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.     

Experimental scrapie in mice: ultrastructural observations

Scrapie, kuru, and Creutzfeldt-Jakob disease are characterized by a similar spongiform pathology, prolonged incubation periods, and an agent with unique physical, chemical, and biological properties. Swiss mice were inoculated with the scrapie agent and sacrificed three to five months later for light and electron microscopy. At three months, small vacuoles were seen within the neuropil of the cerebral cortex and basal ganglia. By the fifth month these vacuoles had increased in number and size and were accompanied by moderate astrocytic proliferation. The brainstem, cerebellum, and spinal cord showed variable changes of much less intensity. Many dilated postsynaptic processes contained osmiophilic particles in random or crystalline arrays. The particles, measuring approximately 23 nm in diameter, appeared consistently in postsynaptic processes of brain from scrapie-infected mice, were lacking in controls, and were a size consistent with sedimentation and filtration data for the scrapie agent. Whether these particles represent the scrapie agent must await further studies.     

Increased blood-brain barrier permeability in scrapie-infected mice

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).     

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

Scrapie is a transmissible neurodegenerative disease of sheep and goats. The neuropathological changes include vacuolation, astrocytosis, the development of amyloid plaques in some instances, and neuronal loss. The mechanisms involved in neuronal cell death in scrapie are not known. Recently, we reported the presence of oxidative stress in the brains of scrapie-infected animals and suggested that this is the main mechanism that induces neuronal cell loss. It is known that oxidative stress induced by free radicals is associated with iron accumulation; this association led to an examination of the levels of iron (total iron, Fe(2+) and Fe(3+)) in the brains of control and scrapie-infected mice by biochemical methods. In the scrapie-infected group, both the level of total iron and the Fe(3+) level were significantly increased in cerebral cortex, striatum, and brainstem as compared to the values in the control group. A shift in the ratio of Fe(2+)/Fe(3+) was observed in the same regions of infected mice. Additionally, in this scrapie model, we confirmed the presence of oxidative stress, as evidenced by the increase of free malondialdehyde. These results suggest that iron metabolism is changed and that iron-induced oxidative stress partly contributes to neurodegeneration in scrapie infection.     

Differential expression of Bax and Bcl-2 in the brains of hamsters infected with 263K scrapie agent

To study the mechanism(s) of neuronal cell death during scrapie infection, we investigated the expression of Bax and Bcl-2 in brains of hamsters infected with 263K scrapie agent. The expression of Bcl-2 mRNA was significantly decreased in the brains of 263K scrapie-infected hamsters compared with controls, whereas the expression levels of Bax mRNA were significantly increased in scrapie-infected brain. The levels of Bax and Bcl-2 proteins in brains of scrapie and control animals reflected the difference in mRNA levels. Immunoreactivity for Bax and Bcl-2 were found predominantly within neurons. In scrapie-infected brains, the number of neuronal cells positive for Bcl-2 was significantly lower in the hippocampal CA3 region and was decreased in the cerebral cortex, whereas the number of neuronal cells positive for Bax was significantly increased in both regions. The possibility that differential regulation of Bax and Bcl-2 expression may play an important role in neuronal cell death induced by scrapie infection is discussed.     

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

Scrapie is a transmissible neurodegenerative disease of sheep and goats. The neuropathological changes include vacuolation, astrocytosis, the development of amyloid plaques in some instances, and neuronal loss. The mechanisms involved in neuronal cell death in scrapie are not known. Recently, we reported the presence of oxidative stress in the brains of scrapie-infected animals and suggested that this is the main mechanism that induces neuronal cell loss. It is known that oxidative stress induced by free radicals is associated with iron accumulation; this association led to an examination of the levels of iron (total iron, Fe²⁺ and Fe³⁺) in the brains of control and scrapie-infected mice by biochemical methods. In the scrapie-infected group, both the level of total iron and the Fe³⁺ level were significantly increased in cerebral cortex, striatum, and brainstem as compared to the values in the control group. A shift in the ratio of Fe²⁺/Fe³⁺ was observed in the same regions of infected mice. Additionally, in this scrapie model, we confirmed the presence of oxidative stress, as evidenced by the increase of free malondialdehyde. These results suggest that iron metabolism is changed and that iron-induced oxidative stress partly contributes to neurodegeneration in scrapie infection.     

Alterations in Potassium Currents May Trigger Neurodegeneration in Murine Scrapie

Conventional electrophysiological intracellular recording techniques were used to test the hypothesis that enhanced calcium entry via voltage-gated calcium channels or theN-methyl-d-aspartate (NMDA) subtype of glutamate receptor–channel complex may be a primary pathological mechanism triggering neurodegeneration in scrapie and related diseases. This study was carried out at a time when cell loss is known to occur and when hippocampal pyramidal cells in area CA1 are rendered hyperexcitable following scrapie infection. There was no change to the NMDA receptor-mediated component of the Schäffer collateral evoked excitatory postsynaptic potential (EPSP) or the level of spontaneous firing activity of CA1 cells following addition of the specific NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (APV, 20 μM), to the perfusate in scrapie-infected mice, indicating that the NMDA receptor–channel complex is not compromised by scrapie. There was also no change seen in the non-NMDA mediated component of the EPSP. The calcium spike of CA1 pyramidal cells was not significantly altered by scrapie infection, indicating that high threshold voltage-gated Ca²⁺channel function is not compromised by scrapie. By contrast, cells from scrapie-infected mice fired calcium spikes repetitively and the long, slow AHP, which in control cells inhibited repetitive firing, was absent. Cells from scrapie-infected mice showed more depolarized membrane potentials than controls but this difference in potential was no longer observed after exposure to TEA. These data indicate a loss of TEA-insensitive and TEA-sensitive potassium conductances. We suggest that altered potassium currents rather than increased calcium entry via voltage-sensitive calcium channels or the NMDA receptor complex may be the primary pathological mechanism triggering neurodegeneration in scrapie and related diseases.     

Scrapie infection diminishes spines and increases varicosities of dendrites in hamsters: a quantitative Golgi analysis

An altered morphology of neuronal dendrites has been shown to be associated with many degenerative diseases of the central nervous system (CNS). Scrapie is a CNS degenerative disorder caused by a novel infectious particle or prion. Golgi impregnation studies showed that neurons in the scrapie-infected brains of hamsters contained varicose swellings and diminished numbers of dendritic spines. In order to ascertain whether or not these differences were statistically significant, quantitative methods were applied to brain samples from scrapie-infected hamsters and compared to uninfected controls. Golgi impregnated layer III pyramidal neurons from both motor and visual cortex exhibited two types of changes in infected animals. First, loss of dendritic spines on the apical shaft of both motor and visual neurons were found from 50 to 200 microns from the cell body (p less than 0.001). Second, spherical varicosities on dendritic stalks ranging from 7 to 25 microns in diameter were found. The average number of varicosities per cell was 18.1 in infected animals with varicosities on dendrites of controls numbering less than 3 per cell. Less than 2% of the control cells exhibited these varicosities, while greater than 80% of the scrapie dendrites exhibited varicosities. These changes in scrapie are similar to those reported in Creutzfeldt-Jakob and Alzheimer's disease in human patients.     

Increase of acidic fibroblast growth factor in the brains of hamsters infected with either 263K or 139H strains of scrapie

Scrapie is the archetypal unconventional slow infection disease. It has been shown that hamsters injected intracerebrally with scrapie strains 139H or 263K show extensive astrocytosis and that the induced reactive astrocytes produce a variety of factors that can affect brain function. Acidic fibroblast growth factor (aFGF) belongs to a family of growth factors that show a high affinity for heparin sulfate proteoglycans. In the current study, we have used immunohistochemistry to investigate the distribution of aFGF in scrapie-infected brain; we observed a low level of aFGF immunoreactivity (ir-aFGF) in ependymal cells and in a few neurons in the hypothalamus of control hamsters. In contrast, in scrapie-infected hamsters, there was an increase of ir-aFGF in a number of cell types, including neurons, pericytes, astrocytes, and ependymal cells. In 139H-infected hamsters, ir-aFGF staining in astrocytes, neurons and neuropil areas of the cortex, hippocampus, thalamus, and hypothalamus was greater than the staining in control animals. For 263K animals, astrocytic ir-aFGF staining was significantly greater than in either control or 139H-infected hamsters in the following regions: cortex, putamen, corpus callosum, thalamus, hypothalamus, fimbria, hippocampus, subependymal areas, and amygdala. In addition, there was a significant increase in neuronal ir-aFGF in the CA1 hippocampal area and in the amygdala. Our results suggest that neurons and astrocytes can produce and/or absorb aFGF during scrapie infection. These findings indicate that aFGF might play an important role in neuronal protection and in astrocytosis in scrapie-infected hamsters.     

Intrinsic physiological and morphological properties of principal cells of the hippocampus and neocortex in hamsters infected with scrapie.

Scrapie is a transmissible spongiform encephalopathy, or "prion disease." We investigated the effects of intracerebral Sc237 scrapie inoculation in hamsters on the physiology and morphology of principal cells from neocortical and hippocampal slices. Scrapie inoculation resulted in increased branching of basal dendrites of hippocampal CA1 pyramidal cells (Sholl analysis), reduced amplitudes of medium and late afterhyperpolarizations (AHPs) in CA1 pyramidal cells and layer V neocortical cells, loss of frequency potentiation of depolarizing afterpotentials (DAPs), and double action potentials in synaptically evoked CA1 pyramidal cell responses. Postsynaptic double action potentials could also be evoked in normal hamster CA1 pyramidal cells by acute pharmacological block of AHPs, suggesting that the depressed AHPs in scrapie-infected hamsters caused the action potential doublets. Both the AHP and the DAP potentiations depend on increased intracellular calcium, which suggests that the underlying deficit, in hamsters infected with Sc237 scrapie, may lie in calcium entry and/or homeostasis. Fast IPSPs, passive membrane properties, and density of dendritic spines remained unchanged. These last two results differ markedly from recent studies on mice infected with ME7 scrapie, indicating diversity of pathophysiology in this group of diseases, perhaps associated with the progressive and substantial neuronal loss found in the ME7, and not the Sc237, model.     

SCRAPIE: HOW MUCH DO WE REALLY UNDERSTAND?

Biological studies have produced convincing evidence for different scrapie strains, some of which undergo mutation. This argues strongly in favour of the infectious scrapie agent having a genome. The length of incubation period is influenced by the strain of agent but is also under strict host control. In mice, this control is exerted by a gene called Sinc which affects the overall rate of agent replication in the CNS. After peripheral infection, invasion of the CNS from lymphoreticular sites of agent replication is a key step in pathogenesis. Evidence from one scrapie model indicates spread of infection along autonomic nerves to the thoracic spinal cord and then to other parts of the CNS. Other studies have shown that infection can spread in neurons. There are close relationships between the presence of replicating agent and the development of vacuolation, and also of cerebral amyloid when it occurs. We can, therefore, begin to understand the patterns of lesion development in the brain in terms of the targeting of infection and its replication at certain sites. Structures known as SAF (Scrapie Associated Fibrils) have been discovered in extracts of scrapie brain (but not uninfected brain) and a glycoprotein (PrP 27-30: SAF protein) is a major constituent of purified SAF. The glycoprotein is coded by a single gene which is present in several species and expressed in uninfected brain. The normal protein seems to be modified in scrapie infected brain so that it accumulates as SAF. The modified protein may also be deposited as extracellular amyloid because there appear to be common epitopes between SAF and scrapie amyloid. The biochemical nature of the scrapie agent remains in doubt and the association between infectivity and purified SAF may arise fortuitously from the fact that scrapie agent is 'sticky'.     

Characteristics of scrapie isolates derived from hay mites

Previous epidemiological evidence suggested that in some instances a vector and/or reservoir is involved in the occurrence and spread of transmissible spongiform encephalopathies (TSEs). In a preliminary study, hay mite preparations from five Icelandic farms with a history of scrapie were injected into mice, and some of these mice became sick after long incubation periods. To confirm that the disease was scrapie, subsequent passages in mice were performed. In addition, the characteristics of the disease process in these passages were assessed and the results compared to those findings with standard scrapie strains. As expected for scrapie, subsequent passages in the same host led to shortened incubation periods compared to those in primary isolate mice, and all mice had spongiform changes in brain. Results were similar for three of four isolates with regard to clinical manifestations, the incubation periods in mice of the three scrapie incubation-period genotypes (s7s7, s7p7, p7p7), and the PrPSc Western blot (WB) pattern. The characteristics of the fourth isolate were markedly different from the other three isolates with regard to these parameters. Comparison of the characteristics of standard mouse-adapted scrapie strains and the four isolates revealed differences; these differences were particularly pronounced for the fourth isolate.     

Evaluation of neurodegeneration in scrapie-infected animals by selective methods that detect cellular degeneration

Scrapie is a fatal neurodegenerative disease of sheep and goats. The precise details of neuronal and neurite degeneration in scrapie-infected animals remain unknown. Using specific silver staining methods, we compared the neurodegeneration caused by treatment of rats with kainic acid (KA) or ibogaine (IBO) to the neuropathology observed in mice infected with the C602 strain of scrapie. As reported previously, KA resulted in extensive silver labeling of neurons, especially in the cortex, putamen and hippocampus. IBO silver labeling was observed only in small clusters of Purkinje neurons in the paravermal region of the cerebellum. However, in scrapie-infected mice, a few silver stained neurons (differing from the dark degenerating neurons observed following neurotoxic exposure) were found in layer II of cortex, cingulate cortex, zona incerta, thalamus and hypothalamus. Some silver grains were observed in glial-like cells, especially those in the paraventricular region. Degenerating axons were positive for silver staining and were found in the cortex, cingulate cortex, corpus callosum, habenulae, septum, fornix, thalamus, caudate putamen and a few in fasciculus retroflexus and substantia nigra. Our results suggest that the limbic system is one of the important loci for the neurodegenerative effect of at least some scrapie strains.     

The occurrence of vacuolation, and periodic acid-Schiff (PAS)-positive granules and plaques in the brains of C57BL/6J, AKR, senescence-prone (SAMP8) and senescence-resistant (SAMR1) mice infected with various scrapie strains

Scrapie is a fatal, but slow, infectious disease. C57BL/6J, SAMP8 (a strain that develops early senescence), SAMR1 (a strain that is resistant to senescence) and AKR/J (a progenitor of the SAM strains) mice were infected with 22A, 139A, 22L and ME7 scrapie strains. Histopathological stains included haematoxylin and eosin (HE), and periodic acid-Schiff (PAS). Vacuolation was found in the brains of all scrapie-infected mice. The 22A strain caused more extensive vacuolation in the brains of SAMP8 and SAMR1 mice than in C57BL mice. PAS-positive plaques (PP) were found in 22A-infected mice in cortex, corpus callosum, hippocampus, subependymal zone area and thalamus. PP were significantly increased in 22A-infected SAMR1 mice compared to mice from other scrapie-infected strains. Clusters of small, round, homogeneous PAS-positive granular structures (PGS) were found in all mouse strains, especially in aging control and 22A-infected C57BL mice, predominantly in the stratum radiatum of the CA1, CA2 and CA3 areas of the hippocampus. Some of these structures were also observed in stratum oriens and piriform cortex, and in cerebellar Purkinje cell areas. Some of the PGS were associated with astrocytes and blood vessels. Each granule was 1-5 microm in diameter and there were clusters consisting of several to 40 PGS; the sizes of the clusters ranged from 10 to 80 microm in diameter. There were more PGS clusters in uninfected C57BL and AKR mice than in uninfected SAMP8 and SAMR1 mice. PGS were not increased in scrapie-infected mice. These findings suggest that PGS accumulation was more dependent on the genetic information of the mouse strain, whereas PP and vacuolation patterns depended on the scrapie strain-mouse strain combination.     

Creutzfeldt-Jakob disease and scrapie prions

Creutzfeldt-Jakob disease, kuru, and Gerstmann-Str?ussler syndrome are transmissible degenerative diseases of the central nervous system caused by novel infectious pathogens designated prions. Scrapie is a neurodegenerative disease of sheep and goats and is also caused by prions. Experimental scrapie has been extensively studied in hamsters and mice. The scrapie prion protein (PrPSc) is the only component of the infectious scrapie prion identified, to date. Scrapie infectivity and PrPSc copartition into membranes, rods, and liposomes raising the possibility that only PrPSc might be required for infection; however, a second component such as a small nucleic acid cannot be eliminated. PrPSc is encoded by a single copy cellular gene and not by a hypothetical nucleic acid within purified prion preparations. Normal, uninfected cells express the cellular prion protein (PrPc). Both PrPSc and PrPc appear to be translated from the same 2.1-kb mRNA. The N-terminal amino acid sequences of hamster PrPC and PrPSc are identical; both correspond to that predicted by the translated prion protein (PrP) gene sequence. While the chemical difference between PrPc and PrPSc remains unknown, the organization of the PrP gene argues that it results from a posttranslational event. Six posttranslational modifications of both PrP isoforms have been identified: (1) cleavage of an N-terminal signal peptide, (2) an intramolecular disulfide bond, (3) an N-linked oligosaccharide attached to Asn 181, (4) a second oligosaccharide attached to Asn 197, (5) cleavage of a C-terminal hydrophobic peptide, and (6) a phosphatidylinositol glycolipid attached to the C-terminus. The mouse PrP gene is on chromosome 2 and is linked to a gene controlling the scrapie incubation time (Prn-i). PrP genes from inbred mice with short and long incubation times differ by two amino acids, a finding consistent with but not proving that PrP modulates susceptibility to scrapie. PrPSc stimulation of a posttranslational process which converts PrPc or its precursor into PrPSc is one possible mechanism for prion replication. This is consistent with observations showing that human prion diseases are manifest as infectious, sporadic and genetic disorders.     

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5–10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.     

Ultrastructural evidence that ependymal cells are infected in experimental scrapie

During the last stage of infection in the experimental scrapie-infected hamster model, light microscopy reveals typical immunostaining of PrPsc in the subependymal region and at the apical ependymal cell borders. Whereas the subependymal immuno-staining is known to originate from extracellular amyloid filaments and residual membranes of astrocytes as constituents of plaque-like structures, the ultrastructural correlate of the supraependymal PrPsc staining remains uncertain. To decipher this apical PrPsc immunopositivity and subsequently the ependymocyte-scrapie agent interaction, we employed highly sensitive immuno-electron microscopy for detecting PrPsc in 263K scrapie-infected hamster brains. The results revealed the supraependymal PrPsc signal to be correlated not only with extracellular accumulation of amyloid filaments, but also with three distinct ependymal cell structures: (1) morphologically intact or altered microvilli associated with filaments, (2) the ependymal cell cytoplasm in proximity of apical cell membrane, and (3) intracytoplasmic organelles such as endosomes and lysosomal-like structures. These findings suggest a strong ependymotrope feature of the scrapie agent and recapitulate several aspects of the cell-prion interaction leading to the formation and production of PrPsc amyloid filaments. Our data demonstrate that in addition to neurons and astrocytes, ependymocytes constitute a new cellular target for the scrapie agent. In contrast, the absence of PrPsc labeling in choroid plexus and brain vascular endothelial cells indicates that these cells are not susceptible to the infection and may inhibit passage of the infectious agent across the blood-brain barrier.     

Targeting of scrapie lesions and spread of agent via the retino-tectal projection

Scrapie infectivity and degenerative vacuolation was initially localized within the contralateral superior colliculus following intraocular injection. The time course of these events was prolonged. With the ME7 strain of scrapie in Sincs7 genotype mice, infectivity began to rise in the superior colliculus from about 70 days, followed by the earliest asymmetrical lesions there from 120 days, with death occurring at about 250 days, at which time vacuolar degeneration was widespread in the brain. With other mouse Sinc genotype mouse/agent strain combinations the process was even further prolonged. With 87V scrapie strain in Sincp7 genotype mice the first lesions to appear were in the contralateral tectum at 300 days. It is concluded that scrapie agent can spread within ganglion cell axons.