Brains Of Scrapie-infected Mice Research Articles

Evidence for Oxidative Stress in Experimental Prion Disease

Oxidative stress has been shown to be important in several neurodegenerative disorders. Previous in vitro studies have already demonstrated the ability of a prion protein fragment to induce oxidative stress in cultured cells. By immunohistochemistry for nitrotyrosine (NT) and heme oxygenase-1 as markers for oxidative stress, we found widespread neuronal labeling for NT in scrapie-infected mouse brains, in agreement with peroxynitrite mediated neuronal degeneration. Damage by free radicals is a likely cause for neurodegeneration in prion disease, and antioxidants are a potential therapy of these disorders.

Overexpression of heme oxygenase-1 and ferritin in the brains of scrapie-infected mice

Overexpression of heme oxygenase-1 and ferritin in the brains of scrapie-infected mice

Overexpression of cyclooxygenase-2 MRNA and protein in the brain of scrapie-infected mice

Overexpression of cyclooxygenase-2 MRNA and protein in the brain of scrapie-infected mice

Increased ferric iron content and iron-induced oxidative stress in the brains of scrapie-infected mice

Increased ferric iron content and iron-induced oxidative stress in the brains of scrapie-infected mice

Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapie-infected mice

A number of aspects of the pathogenesis of scrapie remain to be elucidated. The cellular and molecular aspects of the neuropathology in scrapie suggest the possibility that the proinflammatory cytokines could act as pathogenic mediators in this neurodegenerative disease. To understand this possibility, we examined the expression of proinflammatory cytokine genes in brains of IM mice-infected with 87V scrapie agent. Additionally, we also analyzed the activity of nuclear factor-kappa B (NF-κB), which is the major transcriptional activator for inflammatory cytokines, and formation of reactive oxygen species (ROS) as a common upstream messenger for its activation. The induction of mRNAs of the inflammatory cytokines, IL-1α, IL-1β and TNF-α, was detected only in the brains of scrapie-infected mice. The activity of NF-κB was significantly increased in the nuclear extracts from brains of the scrapie-infected group and the immunoreactivity of NF-κB was increased in the hippocampus and thalamus in the brains of scrapie-infected mice. The NF-κB immunoreactivity was observed mainly in GFAP-positive astrocytes and also detected in the PrP-amyloid plaques in the brains of 87V scrapie-infected mice. Gene expression of IL-6 and iNOS, the representative target genes for NF-κB activation, were activated only in the infected group. The production of ROS was significantly increased in the brain mitochondrial fractions of scrapie-infected mice. These results suggest that prion accumulation in astrocytes might activate NF-κB through the increase of ROS generation, and thus alterations in NF-κB-directed gene expression may contribute to both the neurodegeneration and proinflammatory responses which occur in scrapie.

Increased expression of CaM kinase II alpha in the brains of scrapie-infected mice

We investigated the distribution of calcium/calmodulin-dependent protein kinase II (CaM kinase II) in the brains of mice infected with ME7 scrapie strain. CaM kinase II is an enzyme that plays a major role in the regulation of long-term potentiation, a form of synaptic plasticity associated with learning and memory. Immunoreactivity of CaM kinase II α, measured by Western blot, increased markedly in scrapie-infected brains compared with control brains. Immunohistochemically, CaM kinase II α immunoreactivity was upregulated in the cerebral cortex and hippocampal CA1 area of scrapie-positive mice infected with ME7 scrapie strain. This result implies that this enzyme is associated with aberrant function of synaptic transmission and LTP of the pyramidal neurons in the hippocampal CA1 area of mice infected with ME7 scrapie strain.

Infectivity of Scrapie Prions Bound to a Stainless Steel Surface

The transmissible agent of Creutzfeldt-Jakob disease (CJD) is not readily destroyed by conventional sterilization and transmissions by surgical instruments have been reported. Decontamination studies have been carried out thus far on solutions or suspensions of the agent and may not reflect the behavior of surface-bound infectivity. As a model for contaminated surgical instruments, thin stainless-steel wire segments were exposed to scrapie agent, washed exhaustively with or without treatment with 10% formaldehyde, and implanted into the brains of indicator mice. Infectivity was estimated from the time elapsing to terminal disease. Stainless steel wire (0.15 x 5 mm) exposed to scrapie-infected mouse brain homogenate and washed extensively with PBS retained the equivalent of about 10(5) LD50 units per segment. Treatment with 10% formaldehyde for 1 hr reduced this value by only about 30-fold. The model system we have devised confirms the anecdotal reports that steel instruments can retain CJD infectivity even after formaldehyde treatment. It lends itself to a systematic study of the conditions required to effectively inactivate CJD, bovine spongiform encephalopathy, and scrapie agent adsorbed to stainless steel surfaces such as those of surgical instruments.

Scrapie-infected mice and PrP knockout mice share abnormal localization and activity of neuronal nitric oxide synthase.

PrP(Sc), the only identified component of the scrapie prion, is a conformational isoform of PrPc. The physiological role of PrPc, a glycolipid-anchored glycoprotein, is still unknown. We have shown previously that neuronal nitric oxide synthase (nNOS) activity is impaired in the brains of mice sick with experimental scrapie as well as in scrapie-infected neuroblastoma cells. In this work we investigated the cell localization of nNOS in brains of wild-type and scrapie-infected mice as well as in mice in which the PrP gene was ablated. We now report that whereas in wild-type mice, nNOS, like PrPc, is associated with detergent-insoluble cholesterol-rich membranous microdomains (rafts), this is not the case in brains of scrapie-infected or in those of adult PrP(0/0) mice. Also, adult PrP(0/0), like scrapie-infected mice, show reduced nNOS activity. We suggest that PrPc may play a role in the targeting of nNOS to its proper subcellular localization. The similarities of nNOS properties in PrP(0/0) as compared with scrapie-infected mice suggest that at least this role of PrPc may be impaired in scrapie-infected brains.

Characterization of the Human Analogue of a Scrapie-responsive Gene

We have recently described a novel mRNA denominated ScRG-1, the level of which is increased in the brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J.-P., Lasmézas, C., Dormont, D., Tovey, M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain follows the accumulation of PrPSc, the proteinase K-resistant form of the prion protein (PrP), and precedes the widespread neuronal death that occurs in late stage disease. In the present study, we have isolated a cDNA encoding the human counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal tissues revealed that the gene was expressed primarily in the central nervous system as a 0.7-kilobase message and was under strict developmental control.

Extensive degeneration of catecholaminergic neurons to scrapie agent 87V in the brains of IM mice

Scrapie is a degenerative disease of the central nervous system of sheep and goats. The causative agent has been passaged to a number of laboratory species, including mice and hamster. Amyloid plaque formation and vacuolation, the signs of senile dementia, are found in the brains of mice infected with 87V scrapie agent. Dopamine (DA) and norepinephrine (NE) concentrations in the brains of scrapie-infected mice were measured with high-performance liquid chromatography-electrochemical detector (HPLC-ECD). A significant decrease in NE level was exhibited in all regions tested, whereas the level of DA decreased significantly only in cerebral cortex. Immunohistochemistry was used to examine immunoreactive catecholamine neurons in substantia nigra and locus ceruleus using antisera against tyrosine hydroxylase (TH). The population of TH-immunoreactive neurons in the substantia nigra and locus ceruleus were significantly decreased in scrapie-infected mice compared to controls. These data suggest that both the noradrenergic and dopaminergic system are sensitive to the action of scrapie agent 87V and that changes in the catecholamine levels in the brains of scrapie-infected mice may contribute to some of the clinical symptoms of the diseases, such as ataxia and apraxia.

Gene Expression in Scrapie: CLONING OF A NEW SCRAPIE-RESPONSIVE GENE AND THE IDENTIFICATION OF INCREASED LEVELS OF SEVEN OTHER mRNA TRANSCRIPTS

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

Expression of inducible nitric oxide synthase in the brains of scrapie-infected mice.

The neuronal cell damage caused by inducible nitric oxide synthase (iNOS) in brain has been reported to be associated, at least in part, with many neurodegenerative diseases including Alzheimer's disease. We recently observed vacuolation and astrocytosis in the brains of ME7 scrapie strain-infected C57BL mice. To investigate if these phenomena might have a relationship to iNOS, the level of iNOS expression was measured immunohistochemically and molecular biologically in the brains of scrapie-infected C57BL mice. The number and size of astrocytes were increased and immunoreactivity of glial fibrillary acidic protein (GFAP) was significantly enhanced. iNOS immunoreactivity was observed in the astrocytes of the scrapie-infected group, but not in the control group. iNOS mRNA levels were increased in scrapie-infected mice compared to the levels in non-infected mice of the same age. Our results suggest that iNOS induction in reactive astrocytes is a part of the neurodegenerative mechanisms in scrapie infection.

The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells.

Prions are thought to consist of infectious proteins that cause transmissible spongiform encephalopathies. According to overwhelming evidence, the pathogenic prion protein PrPSc converts its host encoded isoform PrPC into insoluble aggregates of PrPSc, concomitant with pathological modifications (for review, see refs. 1-3). Although the physiological role of PrPC is poorly understood, studies with PrP knockout mice demonstrated that PrPC is required for the development of prion diseases. Using the yeast two-hybrid technology in Saccharomyces cerevisiae, we identified the 37-kDa laminin receptor precursor (LRP) as interacting with the cellular prion protein PrPC. Mapping analysis of the LRP-PrP interaction site in S. cerevisiae revealed that PrP and laminin share the same binding domain (amino acids 161 to 180) on LRP. The LRP-PrP interaction was confirmed in vivo in insect (Sf9) and mammalian cells (COS-7). The LRP level was increased in scrapie-infected murine N2a cells and in brain and spleen of scrapie-infected mice. In contrast, the LRP concentration was not significantly altered in these organs from mice infected with the bovine spongiform encephalopathic agent (BSE), which have a lower PrPSc accumulation. LRP levels, however, were dramatically increased in brain and pancreas, slightly increased in the spleen and not altered in the liver of crapie-infected hamsters. These data show that enhanced LRP concentrations are correlated with PrPSc accumulation in organs from mice and hamsters. The laminin receptor precursor, which is highly conserved among mammals and is located on the cell surface, may act as a receptor or co-receptor for the prion protein on mammalian cells.

Immunocytochemical evaluation of blood-brain barrier to endogenous albumin in scrapie-infected mice.

A quantitative immunocytochemical procedure was used for evaluation of the blood-brain barrier (BBB) to endogenous albumin in plaque-forming (PF) and non-plaque-forming (NPF) groups of scrapie-infected mice at the clinical stage of disease. Ultrathin sections of brain samples (cerebral cortex, hippocampus and cerebellum) embedded in resin (Lowicryl K4M) were exposed to anti-mouse albumin antiserum followed by protein A-gold. Using morphometry, the density of immunosignals (gold particles per microns2) was recorded over four compartments: vascular lumen, endothelium, subendothelial space, and brain parenchyma (neuropil). Morphometric and statistical analyses did not reveal significant differences in the barrier function of the microvasculature of the cerebral cortex and hippocampus in either group of mice, although a slight increase in the number of leaking vessels in the PF group was noted. In contrast, in the cerebellum, the permeability of the microvessels to albumin was significantly higher in the PF than in the NPF mouse group, and this was paralleled by the infiltration of the walls of numerous vascular profiles with amyloid deposits (amyloid angiopathy). These data also indicate the existence of distinct regional differences in BBB function in the brain of scrapie-infected mice. The vascular amyloid deposits and the amyloid plaques present in the cerebral cortex of PF mice were labeled with numerous immunosignals suggesting the affinity of extravasated albumin to these deposits. In conclusion, no convincing evidence was obtained indicating that impairment of the BBB, manifested by increased permeability of vascular segments, is directly related to the deposition of amyloid in the vascular wall and in plaques. Segmental impairment of the barrier function seems to be rather the result of disturbed structural integrity of the components of the vascular wall.

Differential effects of a new amphotericin B derivative, MS-8209, on mouse bse and scrapie: implications for the mechanism of action of polyene antibiotics

Mice were infected intracerebrally with the bovine spongiform encephalopathy (BSE) or the scrapie agent and treated during 8 weeks postinfection to test the protective effect of a new amphotericin B (AmB) derivative, MS-8209, in experimental transmissible spongiform encephalopathies. The results show that (i) the treatment prolonged the incubation period of both BSE-infected and scrapie-infected mice, (ii) MS-8209 and AmB were much more efficient in delaying the onset of scrapie than that of BSE, and (iii) a delay in Prp-res (proteinase K-resistant prion protein) and GFAP (glial fibrillary acidic protein) accumulation was observed in the brains of scrapie-infected mice, but was not significant in BSE-infected mice. The analysis of the molecular and clinical results strongly suggests a common mechanism of action of this category of drugs on the different transmissible spongiform encephalopathy strains. This could be due to an interaction with the PrP transconformation process leading to the formation of PrP-res.

Lipid composition in scrapie-infected mouse brain: prion infection increases the levels of dolichyl phosphate and ubiquinone.

The neutral and phospholipid composition of mouse brain infected with scrapie prions was investigated. During the later stages of this disease, the level of dolichol decreased by 30% whereas the level of dolichyl phosphate increased by 30%. In terminally ill mice, there was also a 2.5-fold increase in both total ubiquinone and its reduced form. Furthermore, alpha-tocopherol was elevated at this stage by 50%. In contrast, no changes were observed in phospholipid amount, in phospholipid composition, and in phosphatidylethanolamine plasmalogen content during the entire disease process. The fatty acid and aldehyde composition of individual phospholipids remained unaltered as well. No modifications could be detected in cholesterol content. Thus, the majority of membrane lipids in scrapie-infected mouse brain are modified in neither quantity nor structure, but specific changes occur to a few polyisoprenoid lipids. This specificity indicates that, although prions accumulate in lysosomes, the infection process is not associated with a general membrane destruction caused by lysosomal enzyme leakage.

103 Perturbation of mitochondrial free radical metabolism in the brains of 87V scrapie-infected mice

103 Perturbation of mitochondrial free radical metabolism in the brains of 87V scrapie-infected mice

The abnormal isoform of the prion protein accumulates in late‐endosome‐like organelles in scrapie‐infected mouse brain

The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

Morphogenesis of amyloid plaques in 87V murine scrapie†

Amyloid plaques of scrapie-infected mouse brains are composed of fibrillar forms of a host coded, cell surface sialoglycoprotein called PrP (prion protein). Serial ultrastructural immunogold staining was performed on plaques identified by light microscopic immunocytochemistry of brains of VM mice infected with the 87V strain of scrapie. Classical plaques, of a kuru-type morphology, were composed of a central core of bundles of amyloid fibrils. Amyloid fibrils of classical plaques were immunoreactive for PrP. In addition, PrP was also found at the plaque periphery, in the absence of fibrils, at the plasmalemma of cell processes and in the associated extracellular spaces. Frequent microglial cells and occasional astrocytes contained PrP within lysosomes. Other plaques with few or no recognizable amyloid fibrils were frequent and were termed primitive plaques. PrP could be demonstrated in a non-fibrillar form at the plasmalemma and in the extracellular spaces between neurites of such plaques. Many primitive plaques showed little or no sub-cellular pathology associated with the PrP accumulation. PrP was closely associated with the plasmalemma of occasional dendrites passing towards the centre of primitive plaques. These results suggest that plaques are formed around one or more PrP releasing dendrites. PrP accumulates in the extracellular spaces adjacent to such processes prior to its spontaneous aggregation into fibrils. Lysosomal accumulation of PrP in microglia and astrocytes located at the periphery of plaques suggest that these cells are involved in the phagocytosis of excess or abnormal PrP.

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