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.     

Ultrastructural study of three cases of encephalopathy with hypsarrhythmia

Summary We report a morphological study of three cases of infantile hypsarrhythmia. The cerebral cortex lesions consisted of a diffuse neuropile microspongiosis, corresponding ultrastructurally to vacuolized and enlarged neuronal processes, particularly postsynaptic bags. The morphological aspects and the high cell fusion index in one case resemble those described in transmissible subacute spongiform encephalopathies.     

Ubiquitin immunocytochemistry in human spongiform encephalopathies

The distribution of ubiquitin was studied by immunocytochemistry in eight cases of human spongiform encephalopathy and compared with the findings in seven age-and sex-matched cases of Alzheimer's disease and six non-demented control cases. The results were also compared with the immunocytochemical distribution of prion protein and the lysosomal aspartic protease cathepsin D. In the human spongiform encephalopathies, ubiquitin immunoreactivity was found in a punctate distribution at the periphery of prion protein amyloid plaques and in a finely granular pattern in the neuropil around and within areas of spongiform change. Cortical nerve cells contained scanty ubiquitinated dot-like inclusions, and occasional microglia around the areas of spongiform change also gave a positive staining reaction for ubiquitin, as did multiple irregular thread-like structures in the neuropil and white matter. The ubiquitin-containing structures at the plaque periphery in human spongiform encephalopathies resemble the neuritic processes at the periphery of the senile plaque in Alzheimer's disease. The granular positivity for ubiquitin associated with areas of spongiform change closely resembles the pattern of immunostaining seen with the antibodies to the prion protein and cathepsin D, consistent with the reported accumulation of ubiquitinated proteins and prion protein in lysosomes in the murine scrapie model. Further studies are required to investigate the role of lysosomes in this group of disorders, and to study the localization of other cell stress proteins and prion protein in spongiform encephalopathies.     

Creutzfeldt-Jakob disease with tubulovesicular structures: an ultrastructural study.

Tubulovesicular structures (TVS) have been consistently observed in brain tissue of the transmissible spongiform virus encephalopathies such as natural and experimental scrapie, bovine spongiform encephalopathy and experimentally induced Creutzfeldt-Jakob disease (CJD). TVS were recently demonstrated in 3 cases of naturally occurring CJD. We report here the presence of TVS in another human brain with CJD, as detected in all 3 specimens by thin section electron microscopy. Their occurrence in all types of spongiform encephalopathies, irrespective of the affected host and the strain of infectious agent, emphasizes their biological significance.     

Tubulovesicular structures in Creutzfeldt-Jakob disease

Summary By electron microscopy tubulovesicular structures (TVS) have been consistently observed in brain tissue of transmissible spongiform encephalopathies such as natural and experimental scrapie, bovine spongiform encephalopathy and experimentally induced, but not naturally occurring, Creutzfeldt-Jakob disease (CJD). For the first time we report here the presence of TVS in human brains with CJD as detected by transmission electron microscopy. TVS were observed in all three CJD specimens (two biopsies, one autopsy), but they were rare and were found only in one or two location(s) per grid. TVS were seen in distended pre- and postsynaptic terminals and measured approximately 35 nm in diameter; they were smaller and of higher electron density than synaptic vesicles. Their occurrence in all types of transmissible spongiform encephalopathies irrespective of the affected host and the strain of the infectious agent suggests their biological significance.Supported by the Austrian Fund for the Advancement of Scientific Research (P8196-MED), and a grant from the Polish Academy of Science (VIII/40) (to P. P. L.)     

Identification of inter-species transmission of prion strains

The concern of the potential transmission of animal spongiform encephalopathies to humans, which arose as soon as the interspecies transmission of these diseases was recognized, has been reinforced with the emergence of bovine spongiform encephalopathy (BSE) in cattle. Recent experimental findings suggest that the infectious agent causing BSE in cattle can lead to the occurrence of a new form of Creutzfeldt-Jakob disease in humans. These findings help us understand how the transmission to humans of an animal disease may be recognized. This can involve an indirect approach through the analysis of neurodegeneration, either in the disease host, or more specifically, in genetically well-defined experimental hosts to which the disease can be transmitted. Recent experimental studies have also shown that the different molecular features of the abnormal form of the prion protein, which accumulates in the infected tissues, can provide important clues to the relationships between different spongiform encephalopathies. However, a better understanding of the molecular features associated with the specific pathogenic behavior of different strains is required. Complex relationships between the infectious agents involved in spongiform encephalopathies and the disease host can make the recognition of a link between animal prion strains and the human disease difficult to establish.     

Cell death and autophagy in prion diseases (transmissible spongiform encephalopathies)

Neuronal autophagy, like apoptosis, is one of the mechanisms of programmed cell death. In this review, we summarize current information about autophagy in naturally occurring and experimentally induced scrapie, Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome against the broad background of neural degenerations in transmissible spongiform encephalopathies (TSEs). Typically a sequence of events is observed: from a part of the neuronal cytoplasm sequestrated by concentric arrays of double membranes (phagophores); through the enclosure of the cytoplasm and membrane proliferation; to a final transformation of the large area of the cytoplasm into a collection of autophagic vacuoles of different sizes. These autophagic vacuoles form not only in neuronal perikarya but also in neurites and synapses. On the basis of ultrastructural studies, we suggest that autophagy may play a major role in transmissible spongiform encephalopathies and may even participate in the formation of spongiform change.     

Topographic distribution of scrapie amyloid-immunoreactive plaques in chronic wasting disease in captive mule deer (Odocoileus hemionus hemionus)

Summary Chronic wasting disease (CWD), a progressive neurological disorder of captive mule deer, blacktailed deer, hybrids of mule deer and white-tailed deer and Rocky Mountain elk, is characterized neuropathologically by widespread spongiform change of the neuropil, intracytoplasmic vacuolation in neuronal perikarya and astrocytic hypertrophy and hyperplasia. We report the topographic distribution of amyloid plaques reactive to antibodies prepared against scrapie amyloid in CWD-affected captive mule deer (Odocoileus hemionus hemionus). Scrapie amyloid-immunoreactive plaques were found in the cerebral gray and white matter, in deep subcortical nuclei, in isolation or in clusters in areas of vacuolation, and perivascularly, in subpial and subependymal regions. In the cerebellum, immunoreactive amyloid plaques were observed in the molecular, pyramidal and granular layers. Scrapie amyloid-immunoreactive deposits were also seen in neuronal perikarya. Furthermore, amyloid plaques in CWD-affected captive mule deer were alcianophilic at 0.3 M magnesium chloride indicating the presence of weakly to moderately sulfated glycosaminoglycans. Our data corroborate that CWD in captive mule deer belongs to the subacute virus spongiform encephalopathies.     

Pathogenetical significance of porencephalic lesions associated with intracerebral inoculation of sheep with the bovine spongiform encephalopathy (BSE) agent

Decreased rates of transmission of transmissible spongiform encephalopathies (TSEs) to sheep have been attributed to some polymorphisms of the prion protein (PrP) and to a 'species barrier' on interspecies experiments. In addition, the blood–brain barrier may be a further impediment to TSE neuroinvasion. The intracerebral (I/C) route is generally considered the most efficient for TSE transmission, as it may help to bypass those factors. Therefore, susceptibility of particular species to specific TSE agents is conducted by this route. Aims: This study characterizes the traumatic brain lesions associated with the I/C injection of the bovine spongiform encephalopathy agent in sheep, assesses the relevance of such lesions in the outcome of clinical disease and provides insight into the mechanisms of PrP^d conversion and amplification following I/C challenge. Methods: A total of 27 hemibrains have been macroscopically and immunohistochemically examined to investigate the presence of lesions compatible with the needle track and the PrP^d distribution, respectively. Results: No residual inoculum was found and the extension and severity of the traumatic brain lesions were unrelated to the clinical outcome. Sheep with PrP^d accumulation in the brain also showed conspicuous focal aggregates in the porencephalic lesions and in the circumventricular organs. In contrast, sheep without PrP^d deposits in the brain were also negative in the traumatic lesions. Conclusion: Overall, these findings suggest that the efficiency of the I/C route is due to effective absorption and blood recirculation of infection, rather than to primary amplification at the site of injection.     

Analysis of sequence variability of the bovine prion protein gene (PRNP) in German cattle breeds

Different alleles of the prion protein gene (PRNP) of human and sheep are known to be associated with varying susceptibilities to transmissible spongiform encephalopathies. However, no polymorphisms in the bovine PRNP gene with an effect on susceptibility to prion diseases have been identified to date. In this study we investigated such polymorphisms in German cattle; 48 healthy animals from six different German cattle breeds and 43 cattle with bovine spongiform encephalopathy (BSE) were analyzed. In contrast to previous studies, all three exons as well as the promoter region of the PRNP gene were investigated. Sequence variants in the bovine PRNP gene could have an impact on the amino acid sequence or the expression level of the prion protein and thus on susceptibility to BSE. We identified a total of 60 polymorphisms in the PRNP gene of German cattle. Of these 60 polymorphisms, 36 were newly identified, whereas 24 of these polymorphisms had been described previously. We did not detect any novel polymorphisms affecting the amino acid sequence of the prion protein. However, we identified a 23-bp insertion/deletion polymorphism in the putative PRNP promoter region that shows a significant association with BSE susceptibility in our animals.Database accession BN000291.     

Microglia and the pathogenesis of spongiform encephalopathies

Alterations in the phenotype and function of microglia, the resident mononuclear phagocytes of the central nervous system, are among the earliest indications of pathology within the brain and spinal cord. The prion diseases, also known as spongiform encephalopathies, are fatal neurodegenerative disorders with sporadic, genetic or acquired infectious manifestations. A hallmark of all prion diseases is the aberrant metabolism and resulting accumulation of the prion protein. Conversion of the normal cellular protein [PrP^c] into the abnormal pathogenic (or disease-causing) isoform [PrP^Sc] involves a conformational alteration whereby the α-helical content is transformed into β-sheet. The histological characteristics of these disorders are spongiform change, astrocytosis, neuronal loss and progressive accumulation of the protease-resistant prion isoform. An additional upregulation in microglial response has been reported in Kuru, Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome (GSS), scrapie, in transgenic murine models and in culture, where microglial activation often accompanies prion protein deposition and neuronal loss. This article will review the roles of microglia in spongiform encephalopathies.     

Prion protein immunocytochemistry: reliable protocols for the investigation of Creutzfeldt–Jakob disease

Current criteria for the histological diagnosis of Creutzfeldt–Jakob disease (CJD) include features such as spongiform change, neuronal loss and reactive gliosis which are shared to a varying extent with other neurodegenerative disorders. Reliable visualization of prion protein (PrP) has substantial potential value in diagnostic practice and as a research tool, since accumulation of the disease–associated isoform of this protein is apparently specific for spongiform encephalopathies. A number of antisera against PrP have previously been employed in conjunction with a range of pre–treatments designed to optimize the specificity of immunostaining; such varied usage makes the comparison and interpretation of results difficult. This study was undertaken to identify optimal combinations of each of three PrP antisera and five pre–treatments designed to specifically demonstrate disease–specific PrP in a series of seven CJD cases, six cases of Alzheimer–type dementia and six non–demented control cases. Specific staining of amyloid plaques, spongiform neuropil, neurons and, occasionally, astrocytes was achieved in CJD cases. Alzheimer and control cases were unstained. Use of formic acid with guanidine thiocyanate, and hydrolytic autoclaving with IB 3 and SP30 antisera proved most effective and can be recommended for future immunocytochemical studies. PrP immunocytochemistry revealed a greater extent of subcortical neural involvement than routine histological techniques in CJD; the relationship between classical neuropathology in CJD and PrP accumulation as revealed by immunocytochemistry is not clear cut and requires further investigation. These findings may help to broaden our understanding of human spongiform encephalopathies, and have implications for diagnostic practices in neuropathology.     

Spongiform encephalopathy mimicking corticobasal degeneration.

The presentation of subacute spongiform encephalopathies (SSE) is varied. The following case of SSE presented clinically similar to corticobasal degeneration. The SSE diagnosis was suspected because of magnetic resonance imaging (MRI) findings and confirmed pathologically.     

Encephalopathy caused by prions

The concept of prion encephalopathy has emerged from such previous notions as slow virus infections, spongiform encephalopathies or transmissible dementias. The term prion (Prusiner, 1982) is now used in preference to unconventional agents. Proteins and genes of prions have recently been identified by molecular biology. Exactly how prion proteins are amplified in cells is still unknown. It has been demonstrated that amyloid deposits in scrapie-infected brain, Creutzfeldt-Jakob, Gerstmann-Straüssler and Kuru diseases are composed of prion proteins. Prion encephalopathies are good models to study some immunopathological mechanisms observed in the central nervous system in degenerative diseases or ageing.     

Quinacrine does not prolong survival in a murine Creutzfeldt-Jakob disease model

Paramount among issues relating to the transmissible spongiform encephalopathies (also known as prion diseases) is the absence of any effective therapy. This need has been heightened by the substantial European and emerging global problem of bovine spongiform encephalopathy and consequent variant Creutzfeldt-Jakob disease. Stimulated by the recent reports of a potent antiprion effect in cell culture-based clearance assays, we studied the utility of quinacrine in a well-characterized in vivo model of mouse-adapted transmissible spongiform encephalopathy. Our results failed to show any evidence that quinacrine is effective when using the simple but objective measure of survival prolongation.     

INTRANUCLEAR VACUOLES IN CREUTZFELDT-JAKOB DISEASE

In an electron microscopic study of brain biopsy material from a case with Creutzfeldt-Jakob disease intranuclear vacuoles containing membrano-vesicular structures were found. To our knowledge this finding has not been previously reported in human spongiform encephalopathies. It may represent a specific alteration associated with Creutzfeldt-Jakob disease, and suggests the possibility of nuclear infection.     

Photoreceptor degeneration in experimental transmissible mink encephalopathy of hamsters

Hamsters were inoculated intracerebrally with the agent of transmissible mink encephalopathy and developed clerical signs of encephalopathy. Photoreceptor degeneration occurred in all animals examined histologically. The changes were similar to those in scrapie, although less extensive. The findings suggest that either transmissible mink encephalopathy is a mink-adapted form of scrapie or, in rodents, photoreceptor degeneration is a characteristic of infection with agents of the spongiform encephalopathies.     

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.     

Abnormal prion protein is associated with changes of plasma membranes and endocytosis in bovine spongiform encephalopathy (BSE)-affected cattle brains

Aims: Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases of man and animals characterized by vacuolation and gliosis of neuropil and the accumulation of abnormal isoforms of a host protein known as prion protein (PrP). It is widely assumed that the abnormal isoforms of PrP (PrP^d, disease-specific form of PrP) are the proximate cause of neurodegeneration. Methods: To determine the nature of subcellular changes and their association with PrP^d we perfusion-fixed brains of eight bovine spongiform encephalopathy (BSE)-affected cows and three control cattle for immunogold electron microscopy at two different neuroanatomical sites. Results: All affected cattle presented plasma membrane alterations of dendrites and astrocytes that were labelled for PrP^d. PrP^d on membranes of dendrites and occasionally of neuronal perikarya was associated with abnormal endocytotic events, including bizarre coated pits and invagination of the plasma membrane. BSE-affected cattle also presented excess and abnormal multivesicular bodies, sometimes associated to the plasma membrane perturbations. In contrast, two TSE-specific lesions, vacuolation and rare tubulovesicular bodies, were not labelled for PrP^d as were a number of other nonspecific lesions, such as autophagy and dystrophic neurites. At least two different morphological pathways to vacuoles were recognized. Conclusions: When compared with other TSEs, these changes are common to those of sheep and rodent scrapie and shows that there are consistent membrane toxicity properties of PrP^d. This toxicity involves an aberration of endocytosis. However, it is by no means clear that the lesions are of sufficient severity to result in clinical deficits.     

Does PRNP gene control the clinical and pathological phenotype of human spongiform transmissible encephalopathies?

BACKGROUND: Human spongiform transmissible encephalopathies (TSE) are a group of neurodegenerative diseases caused by a transmissible not yet recognized agent; their distinctive neuropathological features are astrocytosis, spongiform lesions of the neuropil, neuronal loss and occasionally amyloid plaques in the cortical and subcortical gray matter. TSE are biochemically characterized by the deposition in the nervous system of an amyloid-type protein, PrPres derived from the post-translational modification of a normal protein, PrPsen. The expression of this protein is controlled by the PRNP gene mapped on chromosome 20 in man. A number of point mutations of the PRNP gene have been described in the familial forms of these TSE. Some of these mutations have been associated with differences in the phenotypic expression of the disease. MATERIAL AND METHODS: This study was designed to verify whether it was possible to identify a selective phenotype depending upon a given PRNP modified genotye; for this purpose, a group of familial TSE cases (CJD 210ILE, CJD 201LYS, FFI 178ASN) were selected and their neuropathological profiles have been compared with those of a large series of sporadic CJD cases. RESULTS: No significant differences were found between the topography and severity of lesions in the cerebral cortex, cerebellum, hippocampus, basal ganglia and thalamus between the two groups. Two differences were found: the clinical duration of the disease which appeared significantly (p = 0.02) shorter in the 210ILE-mutated cases compared to that of non-mutated sporadic cases. The highly selective vulnerability of thalamus in FFI showing a severe pathology especially in its dorso-medial part in comparison with that of the sporadic CJD cases. CONCLUSION: The results of this study confirm that the different polymorphism at codon 129 of the PRNP gene, which could be involved in the structural "domains" of human PrP, might modulate the pathological phenotype of TSE.