CNS amyloid proteins in neurodegenerative diseases

The amyloid plaques found in neurodegenerative diseases show considerable morphologic diversity. Two amyloidogenic proteins have been isolated from the brains of humans and animals with neurodegenerative diseases--beta-protein from Alzheimer's disease (AD) and Down's syndrome, and prion protein (PrP) from scrapie and Creutzfeldt-Jakob disease (CJD). Using monoclonal antibodies to a synthetic peptide corresponding to a portion of beta-protein and rabbit antiserum to hamster scrapie PrP 27-30, we examined in situ amyloid plaques on sections from cases of neurodegenerative diseases, including cases with a spectrum of plaque types. Anti-beta-peptide stained cerebrovascular and plaque core amyloid in all AD cases as well as cerebrovascular amyloid and senile plaque core amyloid in five elderly CJD cases. Anti-PrP stained plaques in CJD, kuru, and Gerstmann-Str?ussler syndrome cases but not cerebrovascular amyloid or plaques in AD. Dual localization experiments showed that in cases with a mixture of plaque types, the antibodies identified different populations of plaques that showed anatomic heterogeneity. Colocalization of the two proteins was not observed in any plaque type. The data suggest that in neurodegenerative diseases two major plaque types exist, which have different etiologic origins. Our results emphasize the need for classification of CNS amyloids based not on their morphology but on the macromolecular components comprising these pathologic polymers.     

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.     

Human Prion Diseases: Creutzfeldt-Jakob Disease,Gerstmann-Sträussler Syndrome,and Unknown Dementia

Recent molecular genetic and immunohistochemical studies have revealed that various dementias belong to the group of prion disease. The clinicopathological variabilities of these disorders can be explained by prion protein (PrP) gene polymorphisms. Here we report on the usefulness of classifying prion diseases into plaque types and non-plaque types, based on the distribution of PrP in the central nervous system. The non-plaque type prion diseases are characterized by rapidly progressing dementia, myoclonus, and periodic synchronous discharges in the electroencephalogram, and by the presence of diffuse gray matter accumulations of PrP, including the synaptic strutures. The plaque type prion diseases have a long clinical course without myoclonus and periodic synchronous discharges, and the major PrP accumulation sites are extracellular PrP plaques. The distribution of abnormal PrP accumulations, either plaque type or non-plaque type is influenced by the polymorphism of the PrP gene and in turn the distribution of PrP deposits within the central nervous system influences the clinical and pathological features of prion diseases.     

Lack of TAR-DNA binding protein-43 (TDP-43) pathology in human prion diseases

Aims: TAR-DNA binding protein-43 (TDP-43) is the major ubiquitinated protein in the aggregates in frontotemporal dementia with ubiquitin-positive, tau-negative inclusions and motor neurone disease. Abnormal TDP-43 immunoreactivity has also been described in Alzheimer's disease, Lewy body diseases and Guam parkinsonism–dementia complex. We therefore aimed to determine whether there is TDP-43 pathology in human prion diseases, which are characterised by variable deposition of prion protein (PrP) aggregates in the brain as amyloid plaques or more diffuse deposits. Material and methods: TDP-43, ubiquitin and PrP were analysed by immunohistochemistry and double-labelling immunofluorescence, in sporadic, acquired and inherited forms of human prion disease. Results: Most PrP plaques contained ubiquitin, while synaptic PrP deposits were not associated with ubiquitin. No abnormal TDP-43 inclusions were identified in any type of prion disease case, and TDP-43 did not co-localize with ubiquitin-positive PrP plaques or with diffuse PrP aggregates. Conclusions: These data do not support a role for TDP-43 in prion disease pathogenesis and argue that TDP-43 inclusions define a distinct group of neurodegenerative disorders.     

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.     

Relationship of microglia and scrapie amyloid-immunoreactive plaques in kuru,Creutzfeldt-Jakob disease and Gerstmann-Sträußler syndrome

Kuru, Creutzfeldt-Jakob disease (CJD) and Gerstmann-Sträussler syndrome (GSS) are transmissible dementias affecting humans characterized neuropathologically by intraneuronal vacuolation, spongiform change, astrocytic hypertrophy and hyperplasia and the variable presence of amyloid plaques. It has been suggested that microglia are amyloid-forming cells, which play an essential role in amyloid plaque formation. To study the relationship between microglia and amyloid plaques in kuru, CJD and GSS, cerebellar tissues were examined by the double-immunostaining technique using anti-ferritin antibodies as the microglial marker and anti-scrapie amyloid antibody as plaque marker. Ferritin-immunoreactive microglia were observed interdigitating with and among unicentric, multicentric and diffuse types of scrapie amyloid-immunoreactive plaques and were found to a lesser extent in the neuropil. In kuru and CJD, scrapie amyloid-immunoreactive plaques were predominantly unicentric and were observed in the granular layer. In kuru, 53% of the amyloid plaques were associated with microglia, whereas only 30% of plaques in CJD were. In contrast, scrapie-amyloid-immunoreactive plaques in GSS were of the multicentric type, predominantly observed in the molecular layer, and 90% of these plaques were associated with microglia. Our data indicate that microglia are frequently associated with scrapie amyloid-immunoreactive plaques in GSS, less commonly in kuru and to a much lesser extent in CJD, suggesting that microglia may play a variable but important role in the formation of plaques in the transmissible spongiform encephalopathies.D.C. Guiroy is supported by the Sonderforschungsbereich 194 from the Deutsche Forschungsgemeinschaft; P.P. Liberski is a recipient of a grant from the Kosciuszko Foundation while in USA and the intramural grant from the Medical Academy Lodz, while in Poland     

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.     

Neuritic plaque evolution in Alzheimer’s disease is accompanied by transition of activated microglia from primed to enlarged to phagocytic forms

Activated microglia, overexpressing the potent neuroactive cytokine interleukin-1, have been implicated as a driving force in the evolution of diffuse amyloid deposits into diagnostic neuritic plaques in Alzheimer’s disease. To evaluate this role further, we used double-label immunohistochemistry to classify and quantify plaque-associated and non-plaque-associated activated interleukin-1-immunoreactive microglia in parahippocampal tissue from 11 patients with Alzheimer’s disease. These activated microglia were subclassified as primed (only slightly enlarged), enlarged, or phagocytic (enlarged with heterogeneous cytoplasmic contents). We further determined the distribution of these microglial subtypes among four defined plaque types. Most (84%) primed microglia were not plaque associated, although 13% were present in diffuse non-neuritic plaques and 3% were present in diffuse neuritic plaques. In contrast, most enlarged (55%) and phagocytic (91%) microglia were plaque associated. Of plaque-associated enlarged microglia, most (71%) were found in diffuse neuritic plaques with the remainder evenly distributed between diffuse non-neuritic and dense-core neuritic plaques (15% each). Of plaque-associated phagocytic microglia, a few were present in diffuse non-neuritic plaques (5%), but most were found in diffuse neuritic plaques (62%) and dense-core neuritic plaques (33%). These findings show preferential association of primed microglia with diffuse amyloid deposits and imply that microglial transformation from primed, to enlarged, to phagocytic types occurs in concert with the evolution of amyloid plaques from diffuse amyloid deposits to the neuritic β-amyloid plaque forms in Alzheimer’s disease. Microglial phagocytic activity in neuritic plaques may reflect involvement in the processing of diffuse amyloid into condensed β-amyloid, or in clearance of neuritic debris. Received: 15 January 1997 / Accepted: 17 March 1997     

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.     

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.     

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.     

Scrapie-specific neuronal lesions are independent of neuronal PrP expression

In the transmissible spongiform encephalopathies (TSE), accumulation of the abnormal disease-specific prion protein is associated with neurodegeneration. Previous data suggested that abnormal prion protein (PrP) could induce neuronal pathology only when neurons expressed the normal form of PrP, but conflicting evidence also has been reported. Understanding whether neuronal PrP expression is required for TSE neuropathological damage in vivo is essential for determining the mechanism of TSE pathogenesis. Therefore, these experiments were designed to study scrapie pathogenesis in vivo in the absence of neuronal PrP expression. Hamster scrapie (strain 263K) was used to infect transgenic mice expressing hamster PrP in the brain only in astrocytes. These mice previously were shown to develop clinical scrapie, but it was unclear whether the brain pathology was caused by damage to astrocytes, neurons, or other cell types. In this electron microscopic study, neurons demonstrated TSE-specific pathology despite lacking PrP expression. Abnormal PrP was identified around astrocytes, primarily in the extracellular spaces of the neuropil, but astrocytes showed only reactive changes and no damage. Therefore, in this model the pathogenesis of the disease appeared to involve neuronal damage associated with extracellular astrocytic accumulation of abnormal PrP acting upon nearby PrP-negative neurons or triggering the release of non-PrP neurotoxic factors from astrocytes.     

Targeting prion amyloid deposits in vivo

The diagnosis of prion diseases in humans is challenging due to a lack of specific and sensitive non-invasive tests. Many forms of human prion disease including variant Creutzfeldt-Jakob disease (vCJD), Gerstmann-Str?ussler-Scheinker (GSS) syndrome, and 10% of sporadic CJD cases are associated with amyloid deposition. Several positron emission tomography (PET) ligands have recently been developed to directly image beta-amyloid associated with Alzheimer disease. One of them, methoxy-X04, is a fluorescent derivative of Congo red with high binding affinity toward amyloid fibrils and good blood-brain barrier permeability. Using methoxy-X04, we investigated whether amyloid-targeting ligands can be also employed for direct imaging of amyloid deposits associated with some prion diseases. Such a method could potentially become a novel diagnostic approach for these conditions. Studies were performed on MB mice infected with the 87V mouse-adapted scrapie strain. Labeling of PrP amyloid plaques in brains of presymptomatic and symptomatic mice was demonstrated using in vivo transcranial two-photon microscopy after systemic administration of methoxy-X04. During real-time imaging, PrP amyloid deposits could be clearly distinguished 15 min after intravenous administration of methoxy-X04. The ligand showed rapid clearance from brain areas that did not contain amyloid deposits. PrP amyloid deposits could also be detected by direct application of methoxy-X04 on cerebellar sections from GSS patients. These results suggest that methoxy-X04 or similar derivatives could be used as PET imaging agents to improve the diagnosis of human prion diseases associated with amyloid deposition.     

Autopsied case of non‐plaque‐type dura mater graft‐associated Creutzfeldt‐Jakob disease presenting with extensive amyloid‐β deposition

We present an autopsied case of non‐plaque‐type dura mater graft‐associated Creutzfeldt‐Jakob disease (dCJD) with extensive amyloid‐β (Aβ) deposition in the brain. A 39‐year‐old Japanese woman presented with memory disturbance and abnormal behavior. The patient had a history of craniotomy with dura matter‐graft transplant for a head injury which occurred when she was 19 years old. Magnetic resonance imaging (MRI) showed hyperintensities in the cerebral cortex and striatum on diffusion‐weighted images, particularly on the dura mater‐grafted right side. Her clinical symptoms, including rapidly progressing cognitive impairment, myoclonus, and periodic sharp wave complexes on electroencephalogram, could not be distinguished from typical sporadic CJD cases. The patient died 11 months after symptom onset, and pathological investigations showed extensive spongiform degeneration with prion protein (PrP) deposition without Kuru plaques; these observations were essentially the same as those of typical sporadic CJD cases. Furthermore, Aβ immunohistochemistry showed extensive diffuse staining in the cerebral neocortex, plaque‐type deposition, positive staining in the pia mater, and cerebral amyloid angiopathy. Although the MRI findings suggested that the pathological involvement originated from the dura mater‐grafted right side, the PrP and Aβ depositions showed no apparent regionalization and laterality. Tau‐pathology including neurofibrillary tangles was hardly identified. The proteins phosphorylated α‐synuclein and phosphorylated transactivation response DNA‐binding protein 43 kDa were not detected on immunostaining. Although this report describes only one case, various speculations were made based on detailed clinical and pathological observations in conjunction with previous reports of dCJD. In particular, this report provides significant insight into the characteristics and progression of dCJD pathology and its relationship with Aβ pathology.     

Widespread serum amyloid P immunoreactivity in cortical amyloid deposits and the neurofibrillary pathology of Alzheimer's disease and other degenerative disorders

Amyloid P (AP) component is present in all types of systemic amyloid deposits. Recently, it has been shown to be also present in cerebral amyloid lesions of Alzheimer's disease (AD). In this study, we used immunocytochemical methods to extend these findings at the electron microscope level and characterize the spectrum of AP immunoreactivity in neurofibrillary pathology (NFP) of AD and other neurodegenerative disorders including Down's syndrome (DS), Creutzfeldt-Jakob, Parkinson's, Pick's and diffuse Lewy body diseases and progressive supranuclear palsy. In AD and DS, AP immunoreaction product was evident in all the classical amyloid lesions and NFP in a large sample of all cortical areas examined. The distribution and relative intensity of immunostaining was similar to that of thioflavin S staining in serial sections. In many cases, however, plaques and vessels stained by anti-AP serum were not apparent with thioflavin S. Serial sections immunostained with antiserum to amyloid A, C-reactive protein or to other proteins involved in systemic amyloidoses and the acute phase response showed no evidence of staining in any of the cerebral lesions. Electron microscopy confirmed that AP immunoreactivity was associated with the abnormal filaments characteristic of NFP as well as amyloid fibrils found in plaques and vessels showing congophilic amyloid angiopathy. Plaques of Creutzfeldt-Jakob disease, Pick bodies of Pick's disease, tangles and Lewy bodies in Parkinson's disease and a subpopulation of Lewy bodies in the diffuse Lewy body disease coexistent with AD were also stained. With the exception of vessels in two of the five cases, AP was not detected in age-matched controls. Our observations indicate AP to be a consistent feature of cerebral NFP and amyloid deposits.     

Interleukin-6 is present in early stages of plaque formation and is restricted to the brains of Alzheimer's disease patients

Interleukin-6 (IL-6) immunoreactivity has previously been shown in plaques in Alzheimer's disease (AD) and elevated IL-6 concentrations have been measured biochemically in brains of AD patients. In this study, we investigated the appearance of IL-6 immunoreactivity in AD plaques according to the stage of plaque formation. Using the Bielschowsky silver-staining method, we were able to differentiate between four types of plaques described earlier: diffuse, primitive, classic and compact. While diffuse plaques represent the early stage of plaque formation, primitive and classic plaques are thought to represent later stages of plaque development. We investigated serial sections of paraffin-embedded cortices of ten clinically diagnosed and histopathologically confirmed AD patients and ten patients with no clinical history of dementia. We found plaques in the brains of both nondemented and demented persons using the silver staining method or immunohistochemistry with antibodies against the amyloid precursor protein. In the group of clinically nondemented persons, diffuse plaques were the predominant plaque type, whereas primitive plaques formed the larger portion of lesions in the group of AD brains. IL-6 could not be detected in plaques of patients without dementia. Many IL-6-positive plaques were found in six of the AD brains and to a smaller extent in the other four AD cases. In the six cases with a large number of IL-6-positive plaques, IL-6 was found in a significantly higher ratio of diffuse plaques than expected from a random distribution of IL-6 in all plaque types. We conclude from these findings that IL-6 immunoreactivity correlates with clinical dementia and that in AD patients, an IL-6-related immunological event may contribute to plaque formation. IL-6 might be involved both in the transformation from diffuse to primitive plaques in AD as well as in the development of dementia.     

Alpha-synuclein-immunoreactive deposits in human and animal prion diseases

Prion related disorders are associated with the accumulation of a misfolded isoform (PrPsc) of the host-encoded prion protein, PrP. There is strong evidence for the involvement of unidentified co-factors in the PrP to PrPsc conversion process. In this study, we show alpha-synuclein-immunoreactive deposits in the central nervous system of various prion diseases (sporadic, iatrogenic and new variant Creutzfeldt-Jakob diseases, and experimental scrapie of hamsters). alpha-Synuclein accumulated close to PrPsc deposits but we did not observe strict colocalization of prion protein and alpha-synuclein immunoreactivities particularly in PrPsc plaques. alpha-Synuclein is thought to be a key player in some neurodegenerative disorders, is able to interact with amyloid structures and has known chaperone-like activities. Our results, in various prion diseases, suggest a role for alpha-synuclein in regulating PrPsc formation.     

Electron microsocopy of brain amyloid plaques from a patient with new variant Creutzfeldt-Jakob disease

Cerebral cortex biopsy from a patient with new variant Creutzfeldt-Jakob disease (nvCJD) has been examined at the electron microscope level. Spongiform changes corresponded mostly to distended neurites scattered in the neuropil or surrounding amyloid plaques. These latter exhibited heterogeneous submicroscopic morphology including variable amount of loosely interwoven amyloid fibrils admixed in a cellular-rich environment constituted essentially by abnormal neuronal processes. By immunoelectron microscopy, fibrils and some membrane structures reacted with anti-prion protein (PrP) antibodies. One striking aspect was the presence of many small dystrophic neurites without paired helical filaments. Moreover, amyloid fibrils showed unexpected intimate association with abnormal membranes, suggesting a relationship between PrP fibrillogenesis and membrane alteration. These ultrastructural findings provide an additional criterion to distinguish nvCJD- from sporadic CJD-type plaques and reinforce the hypothesis that nvCJD brain is infected by a distinctive strain of the transmissible agent encephalopathy. Received: 7 June 1999 / Accepted: 21 September 1999     

Serial reconstruction of beta-protein amyloid plaques: relationship to microvessels and size distribution.

The suggestion that the amyloid plaques in Alzheimer disease are formed by abnormal leakage from microvessels is mainly based on the finding that many plaques are topographically associated with microvessels. However, because the microvessel network is dense and amyloid plaques are numerous, the frequently observed association may result from chance contact, especially for larger plaques. Therefore, we determined the frequency of this association as a variable of plaque size. If all the amyloid plaques are associated with microvessels, a constant and high rate of association would be expected for all plaque sizes. On the other hand, if the association is a chance contact, larger plaques would show more frequent contact than smaller ones. Sections were double-immunostained for amyloid plaques and microvessels with antibodies raised against beta-protein and collagen type IV, respectively. Amyloid plaques were reconstructed using 12 serial sections (7 microns thick) from the entorhinal cortex of two Alzheimer patients. With reconstruction we determined the size distribution of amyloid plaques as well as the influence of size on vascular association. All the amyloid plaques larger than 42 microns were associated with microvessels, however, the smaller the amyloid plaques, the less frequently they were associated with microvessels. Interestingly, although diffuse amyloid plaques occur in all size classes, core-containing amyloid plaques have a more discrete size. We conclude that the topographical relationship between amyloid deposition and capillaries does not support the leakage theory for amyloid plaque formation.     

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.