Altered plasma membranes in experimental scrapie

Summary The status spongiosus in the cerebral cortex of mice affected with two different strains of scrapie virus corresponded to focally swollen perikaryal cytoplasm of nerve cells and astrocytes, to swollen neuronal and astrocytic processes and to membrane-bounded vacuoles within pre- and postsynaptic neuronal terminals. The swollen cytoplasm contained uniformly dispersed, finely granulo-filamentous material. A few enlarged dendrites were filled with fragments of membranes or 350 Å wide vesicular and tubular structures suggestive of virus particles. Ruptured plasma membranes and curled fragments of membranes were seen around cleared cytoplasmic regions and within membrane-bounded vacuoles. Neurons or astrocytes that lined affected cells or processes frequently showed similar changes. Confluence of swollen cells or processes occurred after dissolution of their adjacent plasma membranes. Astrocytes reacted to the injury by proliferation whereas nerve cells degenerated. The findings are compared to those seen in other subacute spongiform virus encephalopathies, i. e., mink encephalopathy, Kuru and Creutzfeldt-Jakob disease. The characteristic vacuolar degeneration of nerve cells in these diseases which is associated with fragmentation and accumulation of plasma membranes is discussed with reference to the peculiar properties of the scrapie virus.This investigation was supported in part by United States Public Health Research Grant NS-09053 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, Bethesda, Maryland.     

Comparison of spongiform lesions in experimental scrapie and rabies in skunks

Summary Striped skunks were inoculated intracerebrally with the scrapie agent (suspension of brain from a naturally infected Suffolk sheep) or intramuscularly with street rabies virus (suspension of salivary glands from naturally infected skunks). Those given the scrapie agent developed clinical signs of weakness, posterior ataxia, and emaciation after incubation periods of 8 to 23 months. Those inoculated with rabies virus developed clinical signs of rabies (aggressive behavior, hyperexcitability, ataxia and paralysis) after incubation periods of 20 to 62 days. The gross lesions in the brains of the skunks given the scrapie agent consisted of marked atrophy of the thalamus and moderate atrophy of the cerebrum. No gross lesions occurred in the rabid skunks. Histologically, the type of spongiform lesion in rabies was the same as that in scrapie. However, spongiform change of rabies infected brains was less extensive (only rarely affected the basal ganglia, hippocampus or hypothalamus) than that of brains infected with the scrapie agent and was characterized by fewer numbers of small vacuoles (as a proportion of total number of vacuoles) than occurred in scrapie spongiform change.     

Scrapie inoculation of mice: light and electron microscopy of the superior colliculi

Summary Ultrastructural examination of the superior colliculi of mice intraocularly inoculated with the ME7 strain of scrapie showed vacuolation early in the course of infection. Brains were examined between 85–260 days after monocular inoculation with scrapie. The mean incubation period for the development of clinical disease was 302 days. Vacuolation was seen initially in the contralateral superior colliculus and subsequently in the ipsilateral colliculus. In coded trails light microscopical vacuolation was seen from 218 days but ultrastructural examination showed that sparse vacuoles were inconsistently present in either or both of the ipsilateral and contralateral colliculi from 85 days; frequent vacuoles were seen from 190 days. Scrapie-induced vacuoles were differentiated from vacuoles present in control tissue by the presence of loculation or by a limiting double membrane which showed protrusion or proliferation of the innermost lamella. Vacuolation was seen in neuronal perikarya, myelinated fibres, dendrites and axonal presynaptic terminals. Vacuoles of myelinated fibres were observed within myelin and possibly also in the inner tongue of oligodendroglial cytoplasm. Whorled membrane configurations were also seen. Tubulovesicular particles, 40 nm in diameter, were recognised in two scrapie-infected mice. It is suggested that some scrapie vacuoles arise as a result of incorporation of abnormal membrane into organelles, possibly mitochondria, in neuronal perikarya and neurites and probably also within oligodendroglial cytoplasm and myelin.     

An electron microscopic study of inclusion bodies in synaptic terminals of scrapie-infected animals

Summary Inclusion bodies consisting of vesicles of about 25 nm diameter and occurring in the synaptic terminals of scrapie-infected animals have been described by a number of people. In the present study these inclusion bodies were looked for in the neocortex, hippocampus and corpus callosum in a variety of strains of mice (C3H, LM, RIII, IM, VL) infected with different strains of scrapie agent (22C, 79A, ME7, 87V) after intracerebral inoculation. In plaque-bearing models of scrapie, terminals containing synaptic inclusion bodies were frequently found surrounding the amyloid plaque cores in the neocortex but not in the corpus callosum. In non-plaque-bearing models, terminals containing synaptic inclusion bodies were found in the neuropil of the neocortex and hippocampus. For all models, these bodies were either presynaptic or postsynaptic but were not, as a rule, found on both sides of the same synapse. Fibrillary material was frequently seen in the postsynaptic terminals containing the inclusion bodies in both the plaque- and non-plaque-bearing models. On one occasion fibrillary material was seen, together with the inclusion bodies, in a neuron cell body. Inclusion bodies were also seen in the neocortex of hamsters infected with the 263K strain of scrapie agent and a Cheviot sheep infected with the ME7 strain of agent. The inclusion bodies and the fibrillary material were thought to be derived from the breakdown of neurotubules.     

Spongiform encephalopathy in mice inoculated with scrapie material of sheep origin

The authors report spongy degeneration in experimental scrapie (second passage) in mice. The scrapie agent was originally isolated from Suffolk sheep imported from Canada and diagnosed histopathologically to be infected with scrapie by intracerebral inoculation into JCL/ICR mice. Ten female SIc/ICR mice, 4 weeks of age, were injected intracerebrally in the right frontal lobus with 20 microliter of 10(-1) or 10(-4) dilution of JCR/ICR mice brain homogenate involving scrapie agent. All animals showed signs of the advanced stages of the disease, clinically manifested by lassitude, arched backs, lethargy and paresis of hind quarters. They were sacrificed five to six months post inoculation, and sections of the brain and spinal cord were examined by light and electron microscopy. Focal symmetrical spongiform lesions were seen light microscopically in the cerebral mantle, thalamus, hypothalamus, midbrain, medulla oblongata, cerebellum and cervical mark. There was evidence that these lesions tended to be more intense in the mice inoculated a higher concentration of scrapie agent. Astrocytic proliferation was present in the deep layer of cerebral gray matter, white matter, corpus callosum, dorsal part of hippocampus and thalamus. No leukocytic infiltration was observed. Electron microscopically, the spongiform lesions were shown to be caused by vacuolation or swelling within the neuropil, and vacuolation and focal swelling in the neuronal perikaryon. The changes in the neuronal perikaryon were caused by enlargement of endoplasmic reticulum and cisterns of the Golgi apparatus, accompanied by spherical swelling of a part of the cytoplasm. The vacuolation near or within the neuron produced deformation of the cell contours and displacement of the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transmission of chronic spongiform encephalopathy with kuru plaques from humans to small rodents.

An unusual case of spongiform encephalopathy was transmitted directly from a human to rats and mice. After serial passages, incubation periods were shortened to about six months in rats and four months in mice. Clinical symptoms were similar in rats and mice, including ruffled fur, arched back, bradykinesia, and hind limb paralysis. Pathologically, a spongy state, proliferation of astrocytes, and neuronal changes were observed. Electron microscopic observation of the parietal cortex of rats and mice disclosed many membrane-bound vacuoles in the neuropil, predominantly in dendrites. In the pons of mice, intramyelinic vacuoles and accumulation of extracellular fluid were prominent. The clinicopathological symptoms of the affected animals resembled those in other experimental spongiform encephalopathies, especially scrapie.     

EVIDENCE THAT TRANSMISSIBLE MINK ENCEPHALOPATHY AGENT IS BIOLOGICALLY INACTIVE IN MICE

Transmissible mink encephalopathy (TME) is probably a form of the sheep disease, scrapie, introduced by accidentally feeding mink with scrapie-infected sheep tissues. Although no successful transmissions of TME to mice have been achieved previous work has involved various limitations. To maximize the possibility of transmission, 176 mice, representing 14 different genotypes mostly not previously tested with TME, were injected with TME-infected mink brain from three sources with different histories. No scrapie-like disease was detected clinically or histologically in these mice or in a further 111 which were subsequently injected with brain or spleen material from 10 of the TME-injected mice killed when senile. Furthermore, a series of experiments involving seven strains of scrapie, demonstrated that prior injection of mice with TME failed to affect the normal progress of scrapie infection indicating that TME agent had not occupied scrapie replication sites or otherwise influenced the pathogenesis of scrapie. The overall conclusion from these experiments is that TME is biologically inactive in mice. Although many strains of natural scrapie can be transmitted to laboratory mice, this has not been possible with all strains and it is concluded that one or more of such strains is likely to be the cause of TME in mink.     

Tubulovesicular structures are not labeled using antibodies to prion protein (PrP) with the immunogold electron microscopy techniques

Tubulovesicular structures (TVS) are disease-specific, intraneuronal particles found by thin-section electron microscopy in all of the transmissible spongiform encephalopathies. We used immunogold (both 10 nm immunogold and 1 nm immunogold silver enhanced) methods for ultrastructural localization of prion protein (PrP). In all scrapie models examined (263 K and 22CH in hamsters and 87V and ME7 in mice), TVS-containing processes were readily detected but neither these processes nor TVS themselves were decorated with gold particles. Even when amyloid plaques were observed in a close contact with TVS-containing neuronal processes, the processes remained unstained, while the plaques were decorated with gold particles. TVS located in areas adjacent to plaques in the 87V model and in areas of diffuse PrP immunolabelling in ME7 were also unlabelled with anti-PrP sera. Using immunogold techniques we were unable to label TVS with anti-PrP antibodies. As these technique proved to be sensitive enough to immunolabel not only amyloid plaques but also pre-amyloid accumulations of PrP, we strongly believe that the absence of staining reflects the structure of TVS and that they are not composed of PrP. That TVS are PrP negative may have several important implications for hypotheses about their nature. Principally, it does not support the suggestion that TVS are cross-sections of “thick tubules” visualized by touch-preparations of scrapie-affected mouse and hamster brains. If PrP is the infectious agent, as suggested by the prion hypothesis, the absence of stainable PrP in TVS would indicate that these are not the ultrastructural correlate of the agent. If, however, TVS turn out to be more than merely a useful ultrastructural marker for the whole group of transmissible spongiform encephalopathies, it may suggest that PrP and the agent are two separate entities. Received: 11 March 1996 / Revised: 9 May 1996 / Accepted: 26 September 1996     

Scrg1 is induced in TSE and brain injuries, and associated with autophagy

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.     

Vacuolization, incubation period and survival time analyses in three mouse genotypes injected stereotactically in three brain regions with the 22L scrapie strain

In previous studies we showed that C57BL mice injected stereotactically in the cerebellum with the 22L scrapie strain had a significantly shorter incubation period than those injected with the same agent in other brain regions. In mice injected in the cerebellum, vacuolization was limited to the cerebellum, medulla and mesencephalon, whereas injection into forebrain regions resulted in vacuolization in all brain regions. The studies suggested that the cerebellum had a selective vulnerability for 22L. In this study we examined the interaction between host genotype and selective vulnerability of specific brain regions. The mouse gene that has the most profound effect on pathogenesis, particularly incubation period, is termed Sinc (scrapie incubation). Groups of mice with three genotypes of Sinc (s7s7, p7p7 and their F1 cross, s7p7) were injected with 22L into the cerebral cortex, thalamus or cerebellum. Analysis of incubation periods showed that, regardless of the host genotype, the cerebellum injection group had a significantly shorter incubation period than groups injected in other regions. After cerebellum injection vacuolization was limited to the cerebellum, medulla and mesencephalon in all three host genotypes. The location of vacuoles within the cerebellum differed depending upon the host strain. Vacuolization developed almost exclusively in grey matter in s7s7 mice, mainly in white matter in p7p7 mice, and in both grey and white matter in F1 mice. These results demonstrate that the selective vulnerability of the cerebellum to induction of clinical disease by 22L does not depend on host genotype, but host genotype does affect lesion distribution within the cerebellum.     

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.     

Isolation and characterization of macrophages from scrapie-infected mouse brain

Summary We have isolated and characterized a population of brain macrophages from normal and scrapieinfected 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 that 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.Dedicated to Prof. F. Seitelberger on the occasion of his seventieth birthdaySupported by National Institute on Aging grant no. AG04220     

An electron microscopic study of natural scrapie sheep brain: Further observations on virus-like particles and paramyxovirus-like tubules

Summary Detailed electron microscopic study of cerebellum and medulla of sheepaffected with natural scrapie is reported.Two types of virus-like particles, non-membrane bound and membrane bound occur in neurons, pre-synaptic terminals and axis cylinders. The latter type were seen being apparently formed at the plasma membranes. Their size corresponds with that attributed to the scrapie agent. Three different types of tubules are described which filled a few axis cylinders and pre-synaptic terminals, their structure is compared with tubules found in other conditions.     

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.     

SPONGIFORM POLIOENCEPHALOMYELOPATHY CAUSED BY A MURINE RETROVIRUS. II. ULTRASTRUCTURAL LOCALIZATION OF VIRUS REPLICATION AND SPONGIFORM CHANGES IN THE CENTRAL NERVOUS SYSTEM

The development of murine retrovirus induced spongiform polioencephalomyelopathy was studied sequentially by electron microscopy. During the initial 30 days, viral infection of the central nervous system, as evidenced by viral budding from membranes, was limited to the endothelial cells and pericytes. Viral particles were observed in the lumen of blood vessels, extracellular spaces and astrocytic endfeet surrounding blood vessels, but no morphological evidence of productive infection was found in astrocytes or neurons during early development of vacuolation. The earliest lesions in the neuropil consisted of swelling of astroglia followed by vacuolation, initially in axons and dendrites and later in neuronal and astrocytic soma, where vacuoles appeared to arise from dilated cisternae of the Golgi apparatus. Vacuoles contained only amorphous debris and fragments of membranes. Virions budding aberrantly into vacuoles were seen only in mice surviving beyond 35 days. Numerous reactive astrocytes were observed, but inflammatory cells were absent. The ultrastructural changes were remarkably similar to those described in scrapie, Kuru, and Creutzfeldt-Jakob disease.     

Immunological studies of scrapie infection

Scrapie is a show, degenerative nervous system disease of sheep and goats. No obvious inflammatory or immunological response occurs despite early replication of the srapie agent in the spleen. The extend earlier investigations (Garlin et al. 1978a, b) on the immunorcactivity of lymphocytes during scrapie infection, purified populations of T and B cells were prepared from BALB/c mice. No consistent differences in the response to mitogens of T and B cells from scrapie-infected and control mice were observed. An assessment of T-dependent B cell function was obtained using a hemolytic plaque-forming cell assay, but again no differences between scrapie-infected and control splenocytes could be detected. To study the possible influence of B cell maturation on scrapie infection, incubation periods were measured in CBA/N mice, bearing an X-linked deficiency of mature B cells. Scrapie incubation periods were identical in affected males and unaffected females, indicating that B cell maturation does not influence the course of scrapie. Using highly enriched preparations of the scrapie agent, we attempted to raise antibodies in rabbits using an extensive immunization protocol. Scrapie-specific antibodies could not be detected using either indirect immunofluorescence or enzyme-linked immunosorbent assay (ELISA). Some antisera reacted more strongly with scrapie than with control preparations; however, these differences disappeared when the antisera were     

CEREBRAL AMYLOIDOSIS IN SCRAPIE IN THE MOUSE: EFFECT OF AGENT STRAIN AND MOUSE GENOTYPE

Cerebral amyloid deposits, predominantly in the form of discrete plaques are common in mice infected with certain scrapie isolates. In this paper the incidences of cerebral amyloidois occurring with different combinations of agent strain and mouse genotype are investigated. Seven different agents from four primary sources were studied in three mouse genotypes (C57BL, VM and their F₁ cross). It is shown that the degree of amyloidosis is a passageable characteristic of the agent depending also on host genotype. However, the amyloid incidence is not influenced by the agent and mouse genotype independently but depends on a specific interaction between the two. For two scrapie agents, 22A and ME7 there is a correlation between incubation period and the incidence of cerebral amyloid in the three mouse genotypes. The degree of cerebral amyloid for ME7 remains unaffected by passage through the two genotypes of mouse, C57BL and VM, and through another species, a Cheviot sheep.     

Spontaneous spongy degeneration of the brain stem in SAM-P/8 mice, a newly developed memory-deficient strain

A spontaneous spongy degeneration of the brain stem and spinal cord was discovered in a murine model of accelerated senescence (SAM), cared for under both conventional (SAM-P/8) and specific pathogen-free (SAM-P/8/Ta) conditions. SAM-P/8 and SAM-P/8/Ta showed no clinical neurological abnormalities, yet there was a deterioration in learning and memory abilities. Light microscopic examination revealed a spongy degeneration in the brain stem and spinal cord, in the reticular formation, and proliferation of hypertrophic astrocytes in the spongy area. The spongiform degeneration progressed with advancing age from four to eight months, after which the entire brain was involved. Astrocytosis increased with advancing degeneration. Ultrastructurally, mild dendritic swelling occurred at one month of age. At two months of age, moderate postsynaptic swelling and a widening of intracellular membrane structure were observed, and at age five months there were large vacuoles circumscribed by membranous lamellae, identifiable as myelin. Vacuoles in SAM-P/8 proved to be swollen neuronal processes and oligodendroglial processes. These SAM-P/8 and SAM-P/8/Ta strains of mice are new memory-deficient strains with spontaneous spongy degeneration associated with aging.     

FURTHER OBSERVATIONS ON PARTICULATE STRUCTURES IN SCRAPIE AFFECTED BRAIN

Further observations on particulate structures in scrapie affected brain
A search procedure at the ultrastructural level revealed characteristic particulate structures in mice of two further laboratory stocks experimentally inoculated with scrapie. The structures represent a specific change related to scrapie disease; their relationship to the scrapie agent is discussed.