Ultrastructural Immuno-localization of Synthetic Prion Protein Peptide Antibodies in 87V Murine Scrapie

Disease specific forms of a host encoded cell surface sialoglycoprotein called prion protein (PrP) accumulate during the incubation period of the transmissible spongiform encephalopathies. A 33–35 kDa disease specific form of PrP is partially resistant to protease digestion whereas the normal form of PrP can be completely digested. Proteinase K digestion of the murine disease specific form of PrP produces diverse forms of low molecular weight PrP, some of which are N-terminally truncated at amino acid residue 49 or 57 within the octapeptide repeat segment. Amyloid plaques are a pathological feature of many of the transmissible spongiform encephalopathies and are composed of PrP. Using synthetic peptide antibodies to the N-terminus of PrP (which is not present in truncated disease specific PrP) and antibodies to the protease resistant fraction of PrP we have immunostained plaques and pre-amyloid deposits in the brains of mice, experimentally infected with the 87V strain of scrapie, for examination by light and electron microscopy. Classical fibrillar amyloid deposits in plaques as well as pre-amyloid deposits were both immunostained by antibodies to the N-terminus of PrP and to the protease resistant core of the PrP molecule. This suggests that both N-terminal and core amino acid residues are present in disease specific PrP released from scrapie infected cells in vivo. The results also suggest that N-terminal truncation of PrP may not be essential for formation of amyloid fibrils.     

In vivo toxicity of prion protein in murine scrapie: ultrastructural and immunogold studies

Prion protein (PrP) is a cell surface, host coded, sialoglycoprotein which accumulates in excess in scrapie, Creutzfeldt‐Jakob disease, bovine spongiform encephalopathy and other transmissible spongiform encephalopathies. Infection of mice with the 87 V or ME7 scrapie strains results in distinctive and very different light microscopical patterns of vacuolation and disease specific PrP accumulation. In both of these scrapie strains immunogold electron microscopy was used to locate PrP to the plasmalemma of neurons from where it was released into the neuropil. Initial PrP accumulation around neurons and in early plaques lacking amyloid fibrils was generally not associated with morphological changes either of the neuron or dendrite releasing the PrP or in the adjacent neuropil in which excess PrP accumulated. However, accumulation of pre‐amyloid PrP in some brain areas was associated with specific degeneration of dendritic spines and axon terminals. Initial PrP aggregation into fibrils was also associated with tissue damage with both ME7 and 87 V plaques and diffuse accumulations. Tissue damage associated with fibrillogenesis was localized and would not be expected to have clinical significance. We conclude that pre‐amyloid PrP release and accumulation is not invariably toxic, either to the neuron releasing PrP or to the neuropil into which it is released. However, axon terminal degeneration and dendritic spine loss in some neuroanatomical areas may be indicative of specific PrP toxicity and may be the main cause of neurological dysfunction in murine scrapie.     

Induction of antibodies against murine full-length prion protein in wild-type mice

The causative and infectious agent of the transmissible spongiform encephalopathies, e.g. bovine spongiform encephalopathy in cattle or variant Creutzfeldt-Jakob disease in humans, is a pathogenic form of the scrapie prion protein (PrP(Sc)) generated by a conformational rearrangement in the normal cellular prion protein (PrP(C)). Anti-PrP antibodies have been shown to exert a protective effect against infection with PrP(Sc). However, the generation of anti-PrP antibodies has proven quite difficult in wild-type animals, PrP being a notoriously poor immunogen. We developed a vaccine against PrP by mixing recombinant murine PrP 23-231 with DnaK, an Hsp70 homolog in Escherichia coli, and cross-linking the two proteins by means of glutaraldehyde. After three injections of the vaccine into BALB/c mice at 6, 8 and 9 weeks of age, a low-titer immune response was detected with ELISA in all animals. The specificity of the antibodies for PrP was confirmed with Western blotting. The straightforward procedure might render active immunization against prion infection feasible.     

Antiserum to scrapie-associated fibril protein reacts with amyloid plaques in familial transmissible dementia

Scrapie-associated fibrils (SAF) are disease-specific markers for the unconventional agent-induced, transmissible spongiform encephalopathies. Polyclonal rabbit antiserum to SAF protein was reacted with brain sections from scrapie-infected mice, two familial cases of transmissible dementia, and three cases of Alzheimer's disease (AD). Specific immunostaining of cerebral amyloid plaques occurred in the scrapie-infected mice and in the two familial cases of transmissible dementia. No immunoreactivity was detected in senile plaques or neurofibrillary tangles in the three cases of AD. Our results suggest that SAF, the causative pathogenic agent, and extracellular deposits of amyloid in the brain are closely related. Immunohistochemical detection of SAF protein could serve as a useful diagnostic adjunct in the postmortem evaluation of difficult cases of dementia. The identification of SAF protein in the brains of two affected members of a family combining the clinical and pathological features of Creutzfeldt-Jakob disease (CJD) and the Gerstmann-Straüssler syndrome (GSS) substantiates earlier conclusions of a nosological relationship between the two. Our study provides further evidence of the similarity of SAF protein to prion protein (PrP 27-30).     

Comparison of PrP transcription and translation in two murine myeloma cell lines

The generation of monoclonal antibodies (MAbs) to the prion protein, PrP, is important in order to establish a large repertoire of useful reagents for the diagnosis of transmissible spongiform encephalopathies (TSE), or prion diseases. However, the presence of PrP on the surface of all mammalian cells (PrP^C) causes self-recognition, thereby restricting the ability of mice to produce an immune response to the PrP immunogen. Although this problem has been alleviated with the generation and use of PrP-knockout mice, the production of MAbs has continued to be severely hampered presumably since the fusion partner for spleen-derived lymphocytes was PrP^C-containing myeloma cell lines. The availability of a mouse myeloma cell line expressing little or no PrP^C on the surface would therefore be useful for MAb generation. Our data indicate that cells differ in their levels of PrP^C expression and suggest that not all murine myeloma cell lines are equally useful for obtaining hybridomas secreting anti-PrP MAbs.     

Morphogenesis of amyloid plaques in 87V murine scrapie†

Amyloid plaques of scrapie–infected mouse brains are composed of fibrillar forms of a host coded, cell surface sialoglycoprotein called PrP (prion protein). Serial ultrastructural immunogold staining was performed on plaques identified by light microscopic immunocytochemistry of brains of VM mice infected with the 8 7V strain of scrapie. Classical plaques, of a kuru–type morphology, were composed of a central core of bundles of amyloid fibrils. Amyloid fibrils of classical plaques were immunoreactive for PrP. In addition, PrP was also found at the plaque periphery, in the absence of fibrils, at the plasmalemma of cell processes and in the associated extracellular spaces. Frequent microglial cells and occasional astrocytes contained PrP within lysosomes. Other plaques with few or no recognizable amyloid fibrils were frequent and were termed primitive plaques. PrP could be demonstrated in a non–fibrillar form at the plasmalemma and in the extracellular spaces between neurites of such plaques. Many primitive plaques showed little or no sub–cellular pathology associated with the PrP accumulation. PrP was closely associated with the plasma–lemma of occasional dendrites passing towards the centre of primitive plaques. These results suggest that plaques are formed around one or more PrP releasing dendrites. PrP accumulates in the extracellular spaces adjacent to such processes prior to its spontaneous aggregation into fibrils. Lysosomal accumulation of PrP in microglia and astrocytes located at the periphery of plaques suggest that these cells are involved in the phagocytosis of excess or abnormal PrP.     

Heparan Sulfate Proteoglycan Is Associated with Amyloid Plaques and Neuroanatomically Targeted PrP Pathology throughout the Incubation Period of Scrapie-Infected Mice

Heparan sulfate proteoglycan (HSPG) has been found to be associated with amyloid deposits in a number of diseases including the cerebral amyloid plaques of Alzheimer's disease and the transmissible spongiform encephalopathies (TSEs). The role of HSPG in amyloid formation and the neurodegenerative pathology of these diseases have not been established. We have addressed these questions using a scrapie mouse model which exhibits both amyloid and nonamyloid deposition of abnormal PrP protein, the protein marker of TSE infection. The distribution of HSPG was examined throughout the course of the disease in the brains of experimentally infected mice and compared with the distribution of abnormal PrP. Abnormally high levels of HSPG were associated with most types of PrP pathology including all plaque types and diffuse neuroanatomically targeted forms. Scrapie-associated HSPG was present from 70 days after infection, the earliest time-point examined, in the same target areas as abnormal PrP. The association with amyloid plaques may indicate that HSPG is involved in amyloid plaque formation and/or persistence but involvement with early diffuse forms of PrP suggests a more fundamental role in scrapie pathogenesis.     

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.     

Cerebellar amyloid plaques in Alzheimer's disease

In a consecutive series of 30 brains of demented patients (presenile, senile and familial types) with the histological hallmarks of Alzheimer's disease, cerebellar amyloid plaques and cerebellar amyloid angiopathy were observed in 80% of the cases. These cerebellar amyloid plaques were sometimes centered on a small amyloidotic blood vessel. They were immunostained with A4 antiserum, but they were not surrounded by a crown of swollen neurites as demonstrated with silver impregnation and Tau antiserum. They were not immunostained with SAF antiserum which decorated the cerebellar Kuru-like plaques observed in subacute transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker syndrome. The absence of neuritic changes around the numerous cerebellar amyloid plaques frequently observed in cases of Alzheimer's disease is an interesting feature and will perhaps explain the mechanism of cytoskeleton changes occurring in the neurons of the cerebral cortex.     

Cerebral amyloid in human prion disease

The clinical and neuropathological features of 21 patients with prion disease were reviewed with special reference to the morphology and immunoreactivity of cerebral amyloid. Six cases had a mutation at codon 102 of the prion protein (PrP) gene and in these the characteristic pathology was the formation of multicentric amyloid plaques which were stained with PrP antibody, whereas spongiform changes were absent in one and minimal in two. In one case, with a 216 base-pair insertion in the PrP gene, there was no spongiform encephalopathy (SE) but cerebellar amyloid was a prominent feature of the pathology. One case with a PrP gene mutation at codon 200 had severe SE but no amyloid. Two iatrogenic and 11 sporadic cases had SE and some form of amyloid was identified in all but three. Amyloid angiopathy and senile neuritic plaques, which stained with antibody to β-protein, were present in familial as well as in sporadic cases, including some who were rather young to be regarded as having Alzheimer's disease. Cerebellar amyloid and degeneration of granule and Purkinje cells were particularly common findings in sporadic as well as in genetically determined cases. This study serves to emphasize the association between prion disease and amyloid deposition in the brain. PrP is a component of some amyloid plaques in a high proportion of cases with inherited prion disease but may also be found in cases of sporadic SE without known mutations or base-pair insertions in the PrP gene.     

Temporal and spatial relationship between the death of PrP-damaged neurones and microglial activation

Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.     

Aged PrP null mice show defective processing of neuregulins in the peripheral nervous system

A prion, a protease-resistant conformer of the cellular prion protein (PrP(C)), is the causative agent of transmissible spongiform encephalopathies or prion diseases. While this property is well established for the aberrantly folded protein, the physiological function of PrP(C) remains elusive. Among different putative functions, the non-pathogenic protein isoform PrP(C) is involved in several cellular processes. Here, we show that PrP(C) regulates the cleavage of neuregulin-1 proteins (NRG1). Neuregulins provide key axonal signals that regulate several processes, including glial cells proliferation, survival and myelination. Interestingly, mice devoid of PrP(C) (Prnp?/?) were recently shown to have a late-onset demyelinating disease in the peripheral nervous system (PNS) but not in the central nervous system (CNS). We found that NRG1 processing is developmentally regulated in the PNS and, by comparing wildtype and Prnp?/? mice, that PrP(C) influences NRG1 processing in old, but not in young, animals. In addition, we found that also the processing of neuregulin-3, another neuregulin family member, is altered in the PNS of Prnp?/? mice. These differences in neuregulin proteins processing are not paralleled in the CNS, thus suggesting a different cellular function for PrP(C) between the CNS and the PNS.     

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.)     

Amyloid plaques in Creutzfeldt-Jakob disease stain with prion protein antibodies

Amyloid plaques are found in the brains of some patients with Creutzfeldt-Jakob disease (CJD) and all patients with a related transmissible disorder, Gerstmann-Str?ussler syndrome (GSS). In scrapie, a prion disease of animals, amyloid plaques have been shown to be composed of prion proteins (PrP), which form filaments of relatively uniform diameter. We report here that antisera raised against hamster scrapie PrP specifically stain amyloid plaques in the brains of both humans and rodents with CJD as well as a human subject with GSS. Earlier studies showed that these antibodies react with both rodent and human CJD PrP. The immunostained congophilic amyloid plaques in rodent brains measured 10 to 30 micron in diameter and exhibited a Maltese cross appearance. Limited proteolysis enhanced immunostaining of amyloid plaques in human brain sections from patients with CJD or GSS. Presumably proteolysis increases the exposure of those epitopes shared by human and rodent PrP. The differences in immunoreactivity between rodent and human amyloid plaques are consistent with other findings showing that cellular genes, not infectious purified prions, encode PrP.     

Amyloid and prions: some biochemical investigations of cerebral amyloidosis in mice

Prion-like transmission of protein aggregates or amyloid in several neurodegenerative diseases, such as Parkinson's disease, Huntington's disease and Alzheimer's disease, in addition to the transmissible spongiform encephalopathies (or prion diseases), has been proposed recently. This is a controversial idea and, in this paper, we consider what we mean by a "prion", and by "amyloid", and present some biochemical investigations of cerebral prion amyloidosis in mice.     

Tubulovesicular particles occur early in the incubation period of murine scrapie

Tubulovesicular bodies are structures, apparently specific to the transmissible spongiform encephalopathies, which are of unknown composition and significance. Prion protein (PrP) is absent from tubulovesicular bodies when tissues are examined by immunogold electron microscopy. In the F1 cross of C57 and VM mice (CVF1) infected with ME7 scrapie there is a marked degeneration of hippocampal CA1 neurons. In this model the earliest changes seen, at about 100 days post inoculation (dpi) are a degeneration of axon terminals and synaptic loss. Terminal disease is around 250 dpi. In blind coded trials we counted the number of tubulovesicular particles and estimated their density in 56–76 electron micrographs taken from the stratum radiatum of each of one or two CVF1 ME7-infected mice at 84, 100, 126, 154 and 181 dpi and from four normal brain inoculated control mice. Tubulovesicular particles were present from 98 dpi and the density of particles increased with increasing incubation period. The very early occurrence of tubulovesicular particles, before the presence of significant pathology, argues that tubulovesicular particles are a part of the primary disease and are not epiphenomena. Received: 28 June 1999 / Revised: 30 August 1999 / Accepted: 6 September 1999     

Kuru: genes, cannibals and neuropathology

Kuru was the first human transmissible spongiform encephalopathy (TSE) or prion disease identified, occurring in the Fore linguistic group of Papua New Guinea. Kuru was a uniformly fatal cerebellar ataxic syndrome, usually followed by choreiform and athetoid movements. Kuru imposed a strong balancing selection on the Fore population, with individuals homozygous for the 129 Met allele of the gene (PRNP) encoding for prion protein (PrP) being the most susceptible. The decline in the incidence of kuru in the Fore has been attributed to the exhaustion of the susceptible genotype and ultimately by discontinuation of exposure via cannibalism. Neuropathologically, kuru-affected brains were characterized by widespread degeneration of neurons, astroglial and microglial proliferation, and the presence of amyloid plaques. These early findings have been confirmed and extended by recent immunohistochemical studies for the detection of the TSE-specific PrP (PrP). Confocal laser microscopy also showed the concentration of glial fibrillary acidic protein-positive astrocytic processes at the plaque periphery. The fine structure of plaques corresponds to that described earlier by light microscopy. The successful experimental transmission of kuru led to the awareness of its similarity to Creutzfeldt-Jakob disease and Gerstmann-Str?ussler-Scheinker disease and formed a background against which the recent epidemics of iatrogenic and variant Creutzfeldt-Jakob disease could be studied.     

Spongiform encephalopathy in transgenic mice expressing a point mutation in the β2-α2 loop of the prion protein

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a β-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.