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Regular Article |
From the Departments of Neuropathology*
and
Clinical Neurology,
Institute of Neurology,
University College London; London, United Kingdom; The National
Hospital for Neurology and Neurosurgery,||
London, United
Kingdom; the Neurotoxicology Group,§
MRC
Toxicology Unit, Leicester, United Kingdom; the Department of
Neuroscience,
Institute of Psychiatry, London,
United Kingdom; and the Department of
Pathology,
New York University School of
Medicine, New York, New York
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently it has been demonstrated that a stop codon mutation of the gene BRI, located on chromosome 13, is associated with FBD.6 The wild-type precursor protein (BriPP), which is a type-II, single-spanning transmembrane protein, is composed of 266 amino acids, whereas the mutated, extended precursor protein (ABriPP) has 277 amino acids. Cleavage of 34 amino acids at the C terminal end of ABriPP generates a 4-kd amyloidogenic fragment, ABri, which has been shown to be deposited as amyloid fibrils in leptomeningeal blood vessels.6 The BRI gene is also associated with another familial cerebrovascular amyloidosis, familial Danish dementia, in which a decamer duplication results in the production of a 34-amino-acid long amyloidogenic peptide, ADan with a C-terminus different from that in FBD.7
In this study of five cases of FBD we studied the topographical distribution and patterns of ABri deposition using immunohistochemistry with an antibody recognizing the C-terminus of ABri (Ab 338) alone and in combination with Thioflavin S. The distribution and relationship of NFTs neuropil threads (NTs) and abnormal neurites (ANs) to ABri deposits were investigated with the antibody AT8 alone and together with Ab 338. The astrocytic and microglial response to ABri deposits was studied using glial fibrillary acidic protein (GFAP) and CD68 antibodies, respectively, for double immunohistochemistry. Ultrastructural characteristics of amyloid and diffuse deposits were studied by immunoelectron microscopy using Ab 338. Western blots of brain homogenates from frozen hippocampal tissue were prepared to study the electrophoretic migration pattern of tau in one case.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Brain and spinal cord samples from five cases of FBD, five sporadic AD cases, and three age-matched controls were collected at post mortem. The pathological diagnosis of AD was made using standard criteria.8 Cases 1–3 were from the original pedigree,4 case 4 from a second, probably unrelated pedigree with identical clinical and pathological features,3 and case 5 is from a recently identified third family with typical clinical presentation and mutation of the BRI gene in affected members. For immunohistochemistry tissue samples were fixed in 10% formalin in phosphate-buffered saline (PBS). For immunoelectron microscopy selected tissue samples from the hippocampal formation (cases 1 and 5) and temporal neocortex (case 5) were fixed in 3% buffered glutaraldehyde. For Western blot analysis of tau unfixed tissue samples from case 5 were taken at post mortem (post mortem delay 7 hours), frozen, and stored at -70°C.
Antibodies
A rabbit polyclonal antibody (Ab338) was raised to the C-terminal 10 amino acids of ABriPP and its specificity confirmed by absorption experiments, as previously described.6 Pre-immune serum from the same animal was also used as a control. An anti-CD68 antibody (PG-M1; DAKO, Ely, UK) was used to demonstrate cells of the microglia/macrophage lineage and anti-GFAP antibody (DAKO) for astrocytes. For tau immunohistochemistry, the AT8 antibody (Innogenetics, Ghent, Belgium) was used, whereas for immunoblotting the rabbit, polyclonal antiserum TP709 the phosphorylation-dependent mouse monoclonal antibody PHF1 (a gift from Peter Davies, Albert Einstein College of Medicine, New York, NY) and the AT8, AT180, AT270, and AT100 (all Innogenetics) monoclonal antibodies were used.
Congo Red Staining
Congo red staining was performed using a standard protocol and viewed under polarized light. In selected areas including 1) the hippocampal formation with parahippocampus, 2) amygdala, 3) upper pons with the locus coeruleus, 4) medulla with the inferior olive, and 5) cerebellar cortex including white matter and the dentate nucleus serial, 15-µm sections were cut and alternate sections stained with Congo red or Ab 338.
Immunohistochemistry
Representative areas of formalin-fixed brain and spinal cord tissue were embedded in paraffin wax. Seven-µm sections were deparaffinized in xylene and rehydrated using graded alcohols. Ab 338 immunohistochemistry required pretreatment with 99% formic acid for 10 minutes. Endogenous peroxidase activity was blocked (0.3% H2O2 in methanol, 10 minutes) and nonspecific binding with 10% dried milk solution. Tissue sections were incubated with Ab 338 (1:2,000, 1 hour at room temperature) followed by biotinylated anti-rabbit IgG (1:200, 30 minutes; DAKO), and ABC complex (30 minutes; DAKO). Color was developed with diaminobenzidine/H2O2. For AT8 immunohistochemistry 7-µm sections were pretreated in a microwave oven in sodium citrate buffer for 20 minutes. After washes in PBS and 10% milk, sections were incubated with the AT8 antibody (1:600) overnight at 4°C followed by biotinylated anti-mouse antibody (1:200, 30 minutes; DAKO). The remaining steps were as described above.
For double-staining with AT8 and Ab 338, 15-µm tissue sections were first pretreated in formic acid as described above and then incubated with AT8 (1:600) for 1 hour at room temperature followed by steps as described above. Sections were then incubated with Ab 338 (1:2,000) overnight at 4°C followed by appropriate secondary antibody and color development.
The AD and normal control cases were stained in a similar manner. For negative controls Ab 338 was replaced with the pre-immune serum or the pre-absorbed Ab 338.
Fluorescence Labeling for Confocal Microscopy
Tissue sections from the hippocampal formation, amygdala, cerebellum including dentate nucleus, cerebral cortex, and white matter were prepared using double-staining with Ab 338 and either GFAP or CD68. To determine the relationship between protein deposition and amyloid formation sections were double-stained with Ab 338 and Thioflavin S.
Twenty-µm sections were de-waxed and re-hydrated. Endogenous peroxidase activity was blocked as described above and sections were pretreated with trypsin (0.1%, 10 minutes at 37°C), then blocked with nonfat milk solution for 30 minutes. Sections were incubated with either GFAP (1:1,000, 1 hour at room temperature; DAKO) or CD68 (1:150, 1 hour at room temperature; DAKO), followed by the respective secondary antibodies (DAKO) and ABC complex. The reaction product was visualized using the tetramethylrhodamine signal amplification kit (NEN Life Science Products, Boston, MA). Incubation with the second antibody, Ab 338 (1:2,000) was performed (overnight, 4°C) without the usual formic acid pretreatment. Sections were then treated with biotinylated anti-rabbit antiserum (1:200) followed by the ABC complex. This reaction was visualized using the fluorescein signal amplification kit.
Double-staining with Ab 338 and Thioflavin S was performed by pretreating sections using 70% formic acid for 10 minutes and blocking in nonfat milk for 30 minutes. Ab 338 (1:2,000) was incubated overnight at 4°C followed by biotinylated anti-rabbit antibody (1:200) for 30 minutes, and finally ABC reagent for 30 minutes. Antibody binding was visualized using the tetramethylrhodamine signal amplification kit. Sections were counterstained in aqueous Thioflavin S (1.0%, 7 minutes; Sigma, Poole, UK) and differentiated with ethanol (70%, 5 minutes). The sections were then washed overnight in PBS.
Sections were viewed with a Leica TCS4D confocal microscope using a 3-channel scan head and argon/krypton laser.
Evaluation of 338 and AT8 Immunohistochemistry
The presence and frequency of Ab 338-positive plaques and CAA were evaluated in different anatomical regions using a semiquantitative approach. For CAA a four-tiered scoring system was devised such that score 0 was given for unaffected areas; ‘+’ if a proportion of the arterioles/small arteries were affected, but no or only an occasional capillary was stained; ‘+ +’ if the majority of arterioles as well as a minority of the capillaries were positive; and ‘+ + +’ for areas in which the majority of the arterioles and capillaries were Ab 338-positive.
For the quantitation of Ab 338-positive parenchymal deposits plaques
were defined as Ab 338-, Congo red-, and Thioflavin S-positive
structures with or without an associated blood vessel, whereas diffuse
deposits were often ill-defined, Ab 338-positive, Congo red-negative
structures, which were only weakly stained, if at all, with Thioflavin
S. Using a x10 objective plaques and diffuse deposits were scored
separately in each case following a principle similar to that
recommended by The Consortium to Establish a Registry for Alzheimer’s
Disease (CERAD) for quantitating neuritic plaques in
AD.8
Score 0 described the absence of either plaques or
diffuse deposits, score ‘+’ corresponded to sparse, score ‘+ +’ to
moderate, and score ‘+ + +’ to frequent plaques or diffuse deposits.
Variation between cases was recorded as shown in Tables 1 and 2
.
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Immunoelectron Microscopy
Ultrathin sections were mounted on etched nickel grids, and treated with sodium metaperiodate solution (4.7% for 1 hour). Grids were washed in 0.5 mol/L Tris-buffered saline (TBS) (pH 7.2) containing 1% Triton X-100 and blocked with 0.5 mol/L TBS/bovine serum albumin (BSA) (pH 7.2) containing 0.05% sodium azide. Grids were then incubated in 10% normal goat serum in 0.5 mol/L TBS/BSA for 1 hour followed by Ab 338 (1:20,000) overnight at 4°C. Washes were performed with 0.5 mol/L TBS/BSA before incubation with goat anti-rabbit IgG gold conjugate (1:15) with a particle size of 20 nm (60 minutes at 37°C; Sigma). After further washes in 0.5 mol/L TBS/BSA the grids were postfixed in 2.5% glutaraldehyde in 0.05 mol/L sodium cacodylate buffer (10 minutes), and counterstained with uranyl acetate/lead citrate using a standard protocol.
Western Blotting
Insoluble tau was extracted from fresh, frozen samples of the hippocampus of case 5, as described previously.10 In brief, 0.1 to 0.2 g of brain tissue was hand-homogenized in 50 mmol/L 2-[N-Morpholino]ethanesulphonic acid (MES) buffer, pH 6.5, containing 1 mol/L NaCl, 50 mmol/L imidazole, 0.1 mmol/L phenylmethyl sulfonyl fluoride, 20 mmol/L NaF, 10 mmol/L sodium pyrophosphate, and 25 mmol/L Na{beta}-glycerophosphate and the homogenate was centrifuged at 28,000 rpm for 30 minutes at 4°C. The supernatant was retained and re-centrifuged at 48,000 rpm for 60 minutes at 4°C. The pellet of the second centrifugation step was solubilized in Laemmli sample buffer and analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a discontinuous buffer method.11 The resolved proteins were electroblotted (15 V, 30 minutes) onto nitrocellulose membranes (pore size, 0.45 µm; Schleicher & Schuell, Andermann, London, UK). Blocking was performed with TBS containing 0.2% Tween 20 and 3% dried skimmed milk (TBS-T-M) before incubation overnight with PHF-1 (1:2,000), TP70 (1:100,000), AT8, AT180, AT270, and AT100 (all 1:200) diluted in TBS-T-M. Blots were developed using either horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (1:1,000) as appropriate followed by enhanced chemiluminescence detection as described by the manufacturer (Nycomed-Amersham, Little Chalfont, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Amyloid plaques, which were both Congo red- and Thioflavin
S-positive, were seen in many of the central nervous system areas. Ab
338 stained all previously described plaque types including the large
and small plaques of the hippocampal formation and also the parenchymal
amyloid plaques in the cerebellum and other structures (Figure 1, A
–C).2,5
Ab
338 and Thioflavin S double-staining showed that the staining patterns
overlapped well in the majority of hippocampal plaques of all types and
cerebellar amyloid plaques, although examples were found whose margins,
determined with Ab 338, extended beyond those seen with Thioflavin S
(Figure 2
, A–C). This was also seen in
the cerebellum, particularly in the granular cell layer, where Ab 338
often stained structures more extensively and/or intensely than
Thioflavin S. The same pattern was also apparent in some of the
perivascular plaques (Figure 2
, D–F).
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. We found not only that the
hippocampus and cerebellum were most severely affected by amyloid
plaques, but also that amyloid plaques were present in considerable
numbers in structures such as the hypothalamus, amygdala, locus
coeruleus, inferior olive, and the cerebellar dentate nucleus (Tables 1 and 2)
(Figure 2
N). In contrast the striatum, globus pallidus, and
substantia nigra, which also lacked CAA, were entirely unaffected by
parenchymal deposits whereas the thalamic involvement varied between
cases. Diffuse deposits, defined as Ab 338-positive and Congo
red-negative structures, unstained or only weakly stained with
Thioflavin S, were noted in varying numbers in other areas such as the
entorhinal cortex, neocortex, tectum, periaqueductal gray, the
medullary pyramids, and spinal cord gray and white matter (Figure 1, D, G, and H
; and Figure 2
, K and M). Both amyloid plaques, which were
mostly of the perivascular type, and diffuse deposits were sparse in
the neocortex where they appeared randomly distributed throughout the
cortical laminae. In the cerebral cortices loosely arranged diffuse
deposits were also seen to surround neurons, often with an accentuation
of staining adjacent to the perikaryon. This phenomenon was found at
several other locations, including in the pyramidal cell and granular
cell layers of the hippocampus, around Purkinje cells, anterior horn
cells of the cord, and inferior olivary neurons (Figure 2
, N and O). A
delicate membranous staining pattern highlighting neuronal cell margins
was noted most readily in hippocampal pyramidal cells (Figure 1I)
. In
the CA1 region and subiculum a number of less compact NFTs, not
associated with visible neuronal nucleus or plasma membrane
(extracellular tangles,12,13
), were stained with Ab 338
and a few such structures seemed to have been incorporated into amyloid
plaques (Figure 1J)
.
Between the hippocampus and third temporal gyrus Ab 338-positive
deposits affected all allocortical areas and also the isocortical
fusiform gyrus. An anatomically determined sharp transition between two
staining patterns was noted at the junction between the subiculum and
presubiculum; the subiculum contained a significant number of Ab 338-,
Congo red-, and Thioflavin S-positive amyloid plaques, whereas the
presubiculum and the parasubiculum had an ill-defined diffuse staining
pattern. The size and shape of the Ab 338 positivity closely matched
the anatomical boundaries of the pre- and parasubicular parvopyramidal
cell islands (Figure 1H)
. In the entorhinal cortex the deposits were
predominantly of the diffuse type with a striking bilaminar
distribution. There was an inner wide band, occupying the deeper
cortical laminae, with an ill-defined, confluent staining pattern and a
narrower outer band composed of often better-circumscribed deposits
(Figure 1G)
. The outer Ab 338-positive band closely overlapped with the
Pre-
neuronal clusters and, with the Pre- neurons
themselves,14
gradually descended in the transenthorhinal
cortex to form a single band of well-defined Ab 338-positive, mainly
diffuse deposits in the deeper laminae of the fusiform gyrus.
In many cerebral and cerebellar areas and also in the spinal cord Ab338 stained subpial deposits, which were variably positive on Congo red or Thioflavin S preparations. Ab 338 also stained subependymal areas around the ventricles and cerebral aqueduct in a diffuse pattern. Clearly defined fibrillar staining was also seen associated with the basolateral aspect of the ependyma.
The cerebral, cerebellar, and spinal cord white matter showed mostly
diffuse deposits, although perivascular amyloid plaques were also noted
(Tables 1 and 2)
.
No staining of vascular amyloid, plaques or diffuse deposits of the FBD cases was observed in controls using the pre-immune serum, the Ab 338 pre-absorbed with synthetic peptide or when the primary antibody was omitted. Immunohistochemical staining with Ab 338 of brain tissues from individuals with AD or normal controls were also negative (results not shown).
Ab 338 Immunohistochemistry, Congo Red, and Thioflavin S Staining of Blood Vessels
All affected blood vessels stained with Congo red and Thioflavin S
were also Ab 338-positive. Furthermore on preparations double-stained
with Ab 338 and Thioflavin S there was an overall good overlap between
the different staining patterns, although a smaller proportion of
affected blood vessels, especially those with perivascular plaques were
more extensively stained with Ab 338 (Figure 2
, D–F, G–I). Many of
the leptomeningeal and parenchymal small arteries, arterioles, and
capillaries were stained throughout the central nervous system,
although the severity of CAA varied considerably in the different
areas; the striatum, globus pallidus, and substantia nigra were
entirely unaffected (grade, 0), in the thalamus and pontine base
angiopathy was of moderate severity (grade, + to ++), whereas in other
areas such as the hippocampus and cerebellar cortex it was severe
(grade, + + +). In the cerebellum there was a difference in the
staining pattern of the blood vessels within the white matter in that
the blood vessels of the deep white matter, with the exception of those
found in the vicinity of the dentate nucleus, were more rarely stained
with Ab 338 than those of the white matter in the cerebellar folia.
The majority of the Ab 338-positive blood vessels were arterial, but a
proportion of venous channels was also affected. These latter were more
frequent in the leptomeninges, but were also found in the parenchyma.
Although small arteries with a diameter of up to 1 mm were stained with
the Ab 338, the majority of the affected blood vessels were smaller
than 300 µm. The larger vessels were usually less affected in that Ab
338 primarily stained the abluminal aspect of the media and the
adventitia. In contrast, in many of the small arteries and arterioles
Ab 338 positivity was seen throughout the full thickness of the wall,
often with severe disruption of its normal structure and sometimes
occlusion of the lumen. An accentuation of the staining in the outer
perimeter of some small arteries and arterioles, was noted (Figure 1E)
.
Longitudinal sections of affected arterioles and capillaries showed
that Ab 338 often stained vessel walls in a linear manner although the
full length was not always affected. Other staining patterns included
the presence of globular deposits along vessel walls or fine Ab
338-positive spicules radiating from the walls of affected arterioles
and capillaries (Figure 2
, G–I). In all regions the glia limitans
around many affected vessels was stained with an extension into the
adjacent parenchyma, often giving rise to plaque-like structures,
previously described as ‘perivascular plaques’ (Figure 1F)
.2
There was substantial vascular amyloid in many retinal vessels and, similar to the subpial deposits, there were small amyloid plaques and diffuse deposits along the retinal inner limiting membrane. No gross decrease in cell numbers could be discerned in any of the retinal layers and the photoreceptor outer segments and retinal pigmented epithelial cells appeared identical to age-matched controls. There was an increase in GFAP expression in Müller’s glial cells, which has been previously documented in aging rat retina.15
AT8 Immunohistochemistry and Its Correlation with Ab 338 Immunohistochemistry
Severe involvement by NFTs, and NTs of mediotemporal and other
limbic structures, including the hippocampus, subiculum, entorhinal and
transenthorhinal cortices, amygdala, uncus, cingulum, and insular
cortex was readily noted (Figure 1K)
. Double AT8 and Ab 338
immunohistochemistry also revealed that the amyloid plaques, occurring
in these structures, tended to be surrounded with ANs, many of which
were fine and thread-like and were often incorporated within the large
hippocampal amyloid plaques in a radial pattern. ANs clustering around
an occasional blood vessel with amyloid deposition were also seen in
the hippocampus, amygdala, and temporal neocortex. There was a
strikingly similar distribution of AT8-positive structures and Ab
338-positive amyloid plaques within the amygdala in that both
pathologies affected the basal nucleus more severely than the lateral
nucleus (Figure 1K)
. Occasional NFTs and NTs were seen in the
parvopyramidal clusters of the pre- and parasubiculum. In the
entorhinal cortex, which was rich in nonfibrillar ABri and contained
only relatively few amyloid plaques, the Pre- and Pri- neurons
were severely affected, containing frequent NFTs and NTs, which because
of their large numbers, produced darkly stained bands in both cell
layers. The Pre-{beta} and Pre-
neurons also contained moderate numbers
of NFTs. In the fusiform gyrus, which contained predominantly
nonfibrillar diffuse plaques, there were numerous NFTs and NTs. These
gradually decreased in number toward the neighboring third temporal
gyrus, which, similar to the first and second temporal gyri contained
only sparse NFTs and NTs. Other neocortices, except the cingulum and
insular cortex, were unaffected. The hypothalamus, claustrum, midbrain
tectum, periaqueductal gray, and locus coeruleus contained sparse and
the pontine raphé moderate numbers of NFTs. The cerebellum and
spinal cord were unaffected by tau pathology (Tables 1 and 2)
.
Correlation between Ab 338, CD68, and GFAP Staining of Different Lesion Types
Tissue sections double-stained with Ab 338 and CD68 and examined
by confocal microscopy showed a good co-localization between cells of
microglial/macrophage lineage and both amyloid plaques and vascular
amyloid. However such cells were few in areas in which diffuse deposits
were predominant (Figure 2
, J and K). A similar relationship between
GFAP-positive astrocytes and AB 338-positive structures was seen on
preparations double-labeled with Ab 338 and GFAP (Figure 2
, L and M).
Immunoelectron Microscopy
Ultrathin sections from the hippocampus and temporal neocortex,
labeled with Ab 338 using the immunogold method, were examined. Tissue
preservation was such that blood vessels, neurons, astrocytes, and both
neuronal and glial processes could be identified. In the hippocampus
frequent amyloid plaques were present and many small blood vessels had
amyloid fibrils deposited within their walls thus confirming the
findings of light microscopy. Amyloid plaques varied in size and were
composed of short fibrils of 
10 nm diameter, which were randomly
distributed but varied in density, some plaques being composed of
loosely arranged fibrils whereas others were more compact. The amyloid
fibrils were consistently heavily labeled by Ab 338. Glial processes
containing intermediate filaments were often seen within plaques
(Figure 3, A and B)
.
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Such electron-dense material was not found in association with
hippocampal amyloid plaques.
Small blood vessels showed labeling of the basal lamina by Ab338.
Several vessels had normal segments of basal lamina adjacent to areas
in which a low level of labeling was present in its central part. The
basal lamina then often became expanded into regions with a greater
labeling density in which amyloid fibrils became apparent. In some
vessels the intensity of labeling was greatest on the abluminal aspect
of the basal lamina reflecting the accentuation of Ab 338 staining seen
around the outer perimeter of some vessels using light microscopy. The
accumulation of amyloid fibrils within the basal lamina produced
increased expansion and eventual disruption of the abluminal surface
whereas the luminal aspect of the basal lamina remained intact (Figure 3, D and E)
.
NFTs composed of paired helical filaments were frequently found in both neuronal cell bodies and processes but these were unlabeled by Ab338.
No labeling was found using the pre-immunization rabbit serum or when the primary antibody was omitted. The antibody that had been previously absorbed using the immunizing peptide showed some residual labeling of amyloid fibrils but this was greatly reduced in comparison with untreated Ab338 (not shown).
Tau Immunoblotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
Western blotting analysis of the insoluble tau extracted from the
control case, with the polyclonal tau antiserum TP70 that recognizes
all forms of tau protein, corresponded to the normal tau pattern of
post mortem adult human brain because it consisted of 6 to 8 bands. The
AD case showed the characteristic triplet with T54, T59, and T64 tau
species and a minor T71 band when probed with all of the tau
antibodies. In the FBD case, tau comprised a triplet pattern, which was
indistinguishable from the pattern seen in the AD case (Figure 4)
.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This immunohistochemical study confirmed findings of previous pathological examinations, including ours, showing that in FBD the hippocampus and cerebellum are among the anatomical areas most severely affected by amyloid deposition in both blood vessels and parenchymal plaques.2,5,18,19 Furthermore we were also able to show not only that ABri is widely deposited throughout the central nervous system, but also that the sites affected include other anatomically well-defined structures such as amygdala, hypothalamic nuclei, inferior olive, the dentate nucleus, and the parvopyramidal cell clusters of the pre- and parasubiculum; which may be affected by A{beta} deposition in AD.20-22
The hippocampal amyloid plaques had rarely been found to be associated with surrounding ANs when studied with silver impregnation techniques.2 However, in addition to tau-positive dystrophic neurites, a rich network of fine thread-like processes, permeating the large amyloid plaques was readily detectable when the AT8 antibody, recognizing phosphorylated serine 202/threonine 205 epitopes of tau,23-25 was used.5 In addition to demonstrating the predominant involvement of limbic structures by neurofibrillary pathology in FBD, we also showed that AT8-positive neurites are associated with amyloid plaques and occasionally with vascular amyloid rather than with nonfibrillar diffuse deposits. In contrast, NFTs and NTs occur in association with both amyloid plaques and diffuse deposits. A number of NFTs with appearances of extracellular tangles12,13 were stained with Ab 338 in the hippocampus and subiculum, which are among the structures with the highest parenchymal ABri load. It has been shown that in AD A{beta} deposition within extracellular tangles, taking place in the vicinity of senile plaques, is a secondary phenomenon26 and we suggest that a similar mechanism is likely in FBD.
The predominantly limbic distribution of the tau pathology together with mild involvement of the temporal cortex would suggest that the NFT pathology corresponds to stage IV in the system recommended by Braak and Braak27 for staging NFT pathology in AD. In FBD the severity of the limbic involvement exceeds that expected in stage IV of AD and the presence of NFTs in structures such as the granular cell layer of the dentate fascia5 and substantia nigra suggests that in FBD the progression of NFT pathology may be different from that seen in AD. We have confirmed our previous finding that NFTs in FBD are composed ultrastructurally of paired helical filaments5 and showed, for the first time that insoluble tau has a triplet electrophoretic migration pattern on Western blots using a panel of phosphorylation-dependent and -independent antibodies, one of which (AT100) has been described as PHF tau-specific.28
Although the mechanism of NFT formation in FBD remains to be investigated, the close topographic association of both fibrillar and nonfibrillar ABri with neurofibrillary pathology in limbic structures may support the notion that NFTs are principally because of ABri-mediated neurotoxicity. This hypothesis is supported by our finding of co-localization of reactive astrocytes and microglia with ABri in {beta}-sheet conformation, which is strikingly similar to the findings in AD, in which a relationship between the presence of these cell types and the neurodegenerative events have been suggested.29-31 Despite the close association between neurofibrillary pathology and ABri deposition in limbic structures, the absence of apparent neurodegeneration in some other well-defined protein-bearing regions such as the dentate nucleus and inferior olive, points to the importance of other biological variables in this process.
Ab 338-positive congophilic angiopathy of variable severity was widespread and was only absent in the striatum, globus pallidus, and substantia nigra. In FBD the overall patterns of amyloid deposition in cerebral blood vessels are similar to those seen in congophilic angiopathies related to other amyloid peptides, in particular A{beta}, in which arteries and arterioles are more frequently affected than veins.32
The origin and mechanism of deposition of ABri in plaques and blood vessel walls is not known. One possibility is that ABri is produced by cellular components of the central nervous system itself. In support of such a hypothesis is the finding that BriPP mRNA is expressed at high levels in a number of areas of normal human brain including the hippocampus, amygdala, and cerebellum,6 which we have shown, are those areas most affected by fibrillar and nonfibrillar ABri deposition. Furthermore furin, which may be involved in cleaving both the wild-type and mutant precursor proteins, releasing a 23-amino acid long C-terminal peptide and the amyloidogenic ABri from BriPP and ABriPP, respectively,33 is also widely expressed in both neurons and glial cells in the central nervous system.34 ABri is deposited in areas composed of particular neuronal groups such as the locus coeruleus, inferior olive, cerebellar dentate, or the parvopyramidal neuronal clusters of the pre- and parasubiculum and often shows a striking perineuronal distribution in areas of diffuse plaques and an association with neuronal plasma membranes. These morphological findings are similar to those that linked A{beta} production with neurons,20,22,35,36 and raise the possibility that neurons may be one of the cell types involved in cerebral ABri production/deposition. Both pathological and experimental evidence now exists to suggest that neuronal expression of an amyloidogenic protein can result in both parenchymal deposits and CAA. A{beta} depositing as vascular amyloid may be of cerebral cortical origin and transported along periarterial interstitial fluid drainage pathways in both human AD32 and in a transgenic mouse model of AD (APP23) that overexpresses mutant human amyloid precursor protein driven by the neuronal Thy-1 promoter. In this transgenic model of AD there are both amyloid plaques and significant deposition of A{beta} in the cerebral vasculature.37 In FBD the widespread deposition of ABri in small cerebral arteries and arterioles is also consistent with a nonneuronal, vascular smooth muscle cell, pericytic or even systemic origin of the ABri peptide. Preliminary data indicate that soluble ABri is present in the circulation as has been found for A{beta} in AD38,39 and some vascular amyloid has been found in blood vessels in organs (J Ghiso, unpublished data). The significance of these findings in relation to deposition of ABri in the central nervous system remains to be investigated.
FBD with CAA, parenchymal amyloid plaques, and diffuse deposits as well
as neurofibrillary degeneration mimics important morphological features
of AD, although there are distinct features in both diseases. Limbic
structures are severely affected by both parenchymal amyloid deposition
and neurofibrillary degeneration in both conditions, although in AD
neocortical involvement is much more prominent than in FBD. The
cytoskeletal pathology in FBD, immunohistochemically,
ultrastructurally, and biochemically, is indistinguishable from that
found in AD.10,40
The morphology of limbic plaques shows
some similarities with AD as the large plaques of FBD noticeably
resemble the cotton wool plaques seen in variant AD with spastic
paraparesis associated with PS1
941
or PS1 exon 4 DelIM
mutation.42
However, there are differences between the two
lesions as the cotton wool plaques are mainly Congo red-negative;
whereas the large hippocampal plaques of FBD are Congo red- and
Thioflavin S-positive. CAA resulting in white matter degeneration is
not a unique feature of FBD as this can be associated with a similar
pathological picture in AD.43,44
Therefore the study of
FBD, including future development of different transgenic animal
models, may result in a better understanding of the link between
amyloid deposition and neurodegeneration.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health grants AG05891 and AG08721 and a grant from the Alzheimer Association. T. R. was recipient of a Wellcome Trust Travel Award, and G. G. is supported by a grant from the Wellcome Trust.
Accepted for publication October 5, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-rich dystrophic neurites permeates neocortex and nearly all neuritic and diffuse amyloid plaques in Alzheimer disease. FEBS Lett 1994, 344:69-73[Medline]
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A. Rostagno, T. Revesz, T. Lashley, Y. Tomidokoro, L. Magnotti, H. Braendgaard, G. Plant, M. Bojsen-Moller, J. Holton, B. Frangione, et al. Complement Activation in Chromosome 13 Dementias. SIMILARITIES WITH ALZHEIMER'S DISEASE J. Biol. Chem., December 13, 2002; 277(51): 49782 - 49790. [Abstract] [Full Text] [PDF]
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J. A. Ghiso, J. Holton, L. Miravalle, M. Calero, T. Lashley, R. Vidal, H. Houlden, N. Wood, T. A. Neubert, A. Rostagno, et al. Systemic Amyloid Deposits in Familial British Dementia J. Biol. Chem., November 16, 2001; 276(47): 43909 - 43914. [Abstract] [Full Text] [PDF]
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S.-H. Kim, J. W. M. Creemers, S. Chu, G. Thinakaran, and S. S. Sisodia Proteolytic Processing of Familial British Dementia-associated BRI Variants. EVIDENCE FOR ENHANCED INTRACELLULAR ACCUMULATION OF AMYLOIDOGENIC PEPTIDES J. Biol. Chem., January 11, 2002; 277(3): 1872 - 1877. [Abstract] [Full Text] [PDF]
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