Disease‐specific particles without prion protein in prion diseases – phenomenon or epiphenomenon?

The search for the cause of transmissible spongiform encephalopathies (TSEs) has a long and tortuous history. In a recent paper, 25-nm virus-like particles were identified that were consistently observed in cell cultures infected with Creutzfeldt-Jakob disease (CJD) and scrapie; they are similar to, or even identical with, the virus-like tubulovesicular structures (TVS) found in experimental scrapie as early as in 1968, and subsequently in all naturally occurring and experimentally induced TSEs. These particles have been viewed with caution by the scientific community because of the unverified or uninterpretable record of virus-like structures reported over the years in TSEs. TVS are spherical or tubular particles of approximate diameter 25-37 nm. They are smaller than synaptic vesicles, but larger than many particulate structures of the central nervous system, such as glycogen granules. Their electron density is higher compared with synaptic vesicles, and in experimental murine scrapie, they form paracrystalline arrays. None of these observations distinguish between TVS as an entity critical to the infectious process, or as a highly specific ultrastructural epiphenomenon, but their consistent presence in all TSEs demands further research.     

IMMUNOSTAINING OF SCRAPIE CEREBRAL AMYLOID PLAQUES WITH ANTISERA RAISED TO SCRAPIE–ASSOCIATED FIBRILS (SAF)

Brain sections from 16 different mouse scrapie models were immunostained with antisera to scrapie-associated fibrils (SAF) from three experimental scrapie sources (hamster 263K, mouse ME7 and mouse 22L). These models involved seven strains of scrapie injected intracerebrally or intraperitoneally into a range of inbred mouse strains, producing a wide variety of neuropathological changes. The only brain structures which were positively immunostained were amyloid plaque cores in those models in which plaques could be readily identified using traditional amyloid stains. The intensity of immunostaining correlated with the density of amyloid in the cores, as detected by Congo red and thioflavine S staining. No differences in immunostaining specificity were found between antisera or between plaques in different combinations of scrapie strain and mouse genotype. There were also no differences in immunoreactivity between plaques in different parts of the brain. These results strongly suggest that SAF and histologically detectable amyloid in scrapie mice are derived from the same precursor protein. Scrapie-associated cerebrovascular amyloid and plaques in sheep and goats also gave positive immunostaining with SAF antisera, although the lesions in the natural disease could only be stained after formic acid pretreatment. Senile plaques in Alzheimer's disease and Down's syndrome, although structurally similar to scrapie amyloid plaques, were found to be completely negative for SAF, in agreement with previous biochemical and immunocytochemical findings.     

Scrapie as a model for neuroaxonal dystrophy: ultrastructural studies

Neuritic degeneration is a prominent ultrastructural feature of scrapie in hamsters. To investigate the morphogenesis of neuritic degeneration, we examined brain tissues from hamsters infected with the 263K strain of scrapie virus and from age-matched controls at varying intervals following intracerebral inoculation. Dystrophic neurites--defined as dendrites, axonal preterminals, and myelinated axons containing mitochondria and pleomorphic, electron-dense inclusion bodies--were found as early as 2 weeks postinoculation. Their numbers increased with the incubation period, and their highest density was observed at the terminal stage of disease. Occasionally, small clusters of these structures formed neuritic plaques. Such dystrophic neurites were only rarely seen in brains of uninfected hamsters. Experimental scrapie thus provides an animal model for human neuroaxonal dystrophies. In addition, since this model allows predictable formation of brain amyloid, it may serve as a model for the study of neuronal aging and Alzheimer's disease.     

Immunogold electron microscopy recognizes prion protein-associated particles prepared from scrapie-infected mouse brain

Previous studies have proposed that the disease isoform of prion protein (PrPSc) is particulate. Our purpose was to search by electron microscopy (EM) for particles in fractions of density gradients prepared from differentially centrifuged homogenates of scrapie-infected, normal, and null mouse brain. Only mild detergents were used during the separation process. The low-density fractions derived from scrapie-infected brain were rich in PrP. Three morphologically distinct types of particle were observed. Type 1 particles, measuring approximately 6.8 nm in mean diameter, were found in abundance in the fractions of scrapie-infected brain at the peak PrP concentrations. They were often clumped and adherent to raft-like structures. Type 2 particles, in low-density fractions from normal brain, were similar to type 1 but were smaller, with the mean diameter measuring approximately 5.3 nm. Type 3 particles from null brain differed morphologically from types 1 and 2 and were not clumped. The low density of the particles indicated a lipid component, which was confirmed by lipid analysis. Immunogold EM using Mab 6H4 labeled a portion of the particles from scrapie mouse brain, but not those from normal or null brain. Dimensions of PrP suggest that the labeled particles carry a PrPSc dimer per particle.     

Neural pathogenesis of experimental scrapie after intraocular inoculation of hamsters

Hamsters were inoculated intravitreally with the scrapie agent. All animals developed scrapie and retinal degeneration typical of scrapie. The retinal degeneration was greater in the inoculated eyes than in the uninoculated eyes. Replication of the scrapie agent was rapid in the inoculated eye. Infectivity then spread slowly down the ipsilateral optic nerve to the brain. The replication in the brain paralleled that in the retina of the uninoculated eye. The results support a neural spread of scrapie.     

Neuroinvasion of the 263K scrapie strain after intranasal administration occurs through olfactory-unrelated pathways

The olfactory system has been implicated in the pathogenesis of transmissible spongiform encephalopathies (TSEs). To examine this issue and identify the pattern of TSE agent spread after intranasal administration, we inoculated a high-infectious dose of neurotropic scrapie strain 263K into the nasal cavity of Syrian hamsters. All animals allowed to survive became symptomatic with a mean incubation period of 162.4 days. Analysis at different time points revealed deposition of the pathological prion protein (PrP^TSE) in nasal-associated lymphoid tissues in the absence of brain involvement from 80 days post-infection (50% of the incubation period). Olfactory-related structures and brainstem nuclei were involved from 100 days post-inoculation (62% of the incubation period) when animals were still asymptomatic. Intriguingly, vagal or trigeminal nuclei were identified as early sites of PrP^TSE deposition in some pre-symptomatic animals. These findings indicate that the 263K scrapie agent is unable to effectively spread from the olfactory neuroepithelium to the olfactory-related structures and that, after intranasal inoculation, neuroinvasion occurs through olfactory-unrelated pathways.     

CEREBROVASCULAR AMYLOIDOSIS IN SCRAPIE-AFFECTED SHEEP

Cerebrovascular amyloidosis (CA) was found in the cerebral and cerebellar cortices in 11 of 20 sheep, of six different breeds, with naturally-occurring scrapie. The occurrence of the lesion did not appear to be influenced by the age of the sheep. This was not seen in 20 brains from age- and breed-matched sheep with other conditions. Furthermore, it was not found in 20 sheep experimentally infected with scrapie by sub-cutaneous inoculation, or in 22 sheep with natural scrapie in a closed breeding group, but it was present at low frequency in a production flock, closed apart from the introduction of rams. It is concluded that this lesion is strongly associated with sheep scrapie disease, but that its occurrence varies between different groups of scrapie sheep. A major source of this variation is likely to be the strain of the agent, though other factors may be involved as has been shown for cerebral amyloidosis in experimental mouse scrapie.     

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.     

The sequential development of spongiform change and gliosis of scrapie in the golden Syrian hamster

The lesion profiles of spongiform change and gliosis in the hamster occurring after intracerebral (IC) inoculation of scrapie virus, are calculated and compared to the lesion profile of spongiform change of scrapie in mice and of scrapie and Creutzfeldt-Jakob disease (CJD) in the squirrel monkey. The profile of scrapie in hamsters differs considerably from that of a closely related strain of scrapie in mice, and both differ from scrapie and CJD in the squirrel monkey. These differences emphasize the effect of the host on the distribution of pathological changes in these unconventional virus infections. The sequential development of the lesions in the hamster shows that the earliest changes are detectable before the onset of clinical disease 49-57 days after inoculation, as assessed by light microscopy. Gliosis is detectable by indirect immunofluorescence 35-39 days after inoculation by use of a monoclonal antibody directed against astrocytes.     

Role of phagocytes in experimental scrapie in hamsters

We studied the interactions between scrapie agent and hamster's phagocytic cells. Macrophages which have been in contact with the scrapie agent are able to carry the infectivity of this agent. We induced variations of different parameters involved on fixation and phagocytosis and we did not observe any modification. A depletion of phagocytic cells induced in hamster at the entry of scrapie increase the incubation time of the illness. Furthermore phagocytic cells cocultured with glial cells seem to be able to transfer infectivity to glial cells. So scrapie agent and hamster's reticulo-endothelial system probably interact at three levels: first at the entry, then during haematogenous diffusion of scrapie agent and finally nearly the central nervous system.     

Appearance of tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie preceeds the onset of clinical disease

Summary We have consistently observed tubulovesicular structures in brain tissues during the terminal stages of naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species and virus strain. In NIH Swiss mice inoculated intracerebrally or intraocularly with the Fujisaki strain of Creutzfeldt-Jakob disease (CJD) virus, tubulovesicular structures, measuring 20–50 nm in diameter, were particularly prominent in dilated, pre-and postsynaptic neuronal processes, occasionally being mixed with synaptic vesicles. These structures appeared 13 weeks following intracerebral inoculation, 5 weeks before the onset of clinical signs, when spongiform changes were also detected. The number and density of tubulovesicular structures increased steadily during the course of clinical disease, and were particularly abundant in mice 47 to 51 weeks after intraocular inoculation. In hamsters infected with the 263 K strain of scrapie virus, these structures were initially detected 3 weeks following intracerebral inoculation and increased dramatically at 10 weeks postinoculation. The appearance of tubulovesicular structures before the onset of overt disease in mice inoculated with CJD virus by either the intracerebral or intraocular route, and before the appearance of other neuropathological changes in hamsters infected with scrapie virus, indicate that they represent either a part or aggregate of the infectious virus or a pathological product of the infection.Presented in part at the 64th annual meeting of the American Association of Neuropathologists, held in Charleston, South Carolina, June 9–12, 1988 and at the 7th annual meeting of the American Society for Virology, Austin, Texas, June 12–16, 1988. Dr. Pawel P. Liberski is a recipient of a fellowship from the Fogarty International Center and a grant from the Ministry of Health and Social Welfare, Poland     

Differentiation of prion protein glycoforms from naturally occurring sheep scrapie,sheep-passaged scrapie strains (CH1641 and SSBP1), bovine spongiform encephalopathy (BSE) cases and Romney and Cheviot breed sheep experimentally inoculated with BSE using two monoclonal antibodies

A panel of ruminant brain tissues were subjected to a Western immunoblotting technique using two monoclonal antibodies (mAbs). The resultant prion protein (PrP) glycoforms showed that three distinctions can be made between natural ovine scrapie cases and sheep experimentally inoculated with bovine spongiform encephalopathy (BSE). Differentiation between BSE-infected cattle and natural cases of sheep scrapie was also possible using these two antibodies. There were subtle differences in the molecular weight positions of the di-glycosylated, mono-glycosylated and unglycosylated forms of the abnormal PrP (PrP(Sc)) associated with these ruminant transmissible spongiform encephalopathies. In particular, a distinct difference for the unglycosylated protein band was observed. For ovine scrapie samples, this band was noticeably of a higher molecular weight than that found for brain samples from the Romney and Cheviot breed sheep infected with BSE and, to a lesser degree, higher than that observed for bovine BSE samples. Using the comparison of glycoform ratios, the technique provided a distinction between the sheep experimentally infected with BSE and natural cases of sheep scrapie but did not provide a distinction between natural cases of bovine BSE and ovine scrapie. The sheep-passaged CH1641 scrapie strain gave molecular weights similar to, but not identical to BSE, and a glycoform ratio similar to ovine scrapie cases. The SSBP1 experimental scrapie strain gave molecular weights that were akin to natural scrapie cases but the glycoform ratio was different to that found for all the other samples. When mAb P4 was substituted for mAb 6H4 in the technique, only the natural scrapie samples and SSBP1 gave strong signals. BSE in sheep and the CH1641 strain gave weak reactions and PrP(Sc) from BSE-infected cattle could not be detected at all. The results suggest that this combination of molecular weight and glycoform ratio analyses, and differentiation with two specific antibodies could be used to provide a possible screening test for BSE in the UK sheep flock, if confirmed as accurate by bioassay and lesion profile analysis in mice inoculated with brain tissue from suspect field cases.     

Acceleration of scrapie in neonatal Syrian hamsters

Prions cause Creutzfeldt-Jakob disease, Gerstmann-Str?ussler syndrome, and kuru of humans as well as scrapie of animals. Prolonged incubation periods, from months to decades, precede clinical disease. In studies on the biochemical characteristics of prions, weanling Syrian hamsters have been used extensively because they have relatively short incubation periods. In studies reported here, inoculation of neonatal hamsters significantly shortened the scrapie incubation period even further. Our results show that the scrapie incubation period in hamsters is a function of age. The interval between inoculation and death from scrapie plotted as a function of age (0 to 30 days) gave a correlation coefficient (r) of 0.86. The duration of clinical disease was also shortened in the hamsters inoculated as neonates compared with weanlings. Intraventricular injection of nerve growth factor prior to inoculation of neonates with scrapie significantly diminished the acceleration observed with scrapie alone in neonates. Histopathologic studies of brain from scrapie-inoculated neonates showed more extensive neuronal loss in the hippocampus and neocortex as well as a more profound gliosis in the caudate compared with animals inoculated as weanlings. Our results demonstrate an age-dependent acceleration of scrapie in neonatal hamsters and may provide a new experimental system for defining factors that modify the pathogenesis of prion diseases.     

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'.     

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.     

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.     

Ruthenium red and lanthanum nitrate a possible tracer and negative stain for scrapie “particles”?

Summary The cucumber- or sausage-shaped particles described before in scrapie rat and natural scrapie sheep are also found in scrapie mouse with the use of ruthenium red and lanthanum nitrate. It is suggested that they have a nucleic acid core surrounded by a coat of acid polysaccharide. The plasma membranes are pair of unit membranes.     

Effects of the polyene antibiotic derivative MS-8209 on the astrocyte lysosomal system of scrapie-infected hamsters

Amphotericine B (AmB), a macrolide polyene antibiotic, is one of a few drugs that has shown therapeutic properties in scrapie-infected hamster. Its beneficial effect on survival time is mostly marked when animals are treated with its derivative MS-8209. To explore the MS-8209 effect at the cellular level, we investigated at the light and electron microscopy levels, the sequential appearance and distribution of PrP concurrently with histopathological changes in hamsters that were infected intracerebrally with the 263 K scrapie strain and treated or not with the drug. The first histopathological modifications and PrP immunostaining were observed in the thalamus and at the inoculation site where the drug caused a delay in the appearance of lesions and PrP accumulation. Using immunoelectron microscopy, at 70 d postinfection, the inoculation site of untreated animals showed an accumulation of PrP in plaque areas constitued by filaments mixed with alterated membrane structures and in developed lysosomal system of reactive astrocytes. Most of the numerous lysosomes containing PrP showed intra-organelle filaments. In contrast, in MS-8209 treated animals, the number of lysosomes was significantly lower (p<0.0038), with very few organelles harboring PrP. Our results suggest that in this scrapie model, MS-8209 treatment delays the disease by preventing the replication of the scrapie agent at the inoculation site where the astrocytes appear to be the first cells producing abnormal PrP. The lysosomal system of these astrocytes could constitute a privileged target for MS-8209.     

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.