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From the Department of Neuropathology and Neuroscience,* Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; the Department of Neurology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; the Department of Pathological Sciences, University of Manchester, Manchester, United Kingdom; and the Nathan Kline Institute, New York University, Orangeburg, New York
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Abstract |
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1-antichymotrypsin accelerated the accumulation of ß-amyloid,4
whereas ablation of Apolipoprotein J attenuated it.5
Thus, identification of Aß-associated proteins in SP amyloid and characterization of their pathological functions is important in the elucidation of the pathobiology of ß-amyloid formation and AD.
We have searched for novel components of SP amyloid by raising monoclonal antibodies (mAbs) against crude amyloid fractions extracted from AD brains, and identified a novel protein that we named CLAC (collagenous Alzheimer amyloid plaque component).6
CLAC is derived from the ectodomain of a novel membrane-bound, neuron-specific collagen that we termed CLAC-precursor (CLAC-P) or collagen type XXV, through shedding by furin.6
Recent work suggests that CLAC is identical to the AMY antigen7
that had previously been identified in SP.8,9
In vitro studies show that recombinant CLAC specifically binds aggregated Aß, but not its soluble form.6
Pathologically, CLAC-immunoreactivity (IR) was detected in a subset of SP in AD brains, especially in primitive plaques or in the periphery of typical plaques, whereas amyloid cores, CAA, or diffuse plaques lacked CLAC-IR.6
This selectivity in the distribution of CLAC deposition in a specific subfraction of amyloid deposits is unique, and not observed with any other non-Aß SP component proteins, ie, Apo E, complement component C1q or heparan sulfate proteoglycan (10-12
, Sakakura T, Kowa H, Iwatsubo T, unpublished observations). Other well-known features of SP amyloid exhibiting selective distributions are the heterogenous C termini of Aß, Aß40, and Aß42. Aß exhibits two major C-terminal variants by the heterogeneity in positions of 
-secretase cleavage of ß-amyloid precursor protein (ßAPP): Aß42 with a longer C terminus is a relatively minor secreted species13
but has a higher propensity to aggregate14
and deposits initially and widely in SP.15,16
In contrast, Aß40, a major secreted species,13
accumulates later robustly in a subset of SP as well as in CAA. However, the temporal and spatial relationships between deposition of CLAC, Aß40, and Aß42, each of which shows unique deposition patterns, as well as the pathological significance of CLAC deposition in ß-amyloid formation, remains elusive. In this study, we have examined the relationship between deposition of CLAC and different Aß species in the brains of patients with AD or Down syndrome (DS), as well as those of transgenic (TG) mice developing ß-amyloid plaques, using multiple labeling and morphometric evaluation. We find that CLAC-positive SP and Aß40/thioflavin S (thioS)-positive SP show mostly separate distribution patterns, suggesting a possible role of CLAC binding to prevent further maturation of ß-sheet-rich, dense amyloid deposits.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Blocks from frontal neocortex (Brodmann area 8/9) were obtained at autopsy from 74 patients with AD (male: 37 cases, female: 37 cases; age 44 to 92 years, 71.5 ± 10.0 (mean ± SE)), as well as from 26 patients with DS (age 31 to 71 years, 52.7 ± 10.9). All AD patients had pathologically confirmed AD based on the consensus criteria of the National Institute of Aging, and were at Braak stages 5 and 6. Tissues from 28 AD patients were fixed in 10% formalin for 18 to 24 hours, then maintained in phosphate-buffered saline (PBS) at 4°C. Tissue blocks from the rest of the AD cases, as well as from all DS cases, were fixed in 10% buffered formalin for 2 to 4 weeks, then embedded in paraffin wax, cut in serial sections of 6-µm thickness and immunostained as below. Brains of TG mice that doubly express human K670N/M671L mutant ßAPP gene and M146L mutant PS1 (PSAPP mice17 ) at ages 3, 6, 9, 12, and 19 months (total, 9 animals) were fixed by immersion in 70% ethanol/150 mmol/L NaCl for 2 weeks, then embedded in paraffin as previously described.18
Antibodies, Tissue Processing, and Immunohistochemistry
For CLAC immunostaining in human brains, a mouse mAb 9D2, that was originally developed against a crude amyloid fraction of AD brains and specifically recognizes CLAC, whose epitope is located at the pyroglutamated N terminus of CLAC,6 was used. Rabbit affinity-purified antibodies against synthetic peptides corresponding to the three non-collagenous (NC) domains of CLAC-P (anti-NC2–2, anti-NC3, and anti-NC4), as well as to the pyroglutamated N terminus of CLAC (anti-pyroGlu113) also were used.6 The immunostaining patterns with antibodies to the NC domains of CLAC-P were essentially similar to that obtained with 9D2.6 Detection of deposits of murine CLAC in the brains of TG mice was performed with anti-NC2–2.6 Mouse mAbs BC05 and BA27, which specifically react with the C termini of Aß42 and Aß40, respectively, have been described;15,16 sections were pretreated with 99% formic acid, followed by incubation with 0.1% trypsin at 37°C before immunostaining for BC05 (BA27 immunostaining was performed solely with formic acid pretreatment). 9D2 optimally detects CLAC-positive SP in 50-µm thick unembedded vibratome sections fixed in 10% formalim for 24 hours without pretreatment.6 Therefore, for CLAC immunostaining of formalin-fixed, paraffin-embedded sections, antigen retrieval of deparaffinized sections by microwave treatment (550W, 10 minutes) in citrate buffer (pH 6.0) followed by proteinase K treatment (100 µg/ml, 10 minutes), a methodology developed for immunostaining of AMY antigen,9 was effective and routinely performed. Immunoperoxidase staining using avidin-biotin complex using diaminobenzidine was performed as described.15,16 Fluorescence labeling of amyloid by thioS was performed as described:19,20 briefly, sections were incubated with 1% thioS in distilled water for 10 minutes, followed by brief wash in 50% ethanol, and then a final wash in tap water. For double or triple fluorescence labeling, sections were incubated by mixture of primary antibodies, followed by incubation with secondary antibodies against mouse or rabbit IgG tagged with Alexa fluorophores and observed with Olympus fluoview confocal microscope as described.21 Fluorescence signals obtained by Alexa 488, 594, and 647 were displayed in pseudocolors of green, red, and blue, respectively.
Morphometry
Deposition of CLAC or Aß in peroxidase-labeled sections was evaluated by quantifying the total percentage of cortical surface area covered by CLAC- or Aß-IRs (percent CLAC or amyloid burden) as described.16,18 Images were captured by HC-2500 digital image recording system (Fujix, Tokyo, Japan) mounted on a BX51 microscope (Olympus, Tokyo, Japan) in five randomly selected cortical areas encompassing the entire depth of cortex (0.57 mm2 each; corresponding to 1280 x 1000 pixels), and the percentage of positive areas was calculated by MacSCOPE image analysis software (Mitani Company, Tokyo, Japan). To evaluate the correlation between patient age and CLAC or amyloid burden in DS cases, regression plots were drawn on patient groups younger or older than 50 years of age, using simple regression method on Statview, version 5.0 (SAS Institute Inc., Cary, NC). CLAC/amyloid burden in double fluorescence specimens was analyzed in a representative cortical area (0.13 mm2) in eight representative AD cases. The percentages of single- or double-positive areas for given probes were calculated by MacSCOPE, as above.
Immunoelectron Microscopy
Double immunolabeling for electron microscopic observation of CLAC/Aß42 or CLAC/Aß40 in AD cortices was performed as follows: 50-µm thick, floating sections fixed in 10% formalin for 24 hours were incubated with mAb 9D2 overnight. After washing, sections were incubated with anti-mouse IgG antibody tagged with 1-nm gold particles (Nanoprobe) for 24 hours. After washing in 10 mmol/L phosphate buffer, sections were post-fixed in 2.5% glutaraldehyde for 1 hour, and transferred to 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid(HEPES) buffer (pH 5.8). After washing in distilled water (DW), silver intensification was performed using HQ-silver kit (Nanoprobes) according to manufacturer’s instructions. After stopping the silver intensification by washing in DW, the sections were post-fixed in 2% osmium tetroxide for 1 hour, dehydrated, and embedded in epoxy resin. Subsequent post-embedding immunolabeling for Aß40/42 was performed as described by Yamaguchi et al22 with some modifications. Briefly, ultra-thin sections were cut at 80 nm and treated with 3% H2O2 for 10 minutes followed by 1% sodium periodate for 10 minutes on a nickel grid. After blocking in 3% bovine serum albumin in 100 mM phosphate buffer (pH 7.4) for 30 minutes, sections were incubated with BC05 or BA27, followed by reaction with anti-mouse IgG antibodies tagged with 10-nm gold particles. The sections were double-stained by uranium-acetate and lead-citrate, and viewed in electron microscope (1200EXII, JEOL).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We have previously found that a subset of SP in the cerebral neocortex in AD, especially the primitive or neuritic types, is immunolabeled by anti-CLAC mAb 9D2 in a thick bundle- or coarse granule-like pattern.6
Notably, CLAC-IRs were always accompanied by Aß-IRs. This observation prompted us to further examine the relationship between CLAC-IR and those of the major two Aß C-terminal species, Aß42 and Aß40, which exhibit distinct distributions in AD brain.15,16
Comparison of three serial sections of frontal neocortex from sporadic AD cases immunolabeled for Aß42, CLAC, and Aß40, respectively, showed that most of the CLAC-positive SP was simultaneously positive for Aß42 (Figure 1, A and B
, arrowheads). In contrast, CLAC was often negative in Aß40-positive amyloid deposits that are often uniformly and densely immunostained for Aß40, including the cores of typical SP and blood vessels affected by cerebral amyloid angiopathy (Figure 1, B and C
, arrows). Accordingly, the distribution pattern of Aß42 overlaps and encompasses that of CLAC, whereas that of Aß40 shows only a limited extent of overlap with that of CLAC. We then directly addressed the co-localization of CLAC and Aß42 or Aß40 by double immunofluorescence labeling combined with morphometric analysis. Approximately 
43% of Aß42-positive areas were CLAC-positive, and 93% of the CLAC-positive areas (CLAC burden) were simultaneously Aß42-positive, only 7.2% remaining Aß42-negative (Figure 1, D and F)
. In sharp contrast, only 26% of Aß40-positive areas were CLAC-positive and 32% of CLAC-positive areas were Aß40-positive (Figure 1, E and F)
. We further analyzed the relationship between CLAC and thioS-positive Aß deposits, which are regarded to represent highly ß-sheeted amyloid structures, by multiple fluorescence labeling. The number of thioS-positive SP in frontal neocortex was variable among AD cases. Numerous thioS-positive SP were observed (ie, detectable in every visual field of 3.4 mm2, occupying 5% of total areas) in 40% of the cases. Among these, only 9% of the thioS-positive area was CLAC-positive, and 7% of the CLAC-positive SP area was thioS-positive, showing minimal and even less extent of overlap compared to Aß42/CLAC or Aß40/CLAC (Figure 2, A to C)
. Comparison of Aß40 and thioS reactivities, on serial mirror sections, showed that thioS-positive areas (Figure 2A
, green) were always Aß40-positive (Figure 2B
, red), and accounted for 50% of the Aß40-positive areas within the entire depth of cerebral cortex. Occasional SP showed co-localization of Aß40 and CLAC, in which thioS was invariably negative (Figure 2, A and B
, a SP in the lower left corner).
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The distinct distribution patterns of CLAC- or Aß40/thioS-positive SP led us to examine the ultrastructural characteristics of Aß deposits that are positive or negative for CLAC. To rigorously compare the fine structure of CLAC-positive or negative structures in an identical manner, we used double immunoelectron microscopy, visualizing CLAC-IR by pre-embedding, 1-nm immunogold labeling/silver intensification, and Aß40- or Aß42-IRs by post-embedding with 10-nm gold particles, and compared their ultrastructures within an identical ultra-thin section. Because thioS-labeling cannot be applied to EM, we examined Aß40-positive deposits at the core of dense SP, that are predicted to be thioS-positive at a light microscopic level, as representative areas of highly ß-sheeted deposits. The latter type of deposit showed the typical morphology of amyloid fibrils, ie, dense bundles composed of relatively straight and smooth-surfaced filaments of 10 nm in diameter, that were intensely decorated by an anti-Aß40 antibody (Figure 3, A and B)
. In contrast, CLAC-positive materials were apparently composed of fibrils, although they sometimes formed a mesh-like structure, in which individual fibrils were loosely packed, less electron dense and smaller in diameter, and distributed in rather random directions (Figure 3C)
. These CLAC-positive fibrils were strongly positive for Aß42 (Figure 3D)
. We quantified the density of amyloid fibrils and found that CLAC-positive fibrils were more loosely distributed compared to CLAC-negative ones (4.9 ± 0.50 versus 7.9 ± 0.72 per 100-nm width in amyloid bundles) (Figure 3E)
.
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The pathological findings observed in autopsied AD brains were considered to represent an end-stage of AD pathology, lacking information regarding the temporal sequence of events in the development of AD pathology. To gain insight into the chronological and spatial relationships between CLAC deposition and those of Aß40 and Aß42, we studied the frontal neocortex of patients with Down syndrome (DS), dying at ages ranging from 31 to 71 years, by immunohistochemistry for Aß42, Aß40, and CLAC. Purely diffuse plaques, which predominate in the neocortex in young individuals with DS and which are known to be exclusively positive for Aß42 (Figure 4A)
but negative for Aß40 (Figure 4G)
(see also16
), were CLAC-negative (Figure 4D)
. However, a subset of Aß42-positive SP (Figure 4B)
, especially those of a primitive type, observed in DS individuals dying between ages of 35 and 50 years became CLAC-positive (Figure 4E)
, and the CLAC burden (percentage of CLAC-positive areas) gradually increased, showing a modest correlation with age (r2 = 0.395; blue dotted line in <50-year group) and amounting to 30 to 100% of the Aß42-positive areas (Figure 4K)
; Aß40 was only occasionally present (Figure 4H)
, especially in CLAC-negative SP. Accordingly, Aß42-positive, CLAC-negative diffuse plaques and Aß42/CLAC-positive primitive plaques accounted for most of the SP at this stage. After 50 years of age, the Aß42 burden remained at a relatively constant level (Figure 4C)
, whereas those of Aß40-positive and thioS-positive SP dramatically increased (Figure 4I)
, showing an age-related increase in Aß40 levels (r2 = 0.614). In contrast, the CLAC burden remained at a relatively constant level (3% of total area) (Figure 4F and K)
. The overlap between CLAC-positive and Aß40-positive areas was minimal in the neocortex of older (>50 years) individuals with DS, similar to that seen in the neocortex in patients with AD (Figure 4J)
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To examine if ß-amyloid plaques that appear in the brains of TG mice overexpressing FAD mutant APP and PS genes are associated with endogenous CLAC, and if the separate distributions of CLAC- and Aß40/thioS-positive areas are also present in TG brains, we immunostained the cerebrum and hippocampus of PSAPP TG mice. The huge core-like amyloid plaques, that are Aß40-positive (as described previously18
), and predominate at 3 to 6 months of age, were CLAC-negative (data not shown). After 12 months of age, in addition to the Aß40-predominant huge-cored plaques (Figure 5A)
, small-sized diffuse deposits, positive for Aß42 but not for Aß40, appeared in the periphery of the core-like huge plaques or in the neuropil of the surrounding areas, most prominently in the molecular layer of the dentate gyrus of hippocampus (Figure 5B)
; these were immunoreactive for CLAC (Figure 5, C to E)
. Thus, the predilection for CLAC to deposit in Aß40/thioS-negative Aß plaques was also seen in the brains of TG mice in which Aß deposition is highly accelerated.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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It is well recognized that Aß42 has a higher propensity to aggregate in vitro14 and accumulates earlier than Aß40 in human brains.15,16 However, it has remained unknown why a significant proportion of Aß42-positive SP remain negative for Aß40 and/or thioS, despite the in vitro results that once a "seed" for ß-amyloid is initially formed, a rapid incorporation of Aß (either Aß40 or Aß42) follows.14 Our observation that CLAC- and Aß42-positive SP show a mostly separate distribution from that of Aß40/thioS-positive SP suggests that binding of CLAC may play a mechanistic role in the evolution of SP, possibly by inhibiting incorporation of Aß40, abundantly secreted from neurons,23 thereby precluding the formation of dense, typical amyloid deposits that are positively labeled by amyloid-sensitive dyes like thioS.19 The reason why CLAC preferentially binds to a specific subset of SP, especially Aß42-positive, Aß40-negative primitive type SP, but not to more typical thioS-positive amyloid or to early diffuse plaques composed of amorphous aggregates of Aß,24 is unknown. Our immunoelectron microscopic observations that CLAC- and Aß42-positive deposits in AD brains exhibited the morphology of fibrils, although they were less typical compared to the Aß40/thioS-positive ones, suggest that CLAC preferentially binds to a fibrillar form of Aß that has some structural characteristics in its ß-sheet structure, which does not allow the binding of thioS. Alternatively, it is possible that once CLAC is bound to Aß fibrils, the binding site(s) for thioS is occupied, rendering the fibrils thioS-negative. In either case, CLAC binding may block the binding site on Aß fibrils for further incorporation of Aß1–40 and inhibit growth of amyloid fibrils. Indeed, our preliminary in vitro experiments suggest that co-incubation of recombinant CLAC with synthetic Aß peptides inhibits the formation of Aß fibrils (Nishimura A, Hashimoto T, Iwatsubo T, unpublished observation), supporting the assumption that binding of CLAC plays a "protective" role against deposition of Aß.
Our observation on a series of DS individuals dying at various ages further substantiates this hypothesis. Based on the findings in young DS persons, as well as in non-demented aged individuals, it has been widely believed that one of the major forms of early Aß deposition in human brains are diffuse plaques that are composed of amorphous, non-fibrillar aggregates of Aß, especially of Aß42 species.16 Further accumulation/removal of Aß may gradually remodel the plaques into primitive or classical types comprised of a more fibrillar form of Aß. The pure diffuse plaques in young DS brains were negative for CLAC, whereas SP readily became CLAC-positive as they acquired the morphology of primitive SP during the period between 30 to 50 years of age. Aß40/thioS-positive SP subsequently appeared, forming a distinct population from CLAC-positive SP. Such Aß40-positive SP dramatically increased after 50 years of age, whereas the level of CLAC-positive, Aß40-negative SP remained constant and these SP did not appear to develop into Aß40/thioS-positive SP. A most plausible interpretation of this chronological progression would be that after 50 years of age, some factor in the extracellular space of DS brains is altered in such a way as to lower the threshold for the incorporation of Aß1–40 onto the amyloid seeds, without allowing CLAC to bind and intervene in the rapid formation of Aß40-rich amyloid. In sharp contrast, Aß40-predominant giant-cored plaques appeared early in the brains of PSAPP TG mice, with Aß42- and CLAC-positive small, diffuse deposits only appearing at a later stage. This may be because the level of overproduction of Aß (1–40 as well as 1–42) in the brains of TG mice is so high as to allow the rapid formation of core-like seeds, as well as the incorporation of Aß1–40 into amyloid plaques at an early stage, and that the diffuse deposits that grow slower in areas "resistant" to rapid amyloid deposition "attract" endogenous murine CLAC. In this regard, it is interesting to note that amyloid plaques in PDAPP TG mice were negative for immunostaining with the AMY117 antibody, although the immunostaining patterns in AD and DS brains (eg, precedence of Aß42 deposition as diffuse plaques to the appearance of AMY-IR, absence in vascular amyloid deposits) were very similar.9 The reason for this discrepancy is unknown at present. However, our preliminary results in different lines of APP TG mice (including PDAPP mice; unpublished observations) suggested that a small amount of endogenous murine CLAC deposits in amyloid plaques irrespective of the type of APP transgenic mice. Although it has been clearly shown that AMY antigen is identical to CLAC,7 the sensitivity and specificity of AMY antibodies may be somewhat different from those of our CLAC antibodies.
It will be highly informative to produce TG mice overexpressing human CLAC-P and to cross these with others expressing human ßAPP, to determine the precise mechanistic role of CLAC in the formation of amyloid plaques in vivo. If the appearance of Aß40/thioS-positive plaques is delayed and CLAC/Aß42-positive "primitive" plaques similar to those seen in human AD and DS brains are increased in the double TG mice, the in vivo "protective" effect of CLAC binding against formation of dense, classical amyloid plaques will be confirmed. This may then pave the way toward therapeutics for AD through the modulation of CLAC.
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Acknowledgements |
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Footnotes |
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Supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports for the 21st Century Center of Excellence Programme and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research, Japan.
Accepted for publication March 30, 2004.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-1-antichymotrypsin promotes ß-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer’s disease. J Neurosci 2001, 21:1444-1451-Synuclein is phosphorylated in synucleinopathy lesions. Nature Cell Biol 2002, 4:160-164[Medline]
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