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

Evidence from Mouse Models and Patients
      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).
      • Bernfield M.
      • Gotte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      Functions of cell surface heparan sulfate proteoglycans.
      • Esko J.D.
      • Lindahl U.
      Molecular diversity of heparan sulfate.
      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,
      • Gallagher J.T.
      Heparan sulfate: growth control with a restricted sequence menu.
      bestowing on the chains structural diversity.
      • Nakato H.
      • Kimata K.
      Heparan sulfate fine structure and specificity of proteoglycan functions.
      • Parish C.R.
      The role of heparan sulphate in inflammation.
      • Bishop J.R.
      • Schuksz M.
      • Esko J.D.
      Heparan sulphate proteoglycans fine-tune mammalian physiology.
      HS contains highly sulfated domains and partially sulfated or non-sulfated domains, which are transitional.
      • Gallagher J.T.
      Heparan sulfate: growth control with a restricted sequence menu.
      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 subdomains
      • Dennissen 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.
      • Hossain 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.
      • Hosono-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.
      Trisulfated disaccharides are considered to be key elements in molecular interactions between HS/heparin and many ligands, including growth factors and morphogens.
      • Bernfield M.
      • Gotte M.
      • Park P.W.
      • Reizes O.
      • Fitzgerald M.L.
      • Lincecum J.
      • Zako M.
      Functions of cell surface heparan sulfate proteoglycans.
      • Esko J.D.
      • Selleck S.B.
      Order out of chaos: assembly of ligand binding sites in heparan sulfate.
      Trisulfated disaccharides, as well as the RB4CD12 epitope, are degraded by extracellular sulfatases, Sulf-1, and Sulf-2.
      • Hossain 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.
      • Saad 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.
      • Morimoto-Tomita M.
      • Uchimura K.
      • Werb Z.
      • Hemmerich S.
      • Rosen S.D.
      Cloning and characterization of two extracellular heparin-degrading endosulfatases in mice and humans.
      In the brain, we have shown that the RB4CD12 HS domains are abundantly present in the vasculature
      • Hosono-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.
      and that these domains can be degraded by the Sulfs ex vivo.
      • Hossain 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.
      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,
      • Snow 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.
      and that structural variation of HSPG correlates with amyloid plaque formation in the brains of AD patients.
      • Snow 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.
      HSPG is also known to facilitate cerebral amyloid deposition induced exogenously in a rat model in vivo.
      • Snow 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.
      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.
      • van 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.
      It was reported that the aggregation state of Aβ requires its binding properties to heparin.
      • Watson 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.
      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 Methods

      Materials

      The RB4CD12 phage display-derived anti-heparan sulfate antibody was produced in a vesicular stomatitis virus (VSV)-tag version and purified as previously described.
      • Dennissen 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.
      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).

      Animals

      C57BL/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),
      • Hsiao K.
      • Chapman P.
      • Nilsen S.
      • Eckman C.
      • Harigaya Y.
      • Younkin S.
      • Yang F.
      • Cole G.
      Correlative memory deficits.
      the Swedish and Indiana (V717F) mutations (J20 strain),
      • Mucke 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.
      or the Swedish and London (V717I) mutations (T41 strain)
      • Rockenstein 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).
      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 Tissues

      Patients 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 Sulfate
      Patient 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
       060491FCVCI8
       080593FCVI+CI27
       081080MCVCI15
       081181MCVI8
       081491MCV+5
       082487FBVI9
      Age-matched non-demented controls
       070795FAIIMI4
       071083FAIICH/CI24
       060190FBIIMI4
       080293FAIIICH/CI20
       070484MBIICI3
       080782M0ICH8
       090891MAIINA
       090387F0IICI7
      ATH, atherosclerosis; CH, cerebral hemorrhage; CI, cerebral infarction; F, female; M, male; MI, myocardial infarction; NA, not applicable; NFT, neurofibrillary tangle; PMI, postmortem interval.
      low asterisk 0 = none, A = rare or a few, B = mild or moderate, C = numerous or marked.

      Fractionation of Brain Samples

      A 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.

      Immunohistochemistry

      Fresh 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.
      • Hossain 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.
      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 Microscopy

      Cryostat-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).

      Immunoblots

      The 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 HS

      There 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.
      • Hosono-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.
      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 Synthesis

      Total 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 Analysis

      All 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.

      Results

      Immunoreactivity of RB4CD12 Anti-Heparan Sulfate is Colocalized with Aβ Plaques in Brains of Transgenic Mouse Models of AD

      RB4CD12 scFv antibody recognizes trisulfated disaccharide-containing highly sulfated domains within HS.
      • Hossain 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.
      • Jenniskens 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.
      The RB4CD12 epitope has been shown to be present abundantly in the vasculature of the brain in mice.
      • Hosono-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.
      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.
      • Hsiao K.
      • Chapman P.
      • Nilsen S.
      • Eckman C.
      • Harigaya Y.
      • Younkin S.
      • Yang F.
      • Cole G.
      Correlative memory deficits.
      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 basement membrane (Figure 1E). The RB4CD12 immunoreactive area that is not colocalized with anti-laminin staining signals was determined by fluorescence microscopy and quantified with computer-aided image analysis. In Tg2576 cortex and hippocampus, RB4CD12-positive but laminin-negative area was increased to fourfold to fivefold of that in non-Tg (Figure 2). In contrast, no change was observed in the cerebellum where no Aβ plaques were observed (Figure 2). We noted that laminin-positive vessels had attenuated diameters and a more ragged profile in Tg2576 cortex and hippocampus (Figure 2).
      Figure thumbnail gr1
      Figure 1RB4CD12 anti-heparan sulfate epitope colocalizes with amyloid β plaques in the brain of Tg2576, J20, and T41 mice. A: Cryostat-cut brain sections of 18-month-old non-Tg and Tg2576, 23-month-old J20, and 12-month-old T41 mice were stained with RB4CD12 anti-HS antibody (red) and 82E1 anti-amyloid β (Aβ) antibody (green). Staining signals in vessels (arrows) and amyloid plaques (arrowheads) are shown. B: Expression of the RB4CD12 epitope and Aβ in aging Tg2576 brain. C: Expression of the RB4CD12 epitope, Aβ and laminin, a marker for vessels in the 18-month-old Tg2576 brain. D: Co-staining of RB4CD12 anti-HS antibody (red) with cell type-specific antibodies against glial fibrillary acidic protein astrocytes (left) or Iba1 microglia (right) (green). Co-stained areas are shown in yellow. E: Immunoelectron microscopy for the RB4CD12 epitope in amyloid fibrils in the brain of 18-month-old Tg2576 mouse. Left panel shows RB4CD12 signals in amyloid fibrils indicated by arrowheads. Right panel shows RB4CD12 signals in the basement membrane of vessels. BM, basement membrane. Scale bars (in A, C, and D): 50 μm. Scale bars (in B and E): 20 μm and 500 nm, respectively.
      Figure thumbnail gr2
      Figure 2Quantification of the RB4CD12 epitope in nonvascular parenchyma in the brain of Tg2576 mice. Cryostat-cut brain sections of Tg2576 mice were stained with RB4CD12 (red) and anti-laminin (green) antibodies. Laminin is a marker for brain vessels. Nonvascular amyloid β plaques were stained with RB4CD12 antibody in the cortex and hippocampus. Graphs are of semiquantitative analysis of immunohistochemical pictures of RB4CD12 and laminin. RB4CD12-positive areas that were not colocalized with anti-laminin staining signals were calculated. *P < 0.05, **P < 0.01.

      The RB4CD12 Epitope Is Immunolocalized in Amyloid Plaques in Postmortem Brains of Alzheimer's Disease Patients

      We tested RB4CD12 antibody for staining of brains from NDC individuals and AD patients (Table 1). In NDCs, vessel-staining signals were predominantly observed (Figure 3A). In AD entorhinal cortex, amyloid deposits, as well as vessels, were positive for RB4CD12 (Figure 3B). Amyloid deposits and microvessels were also stained with RB4CD12 in AD hippocampus (Figure 3C). Interestingly, some pyramidal neurons in AD hippocampus showed intracellular granular staining (Figure 3D). These intracellular staining signals were detected in a certain number of cells that were positive for hyperphosphorylated microtubule-associated protein tau as revealed by the AT180 monoclonal antibody (see Supplemental Figure S1 at http://ajp.amjpathol.org).
      Figure thumbnail gr3
      Figure 3RB4CD12 epitope immunolocalizes in amyloid plaques in the brains of Alzheimer's disease patients. Immunoperoxidase staining for RB4CD12 (brown) in the entorhinal cortex (A, B) and hippocampus (C, D). Open arrowheads in AC show vessel-staining signals in brains of non-demented control (NDC) and Alzheimer's disease (AD). In AD, amyloid deposits were also positive for RB4CD12 (arrows in B and C). Intracellular RB4CD12-staining signals are seen in some hippocampal neurons of AD (arrowheads in D). Scale bars: 50 μm.

      Expression of the RB4CD12 Epitope Borne in Molecules with 70–180 kDa Molecular Weights Is Upregulated in the Cortex of Tg2576 Mice

      As an extension of the staining results in the mouse and human brain tissues, we wished to determine which proteins contain the RB4CD12 epitope and were differentially expressed in Tg2576 brains. We performed Western blotting for cortex samples, which were fractionated as TBS-insoluble/1% SDS-soluble. Four non-Tg and five Tg2576 mice (20 months old) were examined. Multiple bands were positive for RB4CD12 antibody in both non-Tg and Tg2576 (Figure 4A). We measured intensities of 460 kDa, 180 kDa, 120 kDa, and 100–70 kDa bands by densitometry. There was a 1.3-fold increase in the intensity of overall RB4CD12 recognition determinants in Tg2576 brain extracts compared with non-Tg controls (Figure 4B). Expression levels of RB4CD12 epitopes in bands of 180 kDa, 120 kDa, and 100–70 kDa were increased 1.1- to 1.5-fold in the cortex of Tg2576 mice (Figure 4B). There was no significant change in the intensity of bands of 460 kDa. Syndecan-3 and glypican-1 are HSPGs expressed in glial cells surrounding Aβ plaques of Tg2576 mice.
      • O'Callaghan P.
      • Sandwall E.
      • Li J.P.
      • Yu H.
      • Ravid R.
      • Guan Z.Z.
      • van Kuppevelt T.H.
      • Nilsson L.N.
      • Ingelsson M.
      • Hyman B.T.
      • Kalimo H.
      • Lindahl U.
      • Lannfelt L.
      • Zhang X.
      Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells.
      To ascertain whether these proteins are HSPGs that contain the RB4CD12 epitope, we then analyzed their expression. Western blotting revealed the protein bands at 250 to 180 kDa for syndecan-3 and 60 kDa for glypican-1 in the cortex of Tg2576 mice (see Supplemental Figure S2A at http://ajp.amjpathol.org). Expression levels of these proteins in the Tg2576 cortex were comparable to those in non-Tg controls (see Supplemental Figure S2B at http://ajp.amjpathol.org).
      Figure thumbnail gr4
      Figure 4Immunoblotting analysis of the RB4CD12 epitope, disaccharide composition analysis of Heparan sulfate (HS) and quantitative real-time PCR analysis of HS enzymes in the brain of Tg2576 mice. A: Tris-buffered saline (TBS)-insoluble/1% SDS-soluble fractions were prepared from tissue homogenates of four 20-month-old Tg2576 (lanes 1–4) and five 20-month-old non-Tg (lanes 5–9) cortices. Immunoblot with RB4CD12 was performed as described in Materials and Methods. B: Relative intensities of bands with molecular weights of 460 kDa, 180 kDa, 120 kDa, and 70 to 100 kDa indicated by open arrowheads in (A) were measured. C: High performance liquid chromatography analysis determined non-sulfated (0S), monosulfated (NS, 6S), disulfated ([N,6]diS, [N,2]diS) and trisulfated ([N,6,2]triS) disaccharide compositions in non-vasculature fractions and vessel-enriched fractions of 18-month-old non-Tg and Tg2576 cortices. The level of total HS was determined by summing amounts of all disaccharides detected in each fraction. The values are representative of two independent experiments. D: HS disaccharide compositions and the level of total HS in the entorhinal cortex of non-demented control (NDC) (n = 8) and Alzheimer's disease (AD) (n = 8) postmortem brains were determined. E: Total-RNA from the cerebral cortices of 18-month-old Non-Tg (n = 3) and Tg2576 mice (n = 3) were prepared and tested. mRNA levels of 10 HS modification enzymes were determined by quantitative real-time PCR. *P < 0.05, ***P < 0.001.

      Disaccharide Compositions of HS and Expression Profiles of HS Enzymes in the Cortex of Tg2576 and Postmortem AD

      We performed structural analysis of HS chains extracted from mouse and human postmortem brains (Table 1). HS was isolated from the cortex of mice or postmortem human entorhinal cortex. HS was depolymerized into its constituent disaccharides by a mix of bacterial heparitinases. The disaccharide compositions of the HS were determined by reversed-phase ion-pair chromatography. We found that the total HS contents and HS disaccharide compositions in vessel-enriched fractions (“vessel-enriched fr”) and non-vessel associated fractions (“non-vasculature fr”) were comparable between non-Tg and Tg2576 mice (Figure 4C). In human, the reduction in the proportion of non-sulfated disaccharides reached statistical significance. Total HS contents and percentages of other sulfated disaccharides were comparable between NDC and AD (Figure 4D). To understand possible mechanisms of upregulation of the RB4CD12 epitope in AD mouse brains, we measured mRNA levels of 10 HS modification enzymes by quantitative real time-PCR. HS enzymes include sulfotransferases and extracellular sulfatases. These enzymes are regarded as key molecules in the regulation of sulfation of HS. The mRNA level of N-deacetylase/N-sulfotransferase 2 were significantly increased in Tg2576 (24%) (Figure 4E). The mRNA levels of Sulf-1 and Sulf-2 were comparable between non-Tg and Tg2576 mice (Figure 4E).

      Sulf-1 and Sulf-2, Extracellular HS Sulfatases, Degrade the RB4CD12 Epitope Accumulated in Amyloid Plaques

      Previously, we showed that the treatment of wild-type mouse brain sections with Sulf-1 or Sulf-2 greatly diminished the RB4CD12 epitope abundant in vasculature.
      • Hossain 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.
      To determine whether the RB4CD12 epitope in amyloid plaques is susceptible to Sulf-1 and Sulf-2 and degraded by these enzymes, we treated cryo-cut brain sections of 18-month-old Tg2576 mice with recombinant Sulf-1, Sulf-2, or conditioned medium of MCF-7 breast cancer cells, which secrete native Sulf-2.
      • Uchimura K.
      • Morimoto-Tomita M.
      • Bistrup A.
      • Li J.
      • Lyon M.
      • Gallagher J.
      • Werb Z.
      • Rosen S.D.
      HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF. FGF-1, and SDF-1.
      These treatments substantially reduced the RB4CD12 epitope in sections of Tg2576 brain ex vivo (Figure 5). A mixture of bacterial heparinases confirmed that the assay is suitable for ex vivo degradation of HS in brain sections and that the observed signals arose from HS (Figure 5). Anti-Aβ staining signals that were colocalized with the RB4CD12 epitope were retained after Sulf treatment (Figure 5).
      Figure thumbnail gr5
      Figure 5The RB4CD12 epitope in amyloid plaques of Tg2576 mouse brains is degraded ex vivo by Sulf-1, Sulf-2 and conditioned medium of Sulf-2-expressing cells. Cryostat-cut consecutive sections of 18-month-old Tg2576 mouse brains were incubated overnight with recombinant human Sulf-1 and Sulf-2 prepared from CM of transfected HEK293 cells (Sulf1, Sulf2), buffer only (Buffer), or CM of MCF-7 human breast cancer cells (MCF-7 CM).
      • Hossain 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.
      • Uchimura K.
      • Morimoto-Tomita M.
      • Bistrup A.
      • Li J.
      • Lyon M.
      • Gallagher J.
      • Werb Z.
      • Rosen S.D.
      HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF. FGF-1, and SDF-1.
      The Ni-NTA resin-bound materials that were prepared from HEK293 cells transfected with the empty vector were eluted and used (control). A mix of bacterial heparinases (heparinases) served as a positive control. RB4CD12 binding was visualized using a Cy3-conjugated anti-VSV tag antibody (red). Treated sections were co-stained with 82E1 anti-Aβ antibody (green). The data are representative of two independent experiments. Arrowheads indicate amyloid plaques. Scale bars: 20 μm.

      Discussion

      In the present study, we showed that the RB4CD12 epitope is colocalized with amyloid plaques in brains of AD mouse models and patients with AD. Consistent with our previous report,
      • Hosono-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.
      the RB4CD12 epitope was also colocalized with laminin-positive vasculature in brains of mouse models of AD. Quantification analysis revealed that the non-vascular RB4CD12-positive area was increased in the cortex and hippocampus of Tg2576, J20, and T41 AD models. In the cerebellum, where no amyloid plaques were observed in these model mice, RB4CD12 staining was comparable to that in the non-Tg. Morphological alterations of the vasculature observed in the cortex and hippocampus of Tg2576 were consistent with the previous report that Aβ aggregates induce the structural and functional disruption of smooth muscle cells in the vasculature.
      • Christie R.
      • Yamada M.
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      • Hyman B.
      Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy.
      Results in aging brains of Tg2576 mice suggested that Aβ and the HS highly sulfated domains start accumulation at the same age. Aβ and other self-aggregating peptides share cationic motifs that may be involved in binding to the negative charges of sulfated glycosaminoglycan.
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      Glycosaminoglycans and beta-amyloid, prion and tau peptides in neurodegenerative diseases.
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      Effect of amino-acid substitutions on Alzheimer's amyloid-beta peptide-glycosaminoglycan interactions.
      HS and other glycosaminoglycan chains can stabilize mature fibrils against proteolytic degradation.
      • Gupta-Bansal R.
      • Frederickson R.C.
      • Brunden K.R.
      Proteoglycan-mediated inhibition of A beta proteolysis A potential cause of senile plaque accumulation.
      HS facilitates the formation of fibrils of amylin,
      • Watson 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.
      apo-serum amyloid A,
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      • Kisilevsky R.
      The heparin/heparan sulfate-binding site on apo-serum amyloid A Implications for the therapeutic intervention of amyloidosis.
      α-synuclein,
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      • Li J.
      • Uversky V.N.
      • Fink A.L.
      Heparin and other glycosaminoglycans stimulate the formation of amyloid fibrils from alpha-synuclein in vitro.
      prion protein,
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      Prion protein conversion in vitro.
      muscle acylphosphatase,
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      • Monsellier E.
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      • Relini A.
      • Chiti F.
      Kinetic analysis of amyloid formation in the presence of heparan sulfate: faster unfolding and change of pathway.
      transthyretin,
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      • Zhang X.
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      • Damas A.M.
      • Dacklin I.
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      • Lundgren E.
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      • Westermark P.
      • Li J.P.
      Heparan sulfate/heparin promotes transthyretin fibrillization through selective binding to a basic motif in the protein.
      Tau,
      • Goedert M.
      • Jakes R.
      • Spillantini M.G.
      • Hasegawa M.
      • Smith M.J.
      • Crowther R.A.
      Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans.
      and Aβ.
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      • Franklin T.
      • Zhang X.
      • Deng J.
      • Fraser P.E.
      Interactions of Alzheimer amyloid-beta peptides with glycosaminoglycans effects on fibril nucleation and growth.
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      • Lukito W.
      • Wight T.N.
      • Snow A.D.
      The sulfate moieties of glycosaminoglycans are critical for the enhancement of beta-amyloid protein fibril formation.
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      • Ventura S.
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      • Fernandez-Busquets X.
      Modulation of Abeta42 fibrillogenesis by glycosaminoglycan structure.
      In vivo fragmentation of heparan sulfate by heparanase overexpression could protect mice from amyloid protein A amyloidosis.
      • Li J.P.
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      • Gong F.
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      In vivo fragmentation of heparan sulfate by heparanase overexpression renders mice resistant to amyloid protein A amyloidosis.
      Importantly, the degree of sulfation is critical for enhancement of fibrillogenesis of Aβ.
      • Castillo G.M.
      • Lukito W.
      • Wight T.N.
      • Snow A.D.
      The sulfate moieties of glycosaminoglycans are critical for the enhancement of beta-amyloid protein fibril formation.
      Pathological effects of heparin in Aβ aggregation assays are dependent on sulfate moieties at N- and O-positions.
      • Timmer N.M.
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      • van Kuppevelt T.H.
      • de Waal R.M.
      • Verbeek M.M.
      Aggregation and cytotoxic properties towards cultured cerebrovascular cells of Dutch-mutated Abeta40 (DAbeta(1–40)) are modulated by sulfate moieties of heparin.
      Our findings of selective accumulation of the RB4CD12 epitope in amyloid plaques suggest that highly sulfated domains of HS could play an important role in the progression of Aβ deposition. HSPG facilitates cerebral amyloid deposition, which can be induced exogenously in a rat model.
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      • Fraser P.
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      • 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.
      Highly sulfated HS chains could be one candidate for heat-resistant materials present in the brain extract that are essential for exogenous induction of cerebral β-amyloidogenesis in mouse models.
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      Exogenous induction of cerebral beta-amyloidogenesis is governed by agent and host.
      Recently, Timmer et al
      • Timmer N.M.
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      • De Waal R.M.
      • Verbeek M.M.
      Limited expression of heparan sulphate proteoglycans associated with Abeta deposits in the APPswe/PS1dE9 mouse model for Alzheimer's disease.
      demonstrated that only a minimal number of Aβ plaques (∼30%) were co-stained with the epitope of JM403, an anti-HS antibody, in aging brains of APPswe/PS1dE9 model mice. JM403 detects HS subdomains containing the positively charged disaccharide [-glucuronic/iduronic acid-N-unsubstituted glucosamine].
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      Novel heparan sulfate structures revealed by monoclonal antibodies.
      Future studies may reveal differential contribution of HS subdomains composed of specific disaccharide structures to AD pathogenesis. Possible involvement of the RB4CD12 epitope existing in laminin-positive vasculature in AD pathogenesis should also be clarified in the future. Interestingly, intraneuronal RB4CD12 staining was observed in the hippocampus of AD patients. Microtubule-associated protein Tau is the major protein subunit of intraneuronal neurofibrillary tangles, another neuropathological hallmark of AD.
      • Skovronsky D.M.
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      Neurodegenerative diseases: new concepts of pathogenesis and their therapeutic implications.
      It has been shown that Tau and HS coexist in nerve cells with overt neurofibrillary lesion.
      • Goedert M.
      • Jakes R.
      • Spillantini M.G.
      • Hasegawa M.
      • Smith M.J.
      • Crowther R.A.
      Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans.
      The filamentous structures induced by heparin are structurally similar to those found in Alzheimer's disease.
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      Pathways of tau fibrillization.
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      Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction.
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      Quantitative characterization of heparin binding to Tau protein: implication for inducer-mediated Tau filament formation.
      We have shown that some of RB4CD12 staining signals were found in cells that were stained with AT180, an antibody against hyperphosphorylated tau. Our results suggest that highly sulfated domains of HS might play a role in the formation of neurofibrillary tangles.
      In immunoblots of brain lysates with the RB4CD12 antibody, we detected several RB4CD12-positive bands in non-Tg and Tg2576 mouse brains and found that 180 kDa, 120 kDa, and 100–70 kDa bands were upregulated in the cortex of Tg2576 mice. There were no significant changes in the intensities of 460 kDa bands. Our previous results showed that the RB4CD12 epitope is abundant in the basement membrane of the brain vessels and that the RB4CD12-positive bands were predominantly 460 kDa bands in brain vessel fractions.
      • Hosono-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.
      In our immunohistochemical studies, non-vascular RB4CD12 staining was increased in Tg2576 mice. These results suggest that upregulation of 180 kDa, 120 kDa, and 100–70 kDa bands could contribute to the RB4CD12 staining colocalized with amyloid plaques in Tg2576 mouse brain. Several HSPGs are known to be localized in amyloid plaques.
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      Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.
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      Agrin in Alzheimer's disease: altered solubility and abnormal distribution within microvasculature and brain parenchyma.
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      Agrin is a major heparan sulfate proteoglycan accumulating in Alzheimer's disease brain.
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      • David G.
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      • Wesseling P.
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      • Verbeek M.M.
      Heparan sulfate proteoglycan expression in cerebrovascular amyloid beta deposits in Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis (Dutch) brains.
      Because of the high molecular weight (>210 kDa) of agrin and perlecan, it is conceivable that the observed signals in immunoblots might have arisen from other molecules. Syndecan-3 and glypican-1 in glial cells were identified as molecules associated with Aβ deposits.
      • O'Callaghan P.
      • Sandwall E.
      • Li J.P.
      • Yu H.
      • Ravid R.
      • Guan Z.Z.
      • van Kuppevelt T.H.
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      • Ingelsson M.
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      • Kalimo H.
      • Lindahl U.
      • Lannfelt L.
      • Zhang X.
      Heparan sulfate accumulation with Abeta deposits in Alzheimer's disease and Tg2576 mice is contributed by glial cells.
      Our Western blotting results suggested that syndecan-3 with the molecular weights of 180 to 250 kDa could be an HSPG that possesses the RB4CD12 epitope. However, we cannot rule out the possibility that degradation products of agrin or perlecan could harbor the RB4CD12 epitope observed in amyloid plaques. We should also pay attention to possible accumulation of HS degradation products catalyzed by nitric oxide.
      • Cappai R.
      • Cheng F.
      • Ciccotosto G.D.
      • Needham B.E.
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      The amyloid precursor protein (APP) of Alzheimer disease and its paralog APLP2, modulate the Cu/Zn-Nitric Oxide-catalyzed degradation of glypican-1 heparan sulfate in vivo.
      Unexpectedly, the trisulfated disaccharide composition was not increased in either Tg2576 or human postmortem AD brains. The mechanisms underlying the accumulation of the RB4CD12 highly sulfated domains within HS polysaccharides in non-vasculature amyloid plaques are not clear. There are two possibilities to explain the mechanisms. First, the N-sulfation of glucosamine residues is the initial HS sulfation and the N-sulfated domains are primary sites for further modification.
      • Carlsson P.
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      Heparin/heparan sulfate biosynthesis: processive formation of N-sulfated domains.
      Consecutive occurrence of N-sulfation could be attributable to the formation of trisulfated disaccharide clusters, namely, highly sulfated domains, within HS chains in non-vasculature spaces. Second, translocation of HS that contains the RB4CD12 highly sulfated domains between brain vasculature and non-vasculature could be an explanation for the accumulation of the RB4CD12 epitope in Tg2576 brain parenchyma. Our findings that comparable levels of disaccharide compositions and HS contents in vessel-enriched fractions and non-vasculature fractions in the cortex of Tg2576 were shown and that the mRNA level of N-deacetylase/N-sulfotransferase 2 was increased in the cortex of Tg2576 mouse could support the former possibility. A previous study by Lindahl et al
      • Lindahl B.
      • Eriksson L.
      • Lindahl U.
      Structure of heparan sulphate from human brain, with special regard to Alzheimer's disease.
      showed altered distribution of N-sulfated glucosamine residues within HS extracted from postmortem AD brain. Highly N-sulfated HS may be involved in the initiation of the aggregation process of Aβ in AD brains.
      • Bruinsma I.B.
      • te Riet L.
      • Gevers T.
      • ten Dam G.B.
      • van Kuppevelt T.H.
      • David G.
      • Kusters B.
      • de Waal R.M.
      • Verbeek M.M.
      Sulfation of heparan sulfate associated with amyloid-beta plaques in patients with Alzheimer's disease.
      These studies also support the former possibility as an explanation of the mechanisms of accumulation of RB4CD12-positive highly sulfated domains in Aβ plaques. We cannot rule out the possibility that the RB4CD12 epitope is a minor component and that the structural analysis we have performed might not fully detect the minor change. Quantitative analysis for the RB4CD12-positive HS in the cortex would make advances in the study of the mechanisms.
      Herein, we found that the RB4CD12 epitope accumulated in amyloid plaques can be degraded by Sulf-1 and Sulf-2 ex vivo. It was suggested that the RB4CD12 highly sulfated domains are localized at the surface of amyloid plaques, as these HS degrading enzymes could access and efficiently degrade the epitope. Although the RB4CD12 epitope in amyloid plaques was degraded by the Sulfs, substantial amounts of Aβ were retained in these plaques. This result suggests that the highly sulfated domains of HS universally associated with amyloid deposits in the brain. Accumulation of the RB4CD12 epitope in amyloid plaques could induce excessive entrapment of growth factors at amyloid plaques, which might lead to an imbalance in homeostasis of the brain microenvironment. Increasing evidence points to vascular damage as an early contributor in Alzheimer pathology.
      • Bailey T.L.
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      • Hof P.R.
      The nature and effects of cortical microvascular pathology in aging and Alzheimer's disease.
      • Meyer E.P.
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      • Staufenbiel M.
      • Krucker T.
      Altered morphology and 3D architecture of brain vasculature in a mouse model for Alzheimer's disease.
      A recent study suggested that angiogenesis might be impaired in AD model mice,
      • Paris D.
      • Ganey N.
      • Banasiak M.
      • Laporte V.
      • Patel N.
      • Mullan M.
      • Murphy S.F.
      • Yee G.T.
      • Bachmeier C.
      • Ganey C.
      • Beaulieu-Abdelahad D.
      • Mathura V.S.
      • Brem S.
      Impaired orthotopic glioma growth and vascularization in transgenic mouse models of Alzheimer's disease.
      despite the fact that the levels of pro-angiogenic growth factors (eg, vascular endothelial growth factor [VEGF]) are elevated in AD brains.
      • Kalaria R.N.
      • Cohen D.L.
      • Premkumar D.R.
      • Nag S.
      • LaManna J.C.
      • Lust W.D.
      Vascular endothelial growth factor in Alzheimer's disease and experimental cerebral ischemia.
      • Tarkowski E.
      • Issa R.
      • Sjogren M.
      • Wallin A.
      • Blennow K.
      • Tarkowski A.
      • Kumar P.
      Increased intrathecal levels of the angiogenic factors VEGF and TGF-beta in Alzheimer's disease and vascular dementia.
      • Siedlak S.L.
      • Cras P.
      • Kawai M.
      • Richey P.
      • Perry G.
      Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer disease.
      VEGF binds to immobilized heparin and can be stored in the extracellular space by binding to HS and HSPG.
      • Uchimura K.
      • Morimoto-Tomita M.
      • Bistrup A.
      • Li J.
      • Lyon M.
      • Gallagher J.
      • Werb Z.
      • Rosen S.D.
      HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF. FGF-1, and SDF-1.
      • Iozzo R.V.
      Matrix proteoglycans: from molecular design to cellular function.
      Heparin-bound VEGF is mobilized by the action of Sulf-2, which exerts pro-angiogenic activity.
      • Uchimura K.
      • Morimoto-Tomita M.
      • Bistrup A.
      • Li J.
      • Lyon M.
      • Gallagher J.
      • Werb Z.
      • Rosen S.D.
      HSulf-2, an extracellular endoglucosamine-6-sulfatase, selectively mobilizes heparin-bound growth factors and chemokines: effects on VEGF. FGF-1, and SDF-1.
      • Morimoto-Tomita M.
      • Uchimura K.
      • Bistrup A.
      • Lum D.H.
      • Egeblad M.
      • Boudreau N.
      • Werb Z.
      • Rosen S.D.
      Sulf-2, a proangiogenic heparan sulfate endosulfatase, is upregulated in breast cancer.
      VEGF is found to be associated with amyloid plaques in AD, but not non-AD brain.
      • Ryu J.K.
      • Cho T.
      • Choi H.B.
      • Wang Y.T.
      • McLarnon J.G.
      Microglial VEGF receptor response is an integral chemotactic component in Alzheimer's disease pathology.
      Our results also suggested that the highly sulfated domains could be involved in sequestration of VEGF within amyloid plaques and vascular damage in AD through perturbation in the supply of pro-angiogenic growth factors. Aberrant angiogenesis could induce neurovascular uncoupling, which ultimately leads to synaptic dysfunction.
      • Zlokovic B.V.
      Neurovascular mechanisms of Alzheimer's neurodegeneration.
      In summary, we provide evidence that highly sulfated domains recognized by RB4CD12 accumulated in amyloid plaques of brains of AD model mice and patients with AD. Further studies to investigate the roles of the highly sulfated HS domains with special regard to angiogenesis in AD pathology will be needed.

      Acknowledgments

      We thank Steven Rosen and Yoshiko Takeda-Uchimura for their helpful suggestions and discussions. We also thank Kuniko Takanose, Noriko Sugaya, and Hudson Johns for their technical assistance. We are grateful to Yoshio Hashizume and Takayuki Yamamoto for their diagnostic examination and support.

      Supplementary data

      • Supplemental Figure S1

        Immunohistochemistry for the RB4CD12 epitope and phosphorylated tau proteins. Postmortem brain sections of Alzheimer's disease (AD) patients (AD1, AD2) were subjected to heat-induced epitope retrieval and co-stained with RB4CD12 and biotinylated anti-paired helical filament (PHF)-tau antibody AT180 as described in Materials and Methods in the article. Primary antibodies were detected with Cy3-conjugated anti-VSV-G (red) and Cy2-conjugated streptavidin (green). Digital images were captured by fluorescent microscopy. Intracellular RB4CD12-staining signals were seen in some AT180-positive hippocampal neurons (arrows). Vessel-staining signals with RB4CD12 were observed (arrowheads). Bars: 25 μm.

      • Supplemental Figure S2

        Immunoblotting analysis of syndecan-3 and glypican-1. A: Tris-buffered saline (TBS)-soluble and TBS-insoluble/1% SDS-soluble fractions were prepared from tissue homogenates of 20-month-old Tg2576 and age-matched non-Tg cortices. Immunoblots with anti-syndecan-3 antibody for TBS-insoluble/1% SDS-soluble fractions (Sdc3) and with anti-glypican-1 antibody for TBS-soluble fractions (Gpc1) were performed as described in Materials and Methods in the article. B: Relative intensities of bands indicated by open arrowheads in (A) were measured. N = 4, Tg2576; N = 5, Non-Tg; NS, not significant.

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