskeletal disruption in neuronal processes caused by liberation of hydrolytic enzymes from lysosomes overloaded with the abnormal isoform of PrP (PrPsc). We suggest that the lysosomal system is probably acting as the bioreactor for processing of normal PrP to the abnormal isoform. The continuous production of increasing quantities of abnormal PrPsc in lysosome-related bodies will eventually cause disruption of the lysosomal membrane with destruction of the neuronal cytoskeleton and the initiation of vacuolation. Later, death of the cell will be associated with release of the PrPsc isoform into the extracellular environment. Repeated rounds of phagocytosis, lysosomal biogenesis of PrPsc, lysosomal membrane rupture, hydrolytic enzyme release, and neuronal lysis will lead to an exponential increase in cell damage and cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

High-voltage electron microscopic studies of the interrelationship between microglial cells and amyloid fibrils in scrapie-infected mouse brains

We have previously demonstrated the usefulness of high-voltage electron microscopy (HVEM) in the study of microvessels and inflammatory cell attachment in the central nervous system (CNS). In the present study, we used HVEM to further explore the interrelationship between microglial cells (MCs) and amyloid deposits in scrapie-infected mice. Scrapie infection in the mouse has been employed as an animal model to study the pathogenesis of amyloid fibril formation. The central question was whether three-dimensional (3-D) stereo-pair reconstruction would offer further insight into amyloid formation by MCs, which is currently the view of our group. Brains or cervical spinal cords from IM mice previously inoculated with 87V scrapie agent were used. One-half-micrometer thick plastic sections were stained with uranyl acetate and lead citrate. Light-microscopy views enabled us to target primary inoculation channels associated with amyloid deposits. Cells located at the periphery of the amyloid were identified as MCs (Fig. 1).

Morphological and biochemical evidence that scrapie-associated fibrils are derived from aggregated amyloid-like filaments

The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected thoughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected hi all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 × 60 nm to more than 4 μm were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10–18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.

Nucleoside diphosphatase (NDPase) activity associated with human ?-protein amyloid fibers

Nucleoside diphosphatase (NDPase) activity was studied by electron microscope cytochemistry in surgical specimens obtained from aged human cerebral cortices. The presence of NDPase activity on the surface of the microglial cells (MCs) and especially within the endoplasmic reticulum (ER) cisternae that are filled with amyloid fibers and that are in continuity with the extracellular amyloid deposits in plaques suggests a possible role of this enzyme in final elaboration of amyloid protein. The close structural relationship between MCs and amyloid plaques, suggesting the participation of these cells in the synthesis or final elaboration of amyloid fibers, was observed. The comparison of these observations with previously reported data on the distribution of NDPase in MCs and amyloid fibers in scrapie-infected mouse brain suggests that presumably similar mechanisms are acting in both cases. These observations, as compared with the results of other cytochemical and biochemical studies, also suggest that co-localization of NDPase activity with newly formed amyloid fibers in plaques can be associated with glycosyltransferase activities engaged in the amyloid or amyloid precursor protein glycosylation.

Ubiquitin conjugate immunoreactivity in the brains of scrapie infected mice

Sections of brain from normal mice or clinically-ill mice infected with either the 87V or the ME7 strains of sheep scrapie were immunostained to show the localization of ubiquitin-protein conjugates or a specific marker of disease, the scrapie-associated fibril protein (PrP). In both scrapie models immunoreactive ubiquitin-protein conjugates were seen in thread-like structures found throughout the neuropil, in inclusion bodies within vacuolated neurones, and in areas surrounding anti-PrP positive amyloid plaques. The PrP protein was visualized in diffuse deposits in highly vacuolated parts of the scrapie-affected brain, and focally in amyloid plaques, microglia and neuronal processes. The ubiquitin-protein conjugate staining of scrapie amyloid plaques is very similar to that seen in the plaques of Alzheimer's disease. The ubiquitinated intraneuronal inclusion bodies seen in scrapie resemble the granulovacuolar lesions also seen in Alzheimer's disease, but appear much larger and possibly correspond to material in giant autophagic vacuoles. We suggest that these inclusions may be the result of ubiquitinated abnormal proteins being directed to the lysosomal system, and that scrapie and Alzheimer's disease share at least some common processes of neurodegeneration.

Ubiquitin conjugate immunoreactivity in the brains of scrapie infected mice : ∗Michael Landon 1, Helen McDermott 1, R. John Mayer 1, James Lowe 2, Moira Bruce 2, Patricia McBride 3, Robert A. Somerville 3, James Hope 3. Departments of Biochemistry 1 and Pathology 2, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, U.K. and AFRC & MRC Neuropathogenesis Unit

Ubiquitin conjugate immunoreactivity in the brains of scrapie infected mice : ∗Michael Landon 1, Helen McDermott 1, R. John Mayer 1, James Lowe 2, Moira Bruce 2, Patricia McBride 3, Robert A. Somerville 3, James Hope 3. Departments of Biochemistry 1 and Pathology 2, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, U.K. and AFRC & MRC Neuropathogenesis Unit

Immunohistochemical localization of prion protein in spongiform encephalopathies and normal brain tissue

We used polyclonal antibodies raised against hamster and mouse PrP27-30 as immunologic probes to study the localization of intracellular and extracellular deposits of prion protein in normal and scrapie-infected mouse and hamster brains and in Creutzfeldt-Jakob disease (CJD)-infected mouse brains. In addition, we examined normal human brain and brain tissues from patients with CJD, kuru, Alzheimer's disease, and idiopathic chronic encephalitis. There was positive staining in the cytoplasm of neurons of normal and scrapie- and CJD-infected mice, and in the neurons of normal and scrapie-infected hamsters. The staining pattern suggests the localization of PrP in an intracellular membrane compartment, most likely the rough endoplasmic reticulum or Golgi apparatus. Antibodies raised against a 15-amino-acid synthetic peptide of the N-terminal of hamster PrP27-30 displayed a similar pattern of staining in mouse brain sections. We observed no intracellular staining in human brain sections obtained at autopsy. Antibodies prepared against mouse and hamster PrP27-30 reacted with amyloid plaques in scrapie-infected mouse and kuru- and CJD-infected human brain sections but not with amyloid plaques in the brain of a patient with Alzheimer's disease.

Changes in the distribution of anionic sites in brain micro-blood vessels with and without amyloid deposits in scrapie-infected mice

Cationic colloidal gold (CCG) and scrapie-infected mouse brain samples embedded in Lowicryl K4M were used for ultrastructural localization of negatively charged microdomains (anionic sites) in the cerebral microvasculature. The distribution of anionic sites on both fronts (luminal and abluminal) of endothelial cells and in the basement membrane (BM) in the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs (especially capillaries), however, located inside the plaque areas and surrounded directly by amyloid fibers contained attenuated endothelium, the luminal surface of which showed a segmental lack or diminution of anionic sites. In these vessels the BM was frequently infiltrated and replaced by the amyloid fibers. In some vessels located mainly in the areas of the neuropil vacuolization deposits of homogenous material causing the thickening of the BM were noted. These changes were accompanied by irregular labeling of the BM with gold particles. At the sites of bifurcation of some MBVs, predominantly in the area of the venular estuary at the mouth of capillary (at capillary-venular connections), a discontinuity in the distribution of anionic sites was noted. The observed disturbances in the distribution of anionic sites can be associated with a previously noted increased permeability of some MBVs in the brains of scrapie-infected mice.

In vitro expression in eukaryotic cells of a prion protein gene cloned from scrapie-infected mouse brain.

It has been proposed that the causative agent of scrapie represents a class of infectious particle that is devoid of nucleic acid and that an altered form of the endogenous prion protein (PrP) is the agent. However, it has been difficult to exclude the possibility that PrP purified from scrapie tissues might be contaminated with a more conventional viral agent. To obtain PrP uncontaminated by scrapie-infected tissues, PrP cDNA cloned from a scrapie-infected mouse brain was expressed in mouse C127 cells in vitro. mRNA and protein encoded by the cloned PrP gene were identified. The expressed PrP polypeptides appeared to be glycosylated and were released from the cell surface into the medium. Homogenates of the cells expressing the cloned PrP gene were inoculated into susceptible mice but failed to induce clinical signs of scrapie. Thus, either PrP is not the transmissible agent of scrapie or the expressed PrP requires additional modification to be infectious.

Effects of the antiserum against a fraction enriched in scrapie-associated fibrils on the scrapie incubation period in mice.

An antiserum against a fraction enriched for scrapie-associated fibrils (SAF), was examined for its effects on scrapie incubation period by inoculating mice either intraperitoneally or intracerebrally with various dilutions of the serum mixed with scrapie-infected mouse brain homogenate. After intraperitoneal inoculation the mean time of the incubation period increased with increasing concentrations of the antiserum in a statistically significant fashion, when the serum dilutions were made with phosphate-buffered saline. After intracerebral inoculation, however, there were no statistically significant differences between the control group and any of the antiserum-groups. When the antiserum dilutions were made with pre-immune serum, the mice inoculated intraperitoneally also showed no significant differences between the two groups. These results indicate that the specific antibodies to SAF have no effect on the scrapie infectivity.

Ultrastructural studies of glycoconjugates in brain micro-blood vessels and amyloid plaques of scrapie-infected mice.

Lectin or glycoprotein-gold complexes and samples of scrapie-infected mouse brain embedded in Lowicryl K4M were used for ultrastructural localization of glycoconjugates. The lectins tested recognize the following residues: beta-D-galactosyl [RCA, Ricinus communis agglutinin (aggl.) 120], N-acetyl and N-glycolyl neuraminic acid (LFA, Limax flavus aggl.), N-acetyl-D-glucosaminyl and sialyl (WGA, Wheat germ aggl.), N-acetyl-D-galactosaminyl (HPA, Helix pomatia aggl., and DBA, Dolichos biflorus aggl.), alpha-D-mannosyl/alpha-D-glucosyl (Con A, Concanavalin A), alpha-D-galactosyl and alpha-D-galactopyranoside (BSA, Bandeirea simplicifolia aggl., izolectin B4). Labeling of the majority of micro-blood vessels (MBVs) located outside the plaque area and in the remaining cerebral cortex was similar to that which has been previously observed in non-infected animals. Some MBVs, however, located inside the plaque area and surrounded directly by amyloid fibers showed attenuation of the endothelium, the surface of which was scarcely and irregularly decorated with RCA, LFA, WGA and Con A. These abnormalities in the composition of glycoconjugates can be associated with previously noted increased permeability of some MBVs in the brains of scrapie-infected mice. Some vessels in the plaque area were encapsulated by perivascular deposits of homogeneous or flocculogranular material containing several glycoconjugates. A very intimate structural relation between reactive (microglial-like) cells and amyloid fibers suggests the participation of these cells in elaboration of plaque material. Labeling of the cell surface and adjacent amyloid fibers with the same lectins (RCA, WGA, DBA, Con A) suggests the possibility that the glycosylation of these fibers occurs extracellularly. Only WGA and DBA were occasionally labeling some Golgi elements of the reactive cells.

Isolation and characterization of macrophages from scrapie-infected mouse brain

We have isolated and characterized a population of brain macrophages from normal and scrapie-infected mice. The cells are phagocytic, possess Fc-IgG receptors, Mac-1 surface antigen and proliferate in the presence of macrophage colony stimulating factor. They resemble microglia in that they have a plasmalemmal distribution of the enzyme nucleoside diphosphatase, a property tht is characteristic of microglia in situ. In two of the three combinations of scrapie agent and mouse strain examined, the number of brain macrophages was several fold higher than in normal control mice. The increase was not observed in mice infected intraperitoneally or in control mice inoculated with normal brain homogenate. The increase is detectable as early as 3-5 weeks postinoculation. The agent/host combination that failed to show an increase in brain macrophages is one that develops large numbers of amyloid plaques. These observations suggest that these cells are closely associated with the scrapie pathogenic process in the CNS. The failure of these cells to increase in the plaque forming model of scrapie disease also suggests that they play a role in the control of CNS amyloidogenesis.

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.