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 plaques in the brains of mice with Creutzfeldt-Jakob disease

By intracerebral inoculation with the brain homogenates from 9 patients with Creutzfeldt-Jakob disease, amyloid plaques were induced in mouse brains after incubation periods of 403 to 835 days. The plaques existed mainly in the cerebral white matter beneath the lateral ventricle walls and were more numerous in the hemisphere where injection was made. Morphological findings of the plaques were almost identical to those seen in patients with kuru and Creutzfeldt-Jakob disease, and in animals with scrapie. They were also similar to the cores of senile plaques seen in patients with Alzheimer's disease.     

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'.     

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).     

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.     

Serum amyloid P immunoreactivity in hippocampal tangles, plaques and vessels: implications for leakage across the blood-brain barrier in Alzheimer''s disease

Serum amyloid P (SAP) has been shown to be consistently present in all types of amyloid deposits except cerebral lesions of neurofibrillary tangles and senile plaques. We used immunohistochemical methods to demonstrate SAP reactivity in both tangles and plaques, as well as vessels, in lightly fixed frozen tissue sections of hippocampus and parahippocampal gyrus from subjects with Alzheimer's disease (AD) and normal controls. As confirmed by thioflavin S staining, heavy deposition of immunoperoxidase reaction product was evident in Sommer's sector (CA1), the subiculum and entorhinal cortex with both the antisera to SAP used. Serial sections immunostained with antiserum to amyloid A or preimmune rabbit serum showed no evidence for staining in plaques or tangles. These observations provide evidence for extravasation of the protein across the blood-brain barrier (BBB) in disease although expression of it by cellular elements within or entering the brain through the BBB cannot be ruled out. Our results also implicate the use of lightly fixed tissue for localization of some antigens by immunohistochemistry in postmortem human brain.     

Genetic control of amyloid plaque production and incubation period in scrapie-infected mice

The extent of amyloid plaque production was investigated in three inbred mouse strains carrying the p7 allele of the scrapie incubation (Sinc) gene (VM, IM and MB). With either ME7 or 87V scrapie, many more plaques were seen in the MB strain than in VM or IM mice. A backcrossing experiment using 87V suggested the involvement of more than one gene. Within this backcrossing experiment there was a positive correlation between mean plaque count and mean incubation period for the various strains and crosses. Also male mice tended to have higher plaque counts and longer incubation periods than female mice of the same genotype. These results suggest that some of the genes controlling minor variation in the incubation period also influence plaque production. This is consistent with previous evidence that the number of amyloid plaques depends, to some extent, on the duration of agent replication within the brain. This study has also identified a high plaque model (MB mice infected with 87V) for future investigation of the nature of the amyloid protein.     

AMYLOID PLAQUES IN THE BRAINS OF MICE INFECTED WITH SCRAPIE: MORPHOLOGICAL VARIATION AND STAINING PROPERTIES

Amyloid plaques in the brains of mice infected with scrapie: morphological variation and staining properties
Cerebral amyloid deposits predominantly in the form of plaques are associated with experimental scrapie produced by particular agents in inbred mice. In this paper the staining properties and variation in the morphology of these deposits are described. At the light microscope level a discretionary classification into six types is made: shadowy plaques; amorphous plaques; stellate plaques; giant plaques; diffuse amyloid deposits; and perivascular amyloid deposits. It is shown that Masson's trichrome technique provides the most efficient staining method for identifying cerebral amyloid of all these types. A preliminary ultrastructural examination of stellate plaques confirms the presence of amyloid on the basis of characteristic fibrils and demonstrates that microglia and distended neurites are involved in the structure of the plaques. The similarities and differences between cerebral amyloid in scrapie and other forms of amyloid deposits in the brain, particularly kuru plaques and senile plaques, are discussed.     

The comparative immunoreactivities of brain amyloids in Alzheimer's disease and scrapie

Summary An antibody was raised to a synthetic peptide corresponding to a published sequence for the first 24 residues of a cerebrovascular amyloid peptide (CVAP). Immunohistochemical staining of tissue sections revealed that the antibody bound extensively to cerebrovascular amyloid in Alzheimer disease (AD/SDAT) and Down's syndrome cases. The antibody bound less extensively to neuritic plaques (primitive and mature) and indetectably to neurofibrillary tangles. The antibody did not label scrapie plaques, scrapie-associated fibrils, or Gerstmann-Sträussler syndrome plaques. Immunoblotting experiments showed that the cerebrovascular amyloid peptide epitopes contaminating the neurofibrillary tangle preparations could be extracted with urea, leaving the neurofibrillary tangles intact. These data confirm that the cerebrovascular amyloid peptide is a component of cerebrovascular amyloid, and suggest that its epitopes are also components of neuritic plaque amyloid. The reduced level of immunostaining on amyloid cores in tissue sections suggests that either the cerebrovascular amyloid peptide epitopes are a minor component of amyloid cores, or that their mode of packing or state of processing in amyloid cores renders them relatively inaccessible to the antibody. We also conclude that the cerebrovascular amyloid peptide is not a component of neurofibrillary tangles. The synthetic cerebrovascular amyloid peptide possesses amyloid-like properties: at neutral pH it forms insoluble aggregates consisting of 5–7-nm fibrils, which form red-green birefringent adducts with Congo red and fluoresce with thioflavine S.Supported by NH Grants Nos. AG04220, NS21349 and GM30800     

EFFECT OF ROUTE OF INFECTION ON THE FREQUENCY AND DISTRIBUTION OF CEREBRAL AMYLOID PLAQUES IN SCRAPIE MICE

Cerebral amyloid plaques are a conspicuous pathological feature in mice infected with certain strains of scrapie. The origin of the amyloid protein in these plaques, whether it is locally or systemically synthesized, and the mechanisms leading to its deposition are not known. The frequency of plaques and their distribution in the brain are greatly influenced by the route of injection of the scrapie inoculum. Intracerebral injection consistently results in greater numbers of plaques than are obtained if the same inoculum is introduced peripherally. Following intracerebral injection, plaques are commonly seen in areas close to the lateral ventricles, for example the corpus callosum and hippocampus, whereas they are absent from these areas with peripheral routes. Furthermore, when left- and right- sided intracerebral injections are compared, plaques are more frequent on the side of injection. These results suggest that the distribution of amyloid plaques is influenced either by the localization of some component of the inoculum or by traumatic damage at injection. The most plausible explanation is that amyloid deposition is associated with local concentrations of scrapie infectivity and that the amyloid protein originates in the brain.     

Ultrastructural morphology of amyloid fibrils from neuritic and amyloid plaques

The structure of partially purified, CNS amyloid fibrils from three different sources have been compared by negative stain EM. The fibrils isolated from brains with senile dementia of Alzheimer type were 4-8 nm in diameter, narrowing every 30-40 nm and apparently composed of two 2-4 nm filaments. The fibrils from a Gerstmann-Str?ussler syndrome brain were 7-9 nm in diameter, narrowing every 70-80 nm and with a suggestion that they are composed of two 3-5 nm filaments. The fibrils isolated from 87V scrapie-affected mouse brains were 4-8 nm in diameter with a twist every 15-25 nm presumably composed of two 2-4 nm filaments. The fibrils from the scrapie brains were usually observed in pairs. The shape of the clusters of the isolated amyloid fibrils observed in each disease was similar in negative stain and thin section EM preparations and was related to the characteristic morphology of the amyloid fibrils in the neuritic and amyloid plaques in situ. The structural differences between the CNS amyloid fibrils from the various diseases studied by us may reflect differences in the polypeptides which comprise the fibril and/or a different pathogenesis in the formation of the amyloid fibrils.     

Abnormal fibrils from scrapie-infected brain

Summary Abnormal fibrillary structures, designated scrapie-associated fibrils (SAF), have been observed using negative stain techniques in subfractions of brains from scrapie-affected animals. SAF have been observed in all combinations of strain of scrapie agent and strain or species of host examined, regardless of their histopathology, in particular the presence or absence of amyloid plaques. SAF consist either of two or four filaments. They are morphologically dissimilar to the normal brain fibrils — microtubules, neurofilaments, glial filaments, and F actin. However, SAF do bear a resemblance to amyloid.     

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.     

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.     

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.     

Immunohistochemical localization of 14.3.3 ζ protein in amyloid plaques in human spongiform encephalopathies

The localization of 14.3.3 proteins was studied in different subtypes of brain amyloid plaques. We examined paraffin-embedded brain sections of sporadic MV2 Creutzfeldt-Jakob disease (sCJD) with Kuru plaques, sporadic VV2 CJD with plaque-like PrP(sc) (the abnornal form of prion protein) deposits, variant CJD (vCJD) with florid plaques, Gerstmann-Straüssler-Scheinker (GSS) with multicentric plaques and of Alzheimer's disease (AD) with senile plaques. Adjacent immunostaining revealed PrP(sc) and 14.3.3 zeta deposits in the same amyloid plaques in all cases of sporadic CJD and vCJD, whereas 14.3.3 zeta was not seen in amyloid plaques of GSS with A117V, P102L and D202N mutations. The same immunostaining method using anti-betaA4 and anti-14.3.3 zeta antibodies revealed no colocalization in patients with AD. Our data suggest that 14.3.3 zeta protein could interact either with PrP or with other components of PrP(sc) deposits in CJD.     

Lysosomal proteinase antigens are prominently localized within senile plaques of Alzheimer's disease: evidence for a neuronal origin

To investigate the role of proteolysis in amyloid formation, we studied the localization of the proteolytic enzymes, cathepsin D and cathepsin B, in the prefrontal cerebral cortex and hippocampus of human postmortem brains from patients with Alzheimer's disease and from individuals free of neurological disease. In control and Alzheimer brains, cathepsin immunoreactivity within cells was localized to lysosome-related structures, which were particularly abundant in neuronal perikarya. In Alzheimer brain, cathepsin immunoreactivity was also heavily concentrated extracellularly within senile plaques. Cathepsin immunoreactivity associated with plaques was not confined to lysosomes and was distributed throughout the plaque. Isolated amyloid cores, however, were not immunostained. Cathepsin-laden perikarya of degenerating neurons were frequently seen within senile plaques and, in the more advanced stages of degeneration, cathepsin immunoreactivity was present throughout the cytoplasm. Other identified constituents of senile plaques appeared to be less significant sources of cathepsin immunoreactivity, including astrocytes, degenerating neurites, microglia and macrophages. These results demonstrate that lysosomal proteinases are major constituents of the senile plaque and that degenerating neuronal perikarya are a principal source of the cathepsin immunoreactivity. We propose that the unregulated action of extracellular cathepsins liberated from degenerating neurons may lead to abnormal processing of the amyloid precursor protein and to the formation of amyloid locally within senile plaques in Alzheimer's disease.     

Immunoreactivity of cerebral amyloidosis is enhanced by protein denaturation treatments

Summary We investigated paraffin-embedded brain sections from three patients with Gerstmann-Sträussler syndrome and three patients with Alzheimer's disease or senile dementia of Alzheimer type using anti-human prion protein antisera and anti-/A4 protein antisera after protein denaturation treatments. After incubation with guanidine-thiocyanate, trichloroacetate, and phenol, the immunoreactivity of kuru plaques and senile plaques was enhanced to the same level as the formic acid treatment. These treatments revealed small compact amyloid deposits, amyloid deposits surrounding the plaque cores, and diffuse plaques. Most of these chemicals changed the congophilia of both amyloids. It is possible that these treatments denature amyloid fibril proteins and break down the structure of amyloid fibrils, thus revealing buried epitopes.Supported by grants (02454245, 03454171) from the Ministry of Education, Science and Culture, the Ministry of Health and Welfare, and the Science and Technology Agency, Japan     

EFFECTS OF AGE ON CEREBRAL AMYLOID PLAQUES IN MURINE SCRAPIE

Bruce M.E. & Fraser H. (1982) Neuropathology and Applied Neurobiology 8, 71–74
Effects of age on cerebral amyloid plaques in murine scrapie
The frequency of cerebral amyloid plaques in murine scrapie was not influenced by the age of the mouse at injection in a study using several combinations of agent strain and mouse strain. In addition amyloid plaques were not seen in a series of extremely old uninfected mice.     

Myelin-associated glycoprotein (MAG) in the CNS of adult shiverer (Shi/Shi) mice

Brain fractions of adult control (+/+) and shiverer (Shi/Shi) mice were investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. Immunostaining with specific antisera against rat brain myelin-associated glycoprotein (MAG) was detected at about the 96-kD region of gels in electrophoresed samples of the total homogenate, low-speed supernatant fraction, and low- and high-speed sedimentable portions of brain from +/+ mice. Reduced immunostaining was observed in the corresponding samples of brain fractions from Shi/Shi mice. The cerebrum, cerebellum, and medulla of +/+ and Shi/Shi mice were examined immunocytochemically for MAG on paraffin-embedded sections. Periaxonal immunostaining for MAG was observed in all the regions and the highest concentrations were in the corpus callosum, in the central cores of cerebellar folia, and in the medulla. Patterns of distribution were similar in +/+ and Shi/Shi mice, although the density of immunostaining around individual axons and the number of immunostained axons were significantly reduced in Shi/Shi mice. In addition, the three brain regions of Shi/Shi mice exhibited oligodendrocyte-like cells that contained immunostaining for MAG in the cytoplasm and periphery of their perikarya. This type of immunostained cell was not observed in +/+ mice. In this study, immunoblotting with brain fractions and immunocytochemistry revealed strong evidence for reduced concentrations of MAG in the CNS of Shi/Shi mice compared to control mice. In addition, there is immunocytochemical evidence for abnormal accumulation of MAG in perikarya of oligodendroglial-like cells, suggesting the possibility of a transport block for myelin proteins in the shiverer mutant.