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Heparan Sulfate Subdomains that are Degraded by Sulf Accumulate in Cerebral Amyloid ß Plaques of Alzheimer's Disease

2012; Elsevier BV; Volume: 180; Issue: 5 Linguagem: Inglês

10.1016/j.ajpath.2012.01.015

1525-2191

Tomomi Hosono‐Fukao, Shiori Ohtake‐Niimi, Hitomi Hoshino, Markus Britschgi, Hiroyasu Akatsu, Md. Motarab Hossain, Kazuchika Nishitsuji, Toin H. Van Kuppevelt, Koji Kimata, Makoto Michikawa, Tony Wyss‐Coray, Kenji Uchimura,

Connective tissue disorders research

Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid β peptide (Aβ). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aβ aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains. Alzheimer's disease (AD) is characterized by extracellular cerebral accumulation of amyloid β peptide (Aβ). Heparan sulfate (HS) is a glycosaminoglycan that is abundant in the extracellular space. The state of sulfation within the HS chain influences its ability to interact with a variety of proteins. Highly sulfated domains within HS are crucial for Aβ aggregation in vitro. Here, we investigated the expression of the sulfated domains and HS disaccharide composition in the brains of Tg2576, J20, and T41 transgenic AD mouse models, and patients with AD. RB4CD12, a phage display antibody, recognizes highly sulfated domains of HS. The RB4CD12 epitope is abundant in the basement membrane of brain vessels under physiological conditions. In the cortex and hippocampus of the mice and patients with AD, RB4CD12 strongly stained both diffuse and neuritic amyloid plaques. Interestingly, RB4CD12 also stained the intracellular granules of certain hippocampal neurons in AD brains. Disaccharide compositions in vessel-enriched and nonvasculature fractions of Tg2576 mice and AD patients were found to be comparable to those of non-transgenic and non-demented controls, respectively. The RB4CD12 epitope in amyloid plaques was substantially degraded ex vivo by Sulf-1 and Sulf-2, extracellular HS endosulfatases. These results indicate that formation of highly sulfated HS domains may be upregulated in conjunction with AD pathogenesis, and that these domains can be enzymatically remodeled in AD brains. Heparan sulfate (HS) is a linear polysaccharide that exists in large quantities in the extracellular space. One or more HS chains are covalently bound to a core protein comprising heparan sulfate proteoglycan (HSPG).1Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2295) Google Scholar, 2Esko J.D. Lindahl U. Molecular diversity of heparan sulfate.J Clin Invest. 2001; 108: 169-173Crossref PubMed Scopus (782) Google Scholar HS chains and heparins, structural analogues of HS chains, are a family of glycosaminoglycans consisting of repeating disaccharide units of glucuronic/iduronic acid and glucosamine. Modification with sulfation as well as elongation of these disaccharides is enzymatic,3Gallagher J.T. Heparan sulfate: growth control with a restricted sequence menu.J Clin Invest. 2001; 108: 357-361Crossref PubMed Scopus (282) Google Scholar bestowing on the chains structural diversity.4Nakato H. Kimata K. Heparan sulfate fine structure and specificity of proteoglycan functions.Biochim Biophys Acta. 2002; 1573: 312-318Crossref PubMed Scopus (130) Google Scholar, 5Parish C.R. The role of heparan sulphate in inflammation.Nat Rev Immunol. 2006; 6: 633-643Crossref PubMed Scopus (381) Google Scholar, 6Bishop J.R. Schuksz M. Esko J.D. Heparan sulphate proteoglycans fine-tune mammalian physiology.Nature. 2007; 446: 1030-1037Crossref PubMed Scopus (1241) Google Scholar HS contains highly sulfated domains and partially sulfated or non-sulfated domains, which are transitional.3Gallagher J.T. Heparan sulfate: growth control with a restricted sequence menu.J Clin Invest. 2001; 108: 357-361Crossref PubMed Scopus (282) Google Scholar Highly sulfated domains are formed by consecutive clusters of sulfated disaccharides. It has been shown that a trisulfated disaccharide structure [-iduronic acid(2S)-Glucosamine(NS,6S)-] occurs within highly sulfated domains. RB4CD12, a phage display anti-HS antibody, has been shown to recognize trisulfated disaccharide-containing HS subdomains7Dennissen M.A. Jenniskens G.J. Pieffers M. Versteeg E.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies.J Biol Chem. 2002; 277: 10982-10986Crossref PubMed Scopus (136) Google Scholar, 8Hossain M.M. Hosono-Fukao T. Tang R. Sugaya N. van Kuppevelt T.H. Jenniskens G.J. Kimata K. Rosen S.D. Uchimura K. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.Glycobiology. 2010; 20: 175-186Crossref PubMed Scopus (64) Google Scholar, 9Hosono-Fukao T. Ohtake-Niimi S. Nishitsuji K. Hossain M.M. van Kuppevelt T.H. Michikawa M. Uchimura K. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.J Neurosci Res. 2011; 89: 1840-1848Crossref PubMed Scopus (5) Google Scholar Trisulfated disaccharides are considered to be key elements in molecular interactions between HS/heparin and many ligands, including growth factors and morphogens.1Bernfield M. Gotte M. Park P.W. Reizes O. Fitzgerald M.L. Lincecum J. Zako M. Functions of cell surface heparan sulfate proteoglycans.Annu Rev Biochem. 1999; 68: 729-777Crossref PubMed Scopus (2295) Google Scholar, 10Esko J.D. Selleck S.B. Order out of chaos: assembly of ligand binding sites in heparan sulfate.Annu Rev Biochem. 2002; 71: 435-471Crossref PubMed Scopus (1226) Google Scholar Trisulfated disaccharides, as well as the RB4CD12 epitope, are degraded by extracellular sulfatases, Sulf-1, and Sulf-2.8Hossain M.M. Hosono-Fukao T. Tang R. Sugaya N. van Kuppevelt T.H. Jenniskens G.J. Kimata K. Rosen S.D. Uchimura K. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.Glycobiology. 2010; 20: 175-186Crossref PubMed Scopus (64) Google Scholar, 11Saad O.M. Ebel H. Uchimura K. Rosen S.D. Bertozzi C.R. Leary J.A. Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase.Glycobiology. 2005; 15: 818-826Crossref PubMed Scopus (84) Google Scholar, 12Morimoto-Tomita M. Uchimura K. Werb Z. Hemmerich S. Rosen S.D. Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans.J Biol Chem. 2002; 277: 49175-49185Crossref PubMed Scopus (341) Google Scholar In the brain, we have shown that the RB4CD12 HS domains are abundantly present in the vasculature9Hosono-Fukao T. Ohtake-Niimi S. Nishitsuji K. Hossain M.M. van Kuppevelt T.H. Michikawa M. Uchimura K. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.J Neurosci Res. 2011; 89: 1840-1848Crossref PubMed Scopus (5) Google Scholar and that these domains can be degraded by the Sulfs ex vivo.8Hossain M.M. Hosono-Fukao T. Tang R. Sugaya N. van Kuppevelt T.H. Jenniskens G.J. Kimata K. Rosen S.D. Uchimura K. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.Glycobiology. 2010; 20: 175-186Crossref PubMed Scopus (64) Google Scholar However, the roles of the RB4CD12 HS domains in pathological and physiological processes in brain vasculature are not known.Alzheimer's disease (AD) is a progressive neurodegenerative disorder. One of the pathological hallmarks of AD is the presence of extracellular amyloid plaques in brain areas that are responsible for cognition and memory functions. The predominant composition of amyloid plaques is fibrils made of amyloid β peptide (Aβ). A great deal of biochemical and genetic evidence has indicated that aggregation and accumulation of Aβ in toxic forms within the extracellular space play a central role in AD pathogenesis. One of the authors previously reported that certain structures of HS chains exist in amyloid plaques of AD brains,13Snow A.D. Mar H. Nochlin D. Kimata K. Kato M. Suzuki S. Hassell J. Wight T.N. The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease.Am J Pathol. 1988; 133: 456-463PubMed Google Scholar and that structural variation of HSPG correlates with amyloid plaque formation in the brains of AD patients.14Snow A.D. Sekiguchi R.T. Nochlin D. Kalaria R.N. Kimata K. Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.Am J Pathol. 1994; 144: 337-347PubMed Google Scholar HSPG is also known to facilitate cerebral amyloid deposition induced exogenously in a rat model in vivo.15Snow A.D. Sekiguchi R. Nochlin D. Fraser P. Kimata K. Mizutani A. Arai M. Schreier W.A. Morgan D.G. An important role of heparan sulfate proteoglycan (Perlecan) in a model system for the deposition and persistence of fibrillar A beta-amyloid in rat brain.Neuron. 1994; 12: 219-234Abstract Full Text PDF PubMed Scopus (277) Google Scholar Functional roles of HS and HSPG in AD pathology are proposed to be acceleration of Aβ fibril formation and protection of the fibril against microglial phagocytosis.16van Horssen J. Wesseling P. van den Heuvel L.P. de Waal R.M. Verbeek M.M. Heparan sulphate proteoglycans in Alzheimer's disease and amyloid-related disorders.Lancet Neurol. 2003; 2: 482-492Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar It was reported that the aggregation state of Aβ requires its binding properties to heparin.17Watson D.J. Lander A.D. Selkoe D.J. Heparin-binding properties of the amyloidogenic peptides Abeta and amylin Dependence on aggregation state and inhibition by Congo red.J Biol Chem. 1997; 272: 31617-31624Crossref PubMed Scopus (122) Google Scholar Pathological correlations between the RB4CD12 HS domains, which are rich in heparin and AD have not been established. Here we present evidence that the RB4CD12 HS domains are accumulated in cerebral amyloid plaques of transgenic AD mouse models and patients with AD, and that these HS epitopes can be degraded by Sulf-1 and Sulf-2 ex vivo.Materials and MethodsMaterialsThe RB4CD12 phage display-derived anti-heparan sulfate antibody was produced in a vesicular stomatitis virus (VSV)-tag version and purified as previously described.7Dennissen M.A. Jenniskens G.J. Pieffers M. Versteeg E.M. Petitou M. Veerkamp J.H. van Kuppevelt T.H. Large, tissue-regulated domain diversity of heparan sulfates demonstrated by phage display antibodies.J Biol Chem. 2002; 277: 10982-10986Crossref PubMed Scopus (136) Google Scholar Alternative nomenclature of RB4CD12 is HS3A8. The following materials were commercially obtained from the sources indicated. Heparinases (I, II and III), polyclonal rabbit anti-laminin antibody (Ab), horseradish peroxidase-conjugated monoclonal anti-VSV Ab, and Cy3-conjugated monoclonal anti-VSV Ab were from Sigma (St. Louis, MO); biotinylated monoclonal anti-amyloid β (N-terminus) Ab (82E1) was from IBL (Gunma, Japan); polyclonal rabbit anti-VSV Ab was from Bethyl Laboratories (Montgomery, TX); Cy2-conjugated goat anti-mouse IgG (H+L), Cy2-conjugated goat anti-rabbit IgG (H+L), Cy2-conjugated goat anti-rat IgG (H+L) Abs, and Cy2-conjugated streptavidin were from Jackson ImmunoResearch Laboratories (West Grove, PA); rabbit anti-Iba1 Ab was from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); rabbit anti-glial fibrillary acidic protein and monoclonal anti-phospho-PHF-tau pThr231 (AT180) Abs were from Thermo Scientific (Rockford, IL); goat anti-mouse syndecan-3 Ab was from R&D Systems, Inc (Minneapolis, MN); rabbit anti-glypican-1 (M-95) Ab was from Santa Cruz Biotechnology, Inc (Santa Cruz, CA); polyclonal goat anti-rabbit IgG Nanogold, ϕ1.4 nm, was from Nanoprobes (Yaphank, NY); and horseradish peroxidase-conjugated goat anti-rabbit IgG was from Cell Signaling Technology, Inc. (Beverly, MA).AnimalsC57BL/6 mice were from Japan SLC Inc. (Hamamatsu, Japan). Heterozygotic transgenic mice that expressed the human amyloid precursor protein bearing the Swedish (K670N, M671L) mutation (Tg2576 strain),18Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Correlative memory deficits.A beta elevation, and amyloid plaques in transgenic mice, Science. 1996; 274: 99-102Google Scholar the Swedish and Indiana (V717F) mutations (J20 strain),19Mucke L. Masliah E. Yu G.Q. Mallory M. Rockenstein E.M. Tatsuno G. Hu K. Kholodenko D. Johnson-Wood K. McConlogue L. High-level neuronal expression of abeta 1–42 in wild-type human amyloid protein precursor transgenic mice: synaptotoxicity without plaque formation.J Neurosci. 2000; 20: 4050-4058Crossref PubMed Google Scholar or the Swedish and London (V717I) mutations (T41 strain)20Rockenstein E. Mallory M. Mante M. Sisk A. Masliaha E. Early formation of mature amyloid-beta protein deposits in a mutant APP transgenic model depends on levels of A beta(1–42).J Neurosci Res. 2001; 66: 573-582Crossref PubMed Scopus (192) Google Scholar were maintained in barrier facilities. Tg2576 mice were purchased from Taconic Farms, Inc., Hudson, NY. J20 mice were from the Jackson Laboratory (Bar Harbor, ME). The National Center of Geriatrics and Gerontology Institutional Animal Care and Use Committee approved the animal studies.Human Postmortem Brain TissuesPatients with sporadic AD received a pathological diagnosis according to the criteria of the Consortium to Establish a Registry for Alzheimer's Disease and the Braak stage. Non-demented controls were elderly patients who were age-matched and without significant neurological disorders. Patients were also cognitively evaluated by neuropsychological tests using the Mini-Mental State Examination and Hasegawa's dementia scale, which is commonly used in Japan. Entorhinal cortex and hippocampus postmortem tissue samples from neurologically unimpaired subjects (non-demented controls [NDCs]) and from subjects with AD were obtained under Committees on Human Research approval of National Center for Geriatrics and Gerontology and Choju Medical Institute of Fukushimura Hospital. Diagnosis of AD was confirmed by pathological and clinical criteria (Table 1). The incidence of vascular risk factors (eg, atherosclerosis, myocardial infarction, and so forth), the sex ratio, age, and the postmortem interval were comparable between NDC and AD (Table 1). Tissue was cut and frozen or fixed with formalin, and then embedded with paraffin. Frozen tissues were subjected to structural analysis of HS. The embedded tissues were cut using a microtome.Table 1Clinical and Neuropathological Characteristics of Alzheimer's Disease and Non-Demented Control Donor Patients used in the Disaccharide Composition Analysis of Heparan SulfatePatient numberAge (years)SexStage of amyloid deposits (0, A, B, C)⁎0 = none, A = rare or a few, B = mild or moderate, C = numerous or marked.NFT stage (I–VI)Cerebral amyloid angiopathyVascular risk factorsPMI (hr)Alzheimer's disease patients 050894FCV+CI43 051283FCVI+ATH2 060491FCV−CI8 080593FCVI+CI27 081080MCV−CI15 081181MCVI−−8 081491MCV+−5 082487FBVI−−9Age-matched non-demented controls 070795FAII−MI4 071083FAII−CH/CI24 060190FBII−MI4 080293FAIII−CH/CI20 070484MBII−CI3 080782M0I−CH8 090891MAII−−NA 090387F0II−CI7ATH, atherosclerosis; CH, cerebral hemorrhage; CI, cerebral infarction; F, female; M, male; MI, myocardial infarction; NA, not applicable; NFT, neurofibrillary tangle; PMI, postmortem interval. 0 = none, A = rare or a few, B = mild or moderate, C = numerous or marked. Open table in a new tab Fractionation of Brain SamplesA snap-frozen mouse cortex (∼25 mg) was placed in a tube containing 600 μL (30 volume of the tissue weight) of ice-cold Tris-buffered saline (TBS) (20 mmol/L Tris and 137 mmol/L NaCl, pH 7.6) and protease inhibitors (complete protease inhibitor cocktail; Roche Diagnostics, Mannheim, Germany). The tube was placed in a water bath of the Bioruptor ultrasonic vibration (CosmoBio, Tokyo). The tissue was fragmented by sonicating the tube for 15 seconds with the maximum ultrasonic wave output power 4 to 5 times until solid materials in the tube became invisible. The material was ultracentrifuged at 100,000 × g for 20 minutes at 4°C. The supernatant was collected and stored frozen as TBS or “TBS soluble fraction.” The resulting precipitate was suspended in 600 μL (the same volume as previously described) of TBS containing 1% SDS. The suspension was centrifuged at 12,000 rpm for 20 minutes at room temperature. The resulting supernatant was collected and stored frozen as TBS or “TBS-insoluble/1% SDS-soluble fraction.” The protein concentrations of both fractions were measured with a BCA Protein Assay Reagent Kit (Thermo Scientific). Brain cortices were dissected out from 3 Non-Tg or 3 Tg2576 18-month-old mice and then snap frozen. Brain samples were put together, placed on a glass Petri dish, and minced with a blade. The tissues were transferred into a tube containing 1 mL of ice-cold TBS. The tissues were homogenized with a Dounce homogenizer. The homogenate was filtered with a 100-μm nylon mesh. The filtered materials on the mesh were collected and then subjected to the structural analysis described as follows (“vessel-enriched fractions”). Materials filtered through the 100-μm nylon mesh were collected and then analyzed (“non-vasculature fractions”). Methylene blue staining and bright field microscopy confirmed cerebral blood vessels on the filters.ImmunohistochemistryFresh mouse brains were embedded in O.C.T. compound (Sakura Finetek, Torrance, CA) and frozen in liquid nitrogen. The brains were stored at −80°C until analysis. Cryostat-cut sections (10-μm thick) were prepared on MAS-coated glass slides (Matsunami, Osaka, Japan), fixed in ice-cold acetone for 15 minutes, and then air-dried for 30 minutes. Sections were incubated with blocking solution (3% bovine serum albumin in PBS) for 15 minutes at RT. Sections were washed twice with PBS and then incubated with a mixture of RB4CD12 (1:100 dilution), rabbit anti-laminin antibody (1:100 dilution, Sigma), and biotinylated 82E1 (1:50 dilution) overnight at 4°C. Then, primary antibodies were detected with Cy3-conjugated monoclonal anti-VSV-G (4 μg/mL), Cy2-conjugated polyclonal goat anti-rabbit IgG (3 μg/mL), and aminomethylcoumarin acetate-conjugated streptavidin (6.8 μg/mL, Jackson ImmunoResearch, West Grove, PA). Sections were mounted in FluorSave Reagent (Merck, Darmstadt, Germany). Digital images were captured by fluorescent microscopy (model BX50, Olympus, Tokyo, Japan) at the same setting for each antibody. The fluorescently stained area was quantitatively determined using Image-Pro Plus software (Media Cybernetics, Bethesda, MD). To determine the effects of the Sulfs and heparinases, 3% bovine serum albumin-blocked sections were pre-treated with 100 μL of a reaction mixture containing 5 μmol HEPES, pH 7.5, 1 μmol MgCl2, and enzymes at 37°C overnight. Recombinant human Sulf-1 (0.4 μg) and human Sulf-2 (0.4 μg) were prepared from conditioned medium of transfected HEK293 cells and used as previously described.8Hossain M.M. Hosono-Fukao T. Tang R. Sugaya N. van Kuppevelt T.H. Jenniskens G.J. Kimata K. Rosen S.D. Uchimura K. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.Glycobiology. 2010; 20: 175-186Crossref PubMed Scopus (64) Google Scholar For pretreatment with heparitinases, a mixture of 1 mU heparinase I, 0.25 mU heparinase II, and 0.1 mU heparinase III were added to the reaction mixture. Cy2-conjugated streptavidin was used to detect bound 82E1. Human brain sections (4-μm thickness) were obtained from paraffin-embedded tissue blocks. After deparaffinization and rehydration, endogenous peroxidase activity was quenched with 3% H2O2 (Sigma). Sections were subjected to heat-induced epitope retrieval followed by IgG blocking using M.O.M. kit (Vector Laboratories Inc., Burlingame, CA). Sections were incubated with RB4CD12 (1:100 dilution) overnight at 4°C. Bound antibody was detected with horseradish peroxidase-conjugated mouse anti-VSV followed by visualization with diaminobenzidine (3,3′-diaminobenzidinetetrahydrochloride) supplied with the EnVision reagent (Dako Japan, Tokyo, Japan).Immunoelectron MicroscopyCryostat-cut sections from 17-month-old Tg2576 mouse brains were prepared on MAS-coated glass slides, fixed in 4% paraformaldehyde for 5 minutes, and then washed with PBS for 1 hour. Sections were incubated with 3% bovine serum albumin for 30 minutes at RT. Diluted RB4CD12 antibody (1:40) was then applied overnight. After washing, diluted rabbit anti-VSV secondary antibody (7.2 μg/mL) was applied for 1 hour. After several washes, diluted goat anti-rabbit IgG antibody coupled with 1.4-nm-diameter tertiary gold particles (1:40) was applied for 30 minutes. The samples were then washed and fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 3 hours, followed by enlargement of the gold particles with an HQ-Silver Enhancement Kit (Nanoprobes). The specimens were examined in a Hitachi H-7600 transmission electron microscope (Hitachi Koki, Tokyo, Japan).ImmunoblotsThe proteins (40 μg per lane) were separated by NuPAGE 3% to 8% polyacrylamide gel electrophoresis (Invitrogen, Carlsbad, CA), and blotted onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was blocked with 5% skim milk/PBS 0.1% Tween for 1 hour at room temperature and then incubated overnight with RB4CD12 antibody (1:500) in TBS 0.1% Tween at 4°C. The membrane was washed and incubated with horseradish peroxidase-conjugated mouse anti-VSV (1:2000) for 1 hour at RT. Bound antibodies were visualized with SuperSignal West Dura Chemiluminescent reagent (Thermo Scientific). Signals were visualized and quantified using a LAS-3000 mini luminescent image analyzer (Fujifilm, Tokyo, Japan).Preparation and Structural Analysis of HSThere were 100 mg of frozen brain tissues or the cortical vessel residue that remained on filters previously described, which was suspended in 2 mL of 0.2N NaOH and incubated overnight at RT. The samples were neutralized with 4 N HCl and then treated with DNase I and RNase A (0.04 mg/mL each) (Roche Diagnostics) in 50 mmol/L Tris-HCl, pH8.0, 10 mmol/L MgCl2 for 3 hours at 37°C. Subsequently, the samples were treated with actinase E (0.08 mg/mL) (Kaken Pharmaceutical Co., Ltd., Tokyo, Japan) overnight at 37°C. The supernatant was collected by centrifugation at 5000 × g at 4°C for 10 minutes after heat inactivation of the enzyme and then mixed with the same volume of 50 mmol/L Tris-HCl, pH 7.2. The HS was purified by DEAE-Sepharose column chromatography.9Hosono-Fukao T. Ohtake-Niimi S. Nishitsuji K. Hossain M.M. van Kuppevelt T.H. Michikawa M. Uchimura K. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.J Neurosci Res. 2011; 89: 1840-1848Crossref PubMed Scopus (5) Google Scholar The disaccharide compositions of the HS were determined by reversed-phase ion-pair chromatography with postcolumn fluorescent labeling.Quantitative Real Time-PCR for Expression of Genes Related to HS SynthesisTotal RNA was extracted from frozen mouse cortices using TRIZol Reagent (Invitrogen). Total RNA (4 μg was used for reverse transcription reaction in 100 μL of buffer with random hexamers, using Superscript II Reverse Transcriptase (Invitrogen). PCR was conducted in duplicate with 20-μL reaction volumes of SYBR Premix Ex TaqII (Takara Bio Inc. Shiga, Japan), 0.2 μmol/L of each primer and 2 μL of the cDNA reaction mixture. PCR was performed using the following parameters: 95°C, 10 seconds, 1 cycle; 95°C, 5 seconds; and 60°C, 30 seconds, 40 cycles. Analysis was performed using sequence detection software supplied with Thermal Cycler Dice Real Time System TP800 (Takara Bio Inc.). mRNA levels of each gene were normalized by comparison to β-actin mRNA levels. Conclusions are drawn from duplicate PCR reactions at least two independent reverse transcription reactions. Primer sequences used in this study are as indicated for Ndst1, 5′-GCAGATGGCCCTGAACAAGAA-3′ and 5′-GCACGTGCACAGGGTACACA-3′; for N-deacetylase/N-sulfotransferase 2 (Ndst2), 5′-TCATCCAGAAGTTCCTGGGTATCAC-3′ and 5′-AGACAGCGAGTCTTACCACCTTCAA-3′; for Ndst3, 5′-TCTGGTGTCAGCTGCTGGAAG-3′ and 5′-CACGTTGTGGTCGCGGTAGTAG-3′; for Ndst4, 5′-TTGTTCCCAAAGCCAAGATCATTAC-3′ and 5′-TCAGGGCAGCTGGATCTTCA-3′; for Hs6st1, 5′-CTGACTGGACCGAACTCACCAA-3′ and 5′-TCTCGCAGCAGGGTGATGTAGTAG-3′; for Hs6st2, 5′-AAACTTCAACTCAGGCGCCAAC-3′ and 5′-CTCCATTCACTCAAGTACCGTGACA-3′; for Hs6st3, 5′-GACTGGACCGAGCTCACCAA-3′ and 5′-CATGCTTCCATTCGCTCAGGTA-3′; for Hs2st1, 5′-GCAAGCACCTCGTTCACCAA-3′ and 5′-CATCTCGTTCCAGGTGGTTATGTTC-3′; for Sulf1, 5′-CCACATGGAGTTCACCAACGTC-3′ and 5′-TAGCCGTGGTCCGCAGTGTA-3′; for Sulf2, 5′-GAGTACCAGACAGCATGCGAACA-3′ and 5′-TTGGGCACCAGGTTGGAGA-3′; and for Actb, 5′-CATCCGTAAAGACCTCTATGCCAAC-3′ and 5′-ATGGAGCCACCGATCCACA-3′.Statistical AnalysisAll data are presented as means ± SD unless noted otherwise. The values were analyzed by unpaired Student's t-test using Prism software (GraphPad Software, La Jolla, CA). P values less than 0.05 were considered to be statistically significant.ResultsImmunoreactivity of RB4CD12 Anti-Heparan Sulfate is Colocalized with Aβ Plaques in Brains of Transgenic Mouse Models of ADRB4CD12 scFv antibody recognizes trisulfated disaccharide-containing highly sulfated domains within HS.8Hossain M.M. Hosono-Fukao T. Tang R. Sugaya N. van Kuppevelt T.H. Jenniskens G.J. Kimata K. Rosen S.D. Uchimura K. Direct detection of HSulf-1 and HSulf-2 activities on extracellular heparan sulfate and their inhibition by PI-88.Glycobiology. 2010; 20: 175-186Crossref PubMed Scopus (64) Google Scholar, 21Jenniskens G.J. Oosterhof A. Brandwijk R. Veerkamp J.H. van Kuppevelt T.H. Heparan sulfate heterogeneity in skeletal muscle basal lamina: demonstration by phage display-derived antibodies.J Neurosci. 2000; 20: 4099-4111Crossref PubMed Google Scholar The RB4CD12 epitope has been shown to be present abundantly in the vasculature of the brain in mice.9Hosono-Fukao T. Ohtake-Niimi S. Nishitsuji K. Hossain M.M. van Kuppevelt T.H. Michikawa M. Uchimura K. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.J Neurosci Res. 2011; 89: 1840-1848Crossref PubMed Scopus (5) Google Scholar We first analyzed expression of the RB4CD12 epitope in the brain of transgenic mouse models of AD. Tg2576 mice express mutated human amyloid precursor protein in the brain and show numerous Aβ plaques in the cortex and hippocampus.18Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Correlative memory deficits.A beta elevation, and amyloid plaques in transgenic mice, Science. 1996; 274: 99-102Google Scholar The localization of the RB4CD12 highly sulfated domains in Aβ plaques was observed in an 18-month-old Tg2576 hippocampus (Figure 1A). The RB4CD12 epitope was immunolocalized in both diffuse and neuritic amyloid plaques in the brain of Tg2576 (Figure 1, A–C). RB4CD12 also detected brain microvessels in Tg2576 mice. No specific staining was observed when RB4CD12 was substituted with MPB49, a non-HS scFv antibody (not shown). We also tested aged J20 and T41, other mouse models of AD. With respect to expression levels of Aβ peptides, Aβ42 is dominant in J20 and T41 mouse brains, whereas Aβ40 is dominant in Tg2576 mouse. We examined brain sections of these model mice immunohistochemically. The RB4CD12 highly sulfated domains were colocalized with Aβ plaques in the hippocampus of 23-month-old J20 and 12-month-old T41 mice (Figure 1A). To analyze age-dependent accumulation of the RB4CD12 epitope in Aβ plaques, we collected Tg2576 brains from 5-, 8.5-, 14- and 17-month-old mice. Aβ plaques were observed in 8.5-, 14- and 17-month-old Tg2576 brains. Cerebral Aβ deposition increases with age. RB4CD12 stained Aβ plaques at these ages (Figure 1B). Next, we investigated vasculature and non-vasculature RB4CD12 epitopes in aged Tg2576 brain by co-staining with antibodies against Aβ and laminin, a marker of vascular basement membranes. Immunoreactivity of RB4CD12 in vascular structure was colocalized with anti-laminin staining signals (Figure 1C). RB4CD12 staining signals that were not associated with signals of anti-laminin antibody predominantly colocalized with anti-Aβ staining signals in the cortex of Tg2576 mice (Figure 1C, upper panels). The RB4CD12 epitope was also observed in the vessel walls of Aβ-positive leptomeningeal vessels (Figure 1C, lower panels). Staining patterns of RB4CD12 were different from the immunoreactivity of glial fibrillary acidic protein, an astrocyte marker, and Iba-1, a microglia marker (Figure 1D). Immunoelectron microscopy confirmed the localization of RB4CD12 epitope within amyloid fibrils and the baseme