Mostly Separate Distributions of CLAC- versus Aβ40- or Thioflavin S-Reactivities in Senile Plaques Reveal Two Distinct Subpopulations of β-Amyloid Deposits
2004; Elsevier BV; Volume: 165; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63295-6
ISSN1525-2191
AutoresHisatomo Kowa, Tomoko Sakakura, Yusuke Matsuura, Tomoko Wakabayashi, David M. A. Mann, Karen Duff, Shoji Tsuji, Tadafumi Hashimoto, Takeshi Iwatsubo,
Tópico(s)Point processes and geometric inequalities
ResumoCollagenous Alzheimer amyloid plaque component (CLAC) is a unique non-Aβ amyloid component of senile plaques (SP) derived from a transmembrane collagen termed CLAC-precursor. Here we characterize the chronological and spatial relationship of CLAC with other features of SP amyloid in the brains of patients with Alzheimer's disease (AD), Down syndrome (DS), and of PSAPP transgenic mice. In AD and DS cerebral cortex, CLAC invariably colocalized with Aβ42 but often lacked Aβ40- or thioflavin S (thioS)-reactivities. Immunoelectron microscopy of CLAC-positive SP showed labeling of fibrils that are more loosely dispersed compared to typical amyloid fibrils in CLAC-negative SP. In DS cerebral cortex, diffuse plaques in young patients were negative for CLAC, whereas a subset of SP became CLAC-positive in patients aged 35 to 50 years, before the appearance of Aβ40. In DS cases over 50 years of age, Aβ40-positive SP dramatically increased, whereas CLAC burden remained at a constant level. In PSAPP transgenic mice, CLAC was positive in the diffuse Aβ deposits surrounding huge-cored plaques. Thus, CLAC and Aβ40 or thioS exhibit mostly separate distribution patterns in SP, suggesting that CLAC is a relatively early component of SP in human brains that may have inhibitory effects against the maturation of SP into β-sheet-rich amyloid deposits. Collagenous Alzheimer amyloid plaque component (CLAC) is a unique non-Aβ amyloid component of senile plaques (SP) derived from a transmembrane collagen termed CLAC-precursor. Here we characterize the chronological and spatial relationship of CLAC with other features of SP amyloid in the brains of patients with Alzheimer's disease (AD), Down syndrome (DS), and of PSAPP transgenic mice. In AD and DS cerebral cortex, CLAC invariably colocalized with Aβ42 but often lacked Aβ40- or thioflavin S (thioS)-reactivities. Immunoelectron microscopy of CLAC-positive SP showed labeling of fibrils that are more loosely dispersed compared to typical amyloid fibrils in CLAC-negative SP. In DS cerebral cortex, diffuse plaques in young patients were negative for CLAC, whereas a subset of SP became CLAC-positive in patients aged 35 to 50 years, before the appearance of Aβ40. In DS cases over 50 years of age, Aβ40-positive SP dramatically increased, whereas CLAC burden remained at a constant level. In PSAPP transgenic mice, CLAC was positive in the diffuse Aβ deposits surrounding huge-cored plaques. Thus, CLAC and Aβ40 or thioS exhibit mostly separate distribution patterns in SP, suggesting that CLAC is a relatively early component of SP in human brains that may have inhibitory effects against the maturation of SP into β-sheet-rich amyloid deposits. Alzheimer's disease (AD) is characterized pathologically by a massive accumulation of amyloid deposits comprised of amyloid β peptides (Aβ) as senile plaques (SP) or cerebral amyloid angiopathy (CAA).1Selkoe DJ Translating cell biology into therapeutic advances in Alzheimer's disease.Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar While genetic, pathological, and biochemical studies have provided firm evidence supporting the causative significance of Aβ deposition in AD,1Selkoe DJ Translating cell biology into therapeutic advances in Alzheimer's disease.Nature. 1999; 399: A23-A31Crossref PubMed Scopus (1515) Google Scholar a number of non-Aβ proteinacious components have been detected in SP amyloid, some of which have been shown to affect Aβ deposition in vivo in the brains of transgenic or knockout mice. For example, the ablation of murine Apolipoprotein E gene (APO E) attenuated the accumulation of Congophilic β-amyloid, whereas transgenic supplementation of human Apolipoprotein E protein (Apo E), especially E4 isoform, restored and promoted β-sheet-rich amyloid deposits;2Holtzman DM Bales KR Tenkova T Fagan AM Parsadanian M Sartorius LJ Mackey B Olney J McKeel D Wozniak D Paul SM Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer's disease.Proc Natl Acad Sci USA. 2000; 97: 2892-2897Crossref PubMed Scopus (716) Google Scholar, 3Irizarry MC Cheung BS Rebeck GW Paul SM Bales KR Hyman BT Apolipoprotein E affects the amount, form, and anatomical distribution of amyloid β-peptide deposition in homozygous APP(V717F) transgenic mice.Acta Neuropathol (Berl). 2000; 100: 451-458Crossref PubMed Scopus (77) Google Scholar overexpression of α1-antichymotrypsin accelerated the accumulation of β-amyloid,4Nilsson LN Bales KR DiCarlo G Gordon MN Morgan D Paul SM Potter H α-1-antichymotrypsin promotes β-sheet amyloid plaque deposition in a transgenic mouse model of Alzheimer's disease.J Neurosci. 2001; 21: 1444-1451PubMed Google Scholar whereas ablation of Apolipoprotein J attenuated it.5DeMattos RB O'Dell MA Parsadanian M Taylor JW Harmony JA Bales KR Paul SM Aronow BJ Holtzman DM Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease.Proc Natl Acad Sci USA. 2002; 99: 10843-10848Crossref PubMed Scopus (267) Google Scholar Thus, identification of Aβ-associated proteins in SP amyloid and characterization of their pathological functions is important in the elucidation of the pathobiology of β-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).6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar 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.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar Recent work suggests that CLAC is identical to the AMY antigen7Soderberg L Zhukareva V Bogdanovic N Hashimoto T Winblad B Iwatsubo T Lee VM-Y Trojanowski JQ Naslund J Molecular identification of AMY, and Alzheimer disease amyloid-associated protein.J Neuropathol Exp Neurol. 2003; 62: 1108-1117PubMed Google Scholar that had previously been identified in SP.8Schmidt ML Lee VM Forman M Chiu TS Trojanowski JQ Monoclonal antibodies to a 100-kd protein reveal abundant Aβ-negative plaques throughout gray matter of Alzheimer's disease brains.Am J Pathol. 1997; 151: 69-80PubMed Google Scholar, 9Lemere CA Grenfell TJ Selkoe DJ The AMY antigen co-occurs with Aβ and follows its deposition in the amyloid plaques of Alzheimer's disease and Down's syndrome.Am J Pathol. 1999; 155: 29-37Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar In vitro studies show that recombinant CLAC specifically binds aggregated Aβ, but not its soluble form.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar 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.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar This selectivity in the distribution of CLAC deposition in a specific subfraction of amyloid deposits is unique, and not observed with any other non-Aβ SP component proteins, ie, Apo E, complement component C1q or heparan sulfate proteoglycan (10Cho HS Hyman BT Greenberg SM Rebeck GW Quantitation of apoE domains in Alzheimer disease brain suggests a role for apoE in Aβ aggregation.J Neuropathol Exp Neurol. 2001; 60: 342-349Crossref PubMed Scopus (93) Google Scholar, 11Stoltzner SE Grenfell TJ Mori C Wisniewski KE Wisniewski TM Selkoe DJ Lemere CA Temporal accrual of complement proteins in amyloid plaques in Down's syndrome with Alzheimer's disease.Am J Pathol. 2000; 156: 489-499Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 12van Horssen J Otte-Holler I David G Maat-Schieman ML van den Heuvel LP Wesseling P de Waal RM Verbeek MM Heparan sulfate proteoglycan expression in cerebrovascular amyloid β deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains.Acta Neuropathol (Berl). 2001; 102: 604-614PubMed Google Scholar, Sakakura T, Kowa H, Iwatsubo T, unpublished observations). Other well-known features of SP amyloid exhibiting selective distributions are the heterogenous C termini of Aβ, Aβ40, and Aβ42. Aβ exhibits two major C-terminal variants by the heterogeneity in positions of γ-secretase cleavage of β-amyloid precursor protein (βAPP): Aβ42 with a longer C terminus is a relatively minor secreted species13Suzuki N Cheung TT Cai XD Odaka A Otvos Jr, L Eckman C Golde TE Younkin SG An increased percentage of long amyloid β protein secreted by familial amyloid β protein precursor (βAPP717) mutants.Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1334) Google Scholar but has a higher propensity to aggregate14Jarrett JT Berger EP Lansbury Jr, PT The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: implications for the pathogenesis of Alzheimer's disease.Biochemistry. 1993; 32: 4693-4697Crossref PubMed Scopus (1729) Google Scholar and deposits initially and widely in SP.15Iwatsubo T Odaka A Suzuki N Mizusawa H Nukina N Ihara Y Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ-monoclonals: evidence that an initially deposited species is Aβ42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1523) Google Scholar, 16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar In contrast, Aβ40, a major secreted species,13Suzuki N Cheung TT Cai XD Odaka A Otvos Jr, L Eckman C Golde TE Younkin SG An increased percentage of long amyloid β protein secreted by familial amyloid β protein precursor (βAPP717) mutants.Science. 1994; 264: 1336-1340Crossref PubMed Scopus (1334) Google Scholar accumulates later robustly in a subset of SP as well as in CAA. However, the temporal and spatial relationships between deposition of CLAC, Aβ40, and Aβ42, each of which shows unique deposition patterns, as well as the pathological significance of CLAC deposition in β-amyloid formation, remains elusive. In this study, we have examined the relationship between deposition of CLAC and different Aβ species in the brains of patients with AD or Down syndrome (DS), as well as those of transgenic (TG) mice developing β-amyloid plaques, using multiple labeling and morphometric evaluation. We find that CLAC-positive SP and Aβ40/thioflavin S (thioS)-positive SP show mostly separate distribution patterns, suggesting a possible role of CLAC binding to prevent further maturation of β-sheet-rich, dense amyloid deposits. 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 βAPP gene and M146L mutant PS1 (PSAPP mice17Holcomb L Gordon MN McGowan E Yu X Benkovic S Jantzen P Wright K Saad I Mueller R Morgan D Sanders S Zehr C O'Campo K Hardy J Prada CM Eckman C Younkin S Hsiao K Duff K Accelerated Alzheimer-type phenotype in transgenic mice carrying both mutant amyloid precursor protein and presenilin 1 transgenes.Nat Med. 1998; 4: 97-100Crossref PubMed Scopus (1145) Google Scholar) 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.18Takeuchi A Irizarry MC Duff K Saido TC Hsiao Ashe K Hasegawa M Mann DMA Hyman BT Iwatsubo T Age-related amyloid β deposition in transgenic mice overexpressing both presenilin 1 and amyloid β precursor protein Swedish mutant is not associated with global neuronal loss.Am J Pathol. 2000; 157: 331-339Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar 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,6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar 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.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar The immunostaining patterns with antibodies to the NC domains of CLAC-P were essentially similar to that obtained with 9D2.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar Detection of deposits of murine CLAC in the brains of TG mice was performed with anti-NC2–2.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar Mouse mAbs BC05 and BA27, which specifically react with the C termini of Aβ42 and Aβ40, respectively, have been described;15Iwatsubo T Odaka A Suzuki N Mizusawa H Nukina N Ihara Y Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ-monoclonals: evidence that an initially deposited species is Aβ42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1523) Google Scholar, 16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar 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.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar 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,9Lemere CA Grenfell TJ Selkoe DJ The AMY antigen co-occurs with Aβ and follows its deposition in the amyloid plaques of Alzheimer's disease and Down's syndrome.Am J Pathol. 1999; 155: 29-37Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar was effective and routinely performed. Immunoperoxidase staining using avidin-biotin complex using diaminobenzidine was performed as described.15Iwatsubo T Odaka A Suzuki N Mizusawa H Nukina N Ihara Y Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ-monoclonals: evidence that an initially deposited species is Aβ42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1523) Google Scholar, 16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar Fluorescence labeling of amyloid by thioS was performed as described:19Dickson DW Farlo J Davies P Crystal H Fuld P Yen SH Alzheimer's disease: a double-labeling immunohistochemical study of senile plaques.Am J Pathol. 1988; 132: 86-101PubMed Google Scholar, 20Schmidt ML Robinson KA Lee VM Trojanowski JQ Chemical and immunological heterogeneity of fibrillar amyloid in plaques of Alzheimer's disease and Down's syndrome brains revealed by confocal microscopy.Am J Pathol. 1995; 147: 503-515PubMed Google Scholar 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.21Fujiwara H Hasegawa M Dohmae N Kawashima A Masliah E Goldberg MS Shen J Takio K Iwatsubo T α-Synuclein is phosphorylated in synucleinopathy lesions.Nature Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (156) Google Scholar Fluorescence signals obtained by Alexa 488, 594, and 647 were displayed in pseudocolors of green, red, and blue, respectively. Deposition of CLAC or Aβ in peroxidase-labeled sections was evaluated by quantifying the total percentage of cortical surface area covered by CLAC- or Aβ-IRs (percent CLAC or amyloid burden) as described.16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar, 18Takeuchi A Irizarry MC Duff K Saido TC Hsiao Ashe K Hasegawa M Mann DMA Hyman BT Iwatsubo T Age-related amyloid β deposition in transgenic mice overexpressing both presenilin 1 and amyloid β precursor protein Swedish mutant is not associated with global neuronal loss.Am J Pathol. 2000; 157: 331-339Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar 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 × 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. Double immunolabeling for electron microscopic observation of CLAC/Aβ42 or CLAC/Aβ40 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 Aβ40/42 was performed as described by Yamaguchi et al22Yamaguchi H Maat-Schieman ML van Duinen SG Prins FA Neeskens P Natte R Roos RA Amyloid β protein (Aβ) starts to deposit as plasma membrane-bound form in diffuse plaques of brains from hereditary cerebral hemorrhage with amyloidosis-Dutch type, Alzheimer disease, and non-demented aged subjects.J Neuropathol Exp Neurol. 2000; 59: 723-732Crossref PubMed Scopus (74) Google Scholar 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). 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.6Hashimoto T Wakabayashi T Watanabe A Kowa H Hosoda R Nakamura A Kanazawa I Arai T Takio K Mann DMA Iwatsubo T CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV.EMBO J. 2002; 21: 1524-1534Crossref PubMed Scopus (171) Google Scholar Notably, CLAC-IRs were always accompanied by Aβ-IRs. This observation prompted us to further examine the relationship between CLAC-IR and those of the major two Aβ C-terminal species, Aβ42 and Aβ40, which exhibit distinct distributions in AD brain.15Iwatsubo T Odaka A Suzuki N Mizusawa H Nukina N Ihara Y Visualization of Aβ42(43) and Aβ40 in senile plaques with end-specific Aβ-monoclonals: evidence that an initially deposited species is Aβ42(43).Neuron. 1994; 13: 45-53Abstract Full Text PDF PubMed Scopus (1523) Google Scholar, 16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar Comparison of three serial sections of frontal neocortex from sporadic AD cases immunolabeled for Aβ42, CLAC, and Aβ40, respectively, showed that most of the CLAC-positive SP was simultaneously positive for Aβ42 (Figure 1, A and B, arrowheads). In contrast, CLAC was often negative in Aβ40-positive amyloid deposits that are often uniformly and densely immunostained for Aβ40, 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 Aβ42 overlaps and encompasses that of CLAC, whereas that of Aβ40 shows only a limited extent of overlap with that of CLAC. We then directly addressed the co-localization of CLAC and Aβ42 or Aβ40 by double immunofluorescence labeling combined with morphometric analysis. Approximately ∼43% of Aβ42-positive areas were CLAC-positive, and ∼93% of the CLAC-positive areas (CLAC burden) were simultaneously Aβ42-positive, only ∼7.2% remaining Aβ42-negative (Figure 1, D and F). In sharp contrast, only ∼26% of Aβ40-positive areas were CLAC-positive and ∼32% of CLAC-positive areas were Aβ40-positive (Figure 1, E and F). We further analyzed the relationship between CLAC and thioS-positive Aβ deposits, which are regarded to represent highly β-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 Aβ42/CLAC or Aβ40/CLAC (Figure 2, A to C). Comparison of Aβ40 and thioS reactivities, on serial mirror sections, showed that thioS-positive areas (Figure 2A, green) were always Aβ40-positive (Figure 2B, red), and accounted for ∼50% of the Aβ40-positive areas within the entire depth of cerebral cortex. Occasional SP showed co-localization of Aβ40 and CLAC, in which thioS was invariably negative (Figure 2, A and B, a SP in the lower left corner).Figure 2A–B: Six-μm thick, mirror-sectioned serial sections from frontal neocortex of AD brain doubly stained for CLAC (blue, anti-pyroGlu113) and thioS (green) (A) or for CLAC (blue, anti-pyroGlu113) and Aβ40 (red, BA27) (B). Note that most of the thioS-positive areas in A are overlapped and included within the Aβ40-positive areas in B, and occasional CLAC/Aβ40-positive SP (visualized in purple in B; eg, a SP in the lower left) are thioS-negative in A. Bar, 50 μm. C: Relative ratios of CLAC-positive, thioS-negative (blue), CLAC- and thioS-positive (light blue) and CLAC-negative, thioS-positive (green) SP areas in sections stained as in A (mean value in eight AD cases in which numerous thioS-positive SP were observed) are shown. The total areas (100%) correspond to those positive for CLAC plus thio-S.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The distinct distribution patterns of CLAC- or Aβ40/thioS-positive SP led us to examine the ultrastructural characteristics of Aβ 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 Aβ40- or Aβ42-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 Aβ40-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 β-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-Aβ40 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 Aβ42 (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). 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 Aβ40 and Aβ42, we studied the frontal neocortex of patients with Down syndrome (DS), dying at ages ranging from 31 to 71 years, by immunohistochemistry for Aβ42, Aβ40, and CLAC. Purely diffuse plaques, which predominate in the neocortex in young individuals with DS and which are known to be exclusively positive for Aβ42 (Figure 4A) but negative for Aβ40 (Figure 4G) (see also16Iwatsubo T Mann DMA Odaka A Suzuki N Ihara Y Amyloid β protein (Aβ) deposition: aβ42(43) precedes Aβ40 in Down syndrome.Ann Neurol. 1995; 37: 294-299Crossref PubMed Scopus (334) Google Scholar), were CLAC-negative (Figure 4D). However, a subset of Aβ42-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 bur
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