0022-1554/92/$3,30 The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society, Inc.
Vol. 40, NO, 1, pp. 101-113, 1992 Printedin l.%S.A.
Original Artide
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Peripheral Distribution of Dermatan Sulfate Proteoglycans (Decorin) in Amyloid-containing Plaques and Their Presence in Neurofibrillary Tangles of Alzheimer’s Disease’ ALAN D. SNOW,2 HENDERSON MAR, DAVID NOCHLIN, HANS KRESSE, and THOMAS N. WIGHT Departments of Pathology (HM,,nVW)and Neuropathology (ADS,DN), University of Wasbington, Seattle, Wasbington, and Inshtute of Physiological Chemistry and Patbobiochemistry, University of Munster; Munster; Germuny (HK). Received for publication April 30, 1991 and in revised form August 14, 1991; accepted August 27, 1991 (lA2315).
We used a polydonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the s m a l l dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristiclesions in Alzheimer’s brain. All antibodies demonstrated positive decorin immunoStainingin both the amyloid depositsof neuritic plaques (NPs) and the filamentous structures within neurofbrillatytangles (NFTs).Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout N P s containing amyloid fibrils, decorin was pri-
Introduction Current studies investigating the pathogenesis of Alzheimer’sdisease (AD) have primarily focused on the identification and characterization of components within the characteristic brain lesions (i.e., the neuritic plaque, the neurofibrillary tangle, and cerebrovascular amyloid deposits). Understanding the nature of these components and determining their precise location within the enveloping lesions should lead to further clarification of initiating and processing events critical to the pathogenesis of this disease. Much research effort has focused on a major protein component present in the amyloid deposits of neuritic plaques (NPs) and in the walls of blood vessels (i.e., congophilic angiopathy), known as the betaamyloid protein (BAP) (Wong et al., 1985; Glenner and Wong, 1984) or A4 (Masters et al., 1985). In addition to the BAP, other protein components present within the NP and/or cerebrovascular amyloid deposits include the serine protease inhibitor, alphalanti-chymotrypsin (Abraham et al., 1988) and amyloid P component (Coria et al., 1988). In associationwith the paired helical and
Supported by the Alzheimer’sDisease Research Program ofthe American Health Assistance Foundation, the French Foundation for Alzheimer’s Research, and NIH grant AGO5136. Correspondence to: Dr. Alan Snow,University of Washington, Dept. of Pathology, Neuropathology Labs RJ-05, Seattle, WA 98195.
marily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorinwas also immunolocalized to the paired helical and straight filaments within NF% and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque. (JIlistochem Cytochem 40:105-1U,195’2) KEY WORDS:Decorin; Dermatan sulfate; Proteoglycans; Amyloid;
Plaques; Alzheimer’s disease; Neurofibrillary tangles.
straight filaments of NFTs, major components identified include microtubule-associatedphosphoproteins, including the tau protein (Lee et al., 1991; Grundke-Iqbal et al., 1986; Kosik et al., 1986), ubiquitin (Mori et al., 1987; Perry et al., 1987). and neurofilaments (Haugh et al., 1986). Previous studies have also identified a specific group of complex anionic macromolecules known as proteoglycans (PGs), specifically localized to the amyloid deposits in both NPs and in the wall of blood vessels (Snow and Wight, 1989; Snow et al., 1987,1988,1989a). The particular class of PG present in the amyloid deposits of these lesions has been identified by immunocytochemistry as a basement membrane-derived heparan sulfate proteoglycan (HSPG) (Snow et al., 1988). In this latter study, antibodies to the protein core of the basement membrane-derived HSPG identified and localized HSPG core protein to the amyloid deposits in NPs and congophilic angiopathy, to “primitive”plaques (those containing BAP immunoreactivity but lacking positive staining with congo red or thioflavin S), and in subsets of some adjacent neurons and astrocytes,whereas HSPG core protein was not immunolocalized to NFTs. However, a recent study (Snow et al., 1990)utilizing a monoclonal antibody (MAb) that recognizes a specific epitope (glucosamine sulfate alpha144 glucuronic acid determinant) in heparan sulfate chains of a basement membrane-derived HSPG identified and immunolocalized heparan sulfate to 10-20% of NFTs. Since a number of histochemical and immunocytochemical 105
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DECORIN IN ALZHEIMER’S DISEASE BRAIN
studies (Siedlak et al., 1991; Stopa et al., 1990; Snow et al., 1987,1989a;1990)suggest that PGs and/or GAGs are associated with NFTs, and since previous studies imply that heparan sulfate is associated only with some NFTs (Siedlak et al., 1991; Snow et al., 1990), it was important to confirm whether other PGs are localized to NFTs, as well as to other characteristic lesions (i.e., amyloid plaques and cerebrovascular amyloid deposits) in AD. In the present investigation, a polyclonal antibody and a mixture of three mAb primarily recognizing the small dermatan sulfate proteoglycan (DSPG) known as “decorin” or “PG-11”(Schmidt et al., 1987; Voss et al.. 1986; Glossl et al., 1984), were employed to determine whether this particular PG is associated with either NPs, NETS, or congophilic angiopathy in Alzheimer’s brain. Decorin, a small PG that decorates collagen fibrils (Scott and Haigh, 1985; Scott and Orford, 1981; Scott, 1980), contains one GAGchain and has a core protein size of 36,500 M W based on deduced amino acid sequence from full-length cDNA clones (Day et al., 1987; Vogel and Fisher, 1986; Rosenberg et al., 1985; Glossl et al., 1984; Fisher et al., 1983). In the present study, decorin was identified as a new component associated with the amyloid deposits of NPs and the filamentous structure within Unlike HSPGs. which tend to be evenly distributed throughout plaques containing amyloid fibrils, decorin was primarily localized to the periphery of the amyloid plaque as a whole and to the edge of amyloid bundles within this periphery. In addition, decorin was associated with the paired helical and straight filaments within
m.
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Materials and Methods Autopsy Material and Tissue Processing. Brain tissue, including hippocampus and amygdala from eight patients with AD (confirmed at autopsy) were used in the present study. Four tissue specimens (an 84-yearold female, a 76-year-old female, a 72-year-old male, a 64-year-old male) were obtained within 2-5 hr after death and were fuced in either 10% formalin for 24 hr or Carnoy’s solution for 4 hr (Gatenby and Cowdry, 1929) before routine paraffin embedding. The brain tissue from the other four patients (an 82-year-old male, a 79-year-old female, a 72-year-old male, and a 63-year-old female) had previously been fixed in 10% formalin and were obtained from the University of Washington Alzheimer’s Disease Research Center. From each block of paraffin-embedded material, 6-pm serial sections were cut and placed on gelatin-coated slides. For electron microscopy, small pieces of hippocampus and amygdala were fixed in a solution of 3% paraformaldehydeand 0.25% glutaraldehyde in 0.05 M phosphate buffer and processed as described previously (Snow et al., 1988). Proteoglycan and Amyloid Protein Antibodies. For the immunohistochemical studies a number of different PG and amyloid protein anti-
bodies were used. These included: (a) an affinity-purified polyclonal antibody (used at a dilution of 1:lO)against the decorin core protein of human skin fibroblasts (Voss et al., 1986; Glossl et al., 1984); (b) a mixture of three MAb (used at dilutions of 1:5 or 1:lO) against the decorin core protein of human skin fibroblasts (Schmidt et al., 1987). The titer of this antibody mixture was 1/20 ofthe polyclonal antibody sera according to ELISA data (not shown); (c) a polyclonal antibody against residues 1-42 of the BAP or A4 protein of AD (used at dilution of 1:100and 1:400) (gift of Dr. Colin Masters, Perth, Australia); (d) a polyclonal antibody (known as antiserum A or ‘Angela”)raised to the native HPLC-purifiedBAP from Alzheimer’s cortex (gift of Dr. Dennis Selkoe, Boston, MA) (Selkoe et al., 1986); and (e) a polyclonal antibody against the AA amyloid protein (used at a dilution of 1:lO) (gift of Dr. Robert Kisilevsky, Kingston, Canada). Histochemical and Immunocytochemical Detection of Amyloid and Decorin in Alzheimer‘s Brain Tissue. Congo red staining (Puchder et al., 1962) was used on paraffin and 1-pm deplasticized sections (Snow et al., 198%) to detect NPs, NFIS, and congophilic angiopathy in cach ofthe cases. Additionally,antibodies raised to the major low molecular weight amyloid protein (Selkoe et al., 1986)or against residues 1-42 of the BAP identified amyloid deposits in NPs and congophilic angiopathy. Adjacent serial sections were immunostained with either the affhity-purified polyclonal antibody against decorin (1:lO dilution) or the mixture of three MAb against decorin (1:5 or 1:lO dilution), enabling us to determine whether decorin was localized to fibrillar amyloid and/or NFIS. Some paraffh sections were also stained for collagen using the Van Gieson stain to determine whether there was an association between DSPGs and collagen (Mallory, 1961). Decorin was also immunolocalized in the hippocampus in six cases of AD, using ultra-thin (1-pm)sectionsafter r e m d of epon (Mar and Wight, 1988,1989). Briefly, before immunostainingslides with 1-2-pm plastic sections were submerged in a solution of saturated sodium hydroxide in absolute alcohol (sodium ethoxide), diluted 1:1with fresh absolute alcohol, for 15-20 min at room temperature, and were processed for immunocytochemical staining as previously described (Snow et al., 1988; Mar et al., 1987). Immunostainingof tissue sections was accomplished using the avidinbiotin-immunoperoxidase method, employing the appropriate biotinlabeled secondary antibodies, followed by incubation with avidin-conjugated horseradish peroxidase complex (Vector Labs; Burlingame, CA). Peroxidase activity was produced by treatment with 3.3-diaminobenzidine as previously described (Snow et al., 1988). For immunocytochemical staining the primary antibody was used initially through a series of dilutions to obtain the best specificity with the least background staining. Only the optimal dilutions of primary antibody are reported. Some tissue sections were also pre-treated for 5 min with 88% formic acid before immunostaining (Kitamoto et al., 1987). In addition, there was the possibility of masking of decorin core protein by dermatan sulfate GAG chains. Therefore, digestion of dermatan sulfate GAG chains was accomplished by incubating some tissue sections with 0.50 units of chondroitinax ABC (Miles Laboratories; Elkhart, IN) in 0.1 M Tris-acetate buffer (pH 7.3) at 37’C for 4 hr. After the incubation period tissue sections were rinsed in buffcr and immunostained using the decorin antibody. ~
Figure 1. Dermatan sulfate proteoglycans(decorin) in neuritic plaques and neurofibrillarytangles in Alzheimer‘s brain tissue. All tissue sections shown are from the hippocampus of either a 72-year-old male or a 74-year-old male with Alzheimer‘s disease. lmmunostaining was accomplished according to the avidin-biotin-peroxidase method on 1-pm deplasticized sections. (A) Positive decorin immunostaining(polyclonal, 1:lO dilution, chondroitinaseABC pre-treatment) of collagen fibrils (arrows) in and surrounding a blood vessel. (6)Positive decorin immunostaining(polyclonal, 1:lO dilution) of two amyloid plaques (arrows). Note that intense immunostaining is primarily localized to the periphery of the plaques. No positive decorin immunostaining is observed in the cell body of the adjacent neuron. Tissue was also treated with 88% formic acid for 5 min before immunostaining. (C) Positive decorin immunostaining(polyclonal, 1:lO dilution) of amyloid plaque (arrows). Strong immunostaining of plaque periphery is obsetved, whereas the center of the amyloid plaque contains little to no positive decorin immunoreactivity. Formic acid pre-treatment. (D) Positive decorin immunostaining (monoclonal, 1 5 dilution) of an NFT (arrowheads). Adjacent serial section demonstrates positive cungo red staining (Snow et al., 198%) of same NFT as viewed under polarized light (not shown). (E) Intense positivedecorin immunostaining(polyclonal, 1:lO dilution)of an NFT (arrowhead). Formicacid pre-treatment. (F) Lack of positivedecorin immunostaining(polyclonal, 1:lO dilution) in nontangle-bearingneuron. Note lack of positive decorin immunostainingof lipofuscin granules (arrowheads) in the cell body of the neuron. Formic acid pre-treatment. Bars: A,D = 11 pm; B,F = 13 pm; C,E = 14 pm.
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To rule out nonspecific binding of the decorin polyclonal antibody, sections were treated with another amyloid protein antibody of the same IgG species known not to be present in AD brain (i.e., the AA amyloid protein) (gift of Dr. Kisilevsky) (used at 1:lO dilution), or with Tris-bufferedsaline (TBS) instead of the primary antibody. In addition, sectionswere immunostained with the decorin antibodies in the presence of excess decorin antigen. With an ELISA assay, the polyclonal decorin antibody demonstrated a loss of binding after pre-absorption with dermatan sulfate antigen. The ELISA procedure for antibody testing was as follows. Decorin was purified (Hausser et al., 1989) and used for coating Nunc immunoplates I1 96F at a concentration of 10 nmol hexuronic acidlml. Fifty p1(in 0.1 M Tris-HC1, pH 7.4. 50 mM NaCI) were added per well. After 90 min at 37T, antigen was removed and the wells were washed three times with 400 ~10.1%Tween 20 in PBS (pH 7.4). The wells were post-absorbed with 400 PI, 1% gelatin/O.l% SDS in PBS for 90 min at 37’C. A series of decorin antibody dilutions in PBS/1% Tween 20 (50 PI) were then allowed to react for 90 min at 37°C. Three washings followed as described above, as well as another blocking step with 400 p1 of 1% gelatin10.1% SDS in PBS for 15 min at 37°C. The secondary antibody goat anti-rabbit IgG-peroxidase conjugate (Sigma; St Louis, MO) was diluted 500-fold with PBS/O.1%Tween 20/3% bovine serum albumin. Color development occurred using 200 p1of 1 mM ABTS/740 FM hydrogen peroxide in 50 mM sodium citrate (pH 4.0). For pre-absorption, 1 ml of antiserum was incubated with purified decorin (0.9 pmol of hexuronic acid) in 0.1 M %is-HC1 (pH 7.4), 1 M NaCl for 48 hr at 4’C under constant mixing. Only 11140 ofthe original titer against decorin was still present after pre-absorption (not shown). Immunodetectionof Decorin at the Ultrastructural Level in Alzheimer‘s Brain. Decorin was also immunolocalized in the hippocampus in two cases of AD at the ultrastructural level. To achieve optimal results, we found it necessary to remove the epon from ultra-thin sections before colloidal gold immunostaining (Mar and Wight, 1988,1989). This technique avoided interference with immunostaining for decorin believed to be due to the resin (Mar and Wight, 1989).For thin-section plastic removal, thin sectionswere etched by submersion in a solution of saturated sodium hydroxidein absolute alcohol, diluted 1:1 with fresh absolute alcohol, for 1.5 min at room temperature, followed by several rinses in absolute alcohol. Grids were then hydrated to water through a graded alcohol series (95%, two times; 7Ooh, 50%, 30%. distilled water, 1 min each). For immunostaining, grids were blocked with 10% normal goat serum in 0.05 M TBS (pH 7.6) for 10 min, followed by running in TBS four times for a total period of 10 min. Primary antisera (i.e., decorin or the AA amyloid antibodies) were diluted 1:10 in filtered TBS just before use. Grids were incubated overnight on droplets of antisera at 4’C. Each grid was then rinsed on 6 droplets of TBS for a total of 15 min before incubation with colloidal gold. Goat anti-rabbit IgG (for the polyclonal antibodies) or goat anti-mouse IgG (for the monoclonal antibodies) conjugated to 10-nm colloidal gold in TBS was used. Grids were incubated on gold solutions for 1 hr before rinsing in 7-10 drops
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of fresh TBS, followed by rinsing in filtered PBS four times for 10 min. This was followed by fixation in 2% glutaraldehyde in PBS for an additional 10 min. Grids were then rinsed three times in distilled water and floated on drops of 2% neutral uranyl acetate for 30 min. For the reembedding of the thin sections, grids were then rehydrated in an alcohol series of30%,50%, 70%. 90%, and 100% (three exchanges for 1 min each). Grids were then immersed in 2% epon in 100% ethanol (2 min) and blotted immediately between a folded piece of No. 50 Whatman filter paper. The grids were set up by their edges on a piece of silicon rubber [back side of an embedding mold from Pelco (Redding, CA) with small shallow slits cut into it]. Polymerizationwas carried out overnight in a covered petri dish at 60°C before removal from slits and counterstaining with 4% aqueous uranyl acetate and lead citrate. Samples were viewed using a JEOLlOOB electron microscope at 60 kV.
Results Amyloid deposits in NPs and in the walls of blood vessels, as well as NFTs in AD brain, were identified either by positive immunostaining with BAP antibodies or by positive congo red staining (as viewed under polarized light) (Snow et al., 1989~).To determine whether decorin was localized to sites of amyloid deposition and/or NFT accumulation, an affinity-purified polyclonal antibody and a mixture of three MAb against decorin were used on serial sections. In both paraffin-embedded and 1-pm deplasticized sections, decorin appeared to be primarily immunolocalized to the collagenous region surrounding small and medium-sized arterioles in AD brain parenchyma (Figure 1A). A similar association of decorin with collagen was also found in normal human brain parenchyma (not shown). These sites were identified as containing collagen because they stained positively with the Van Gieson stain in paraffinembedded sections (not shown). Meningeal vessels that contained amyloid deposits in their walls, as shown by positive congo red staining under polarized light or by positive BAP immunoreactivity, demonstrated decorin primarily localized to collagen in the adventitia of these blood vessels (not shown). In paraffin-embedded sections, “primitive” or “diffuse” plaques, which were negative with congo red on adjacentsections, were found to be negative for decorin immunoreactivity (not shown). No positive immunostainingin these tissue sites described above was observed by use of a polyclonal antibody to the AA amyloid protein or when Tris-bufferedsaline was used instead of the primary antibody. In addition, pre-absorption experiments using the MAb in the presence of excess dermatan sulfate antigen demonstrated no positive immunostaining in these areas.
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Figure 2. Ultrastructuralimmunolocalizationof dermatan sulfate core protein (decorin) in amyloid plaques and congophilic angiopathy. All tissue sections shown are from the hippocampus of a 72-year-old male (same section as in Figure 1) with Alzheimer‘s disease. lmmunogold labeling was accomplished according to the method of Mar and Wight (1988) on deplasticized ultra-thin sections. (A) Unlabeled star-shaped amyloid plaque with radiating bundles of amyloid fibrils. The center (c) and the periphery(p) of the amyloid plaque are shown. (E)When immunolabeled with the decorin polyclonal antibody (1:lO dilution) or MAb (not shown), the periphery (p) of the amyloid plaque shown in A demonstrates decorin primarily localized to the periphery of amyloid bundles (arrowheads). This is in contrast to heparan sulfate proteoglycan core protein which has been previously shown (Snow et al., 1988) to be evenly distributed over amyloid fibrils in plaques. (C) Higher magnification of the center (c) of the amyloid plaque shown in A. Less immunogold labeling of decorin (arrowheads) (polyclonal, 1:lO dilution) in center of plaque in comparisonwith periphery of plaque (E) is observed. This correlates with the lack of positive decorin immunostainingin the center of amyloid plaques demonstrated in 1-pm deplasticized tissue sections (See Figure 1). (0)As a negative control, the adjacent serial section (from A) was immunostainedwith either a polyclonal antibody (1:lO dilution) to the AA amyloid protein or with a mixture of three MAb to decorin in the presence of excess decorin antigen (not shown). Only a few gold particles immunolabel the same plaque as in A. (E) Collagen (c) fibrils (in cross-section) in the adventitia of an arteriole in the brain parenchyma are shown. (F) Higher magnificationof collagen area from E demonstratespositivedecorin immunogoldlabeling(polyclonal, 1:lO dilution) associatedwith collagen fibrils. Dermatan sulfate proteoglycans are known to be associated with collagen fibrils and serve as an internal positive control. Ears: A,E = 1 pm; E = 0.4 pm; C,D = 0.3 pm; F = 0.15 wm.
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Positive decorin immunostaining was also observed in amyloid plaques in 1-pm sections after plastic removal (Figures 1B and IC). Positive immunostaining with the decorin antibodies tended to be primarily localized to the periphery of amyloid plaques (Figure IC), with little to no immunoreactivity in the center of these plaques. Of the 12 amyloid plaques studied, 10 of these demonstrated positive immunostaining primarily localized to the periphery of the plaque, whereas the other two plaques were not immunostained. Amyloid plaques containing dystrophic neurites demonstrated decorin immunoreactivityconfined to the periphery of the amyloid core as described above. No apparent decorin immunolocalization to neurites was observed (not shown). NF?s (Figures 1D and 1E) (identified by positive congo red staining in adjacent serial sections) were also immunostained positively with the decorin antibodies in AD brain. Neurons that did not contain NFTs were not immunostained with the decorin antibody (Fig. IF). Ultrastructural immunolocalization of the decorin antigen was evident only after removal of epon from ultra-thin sections before colloidal gold immunostaining (Mar and Wight, 1988,1989).Nonneuritic star-shaped amyloid plaques contained both a central core region (marked c in Figure 2A) and a periphery (marked p in Figure 2A). Decorin was primarily localized to the periphery of the amyloid plaque, and to the edges of amyloid bundles in the peripheral region (Figure 2B). Little to no immunogold labeling for decorin was found in the center of amyloid plaques (Figure 2C) in comparison with the periphery (Figure 2B). This correlated well with the lack of decorin immunostaining observed in the center of 10 of 12 amyloid plaques demonstrated in 1-pm deplasticized tissue sections (see Figure IC).As negative controls, the adjacent serial thin section from Figure 2A was immunostained with a polyclonal antibody to the AA amyloid protein (Figure 2D), or using TBS instead of the primary antibody (not shown). In addition, no positive immunostaining was observed using the decorin MAb preabsorbed with excess dermatan sulfate antigen. Little immunogold labeling was observed in both the periphery and the center of the identical amyloid plaque. Collagen fibrils present in the adventitia of arterioles in the brain parenchyma (Figure 2E) also demonstrated positive immunogold labeling of decorin (Figure 2F). The association of decorin with collagen indicated that the antibody was active and effectively localized to collagen, as others have demonstrated (Mar and Wight, 1988; Scott and Haigh, 1985;Young, 1985; Scott and Orford, 1981). Decoriri was also immunolocalizedto most of the extraneuronal and intraneuronal NFTs in AD brain at the ultrastructural level (Figures 3A-3C). Immunogold labeling for decorin was primarily associated with bundles of filaments (most likely a mixture of both paired helical and straight filaments) in NF?s (Figures 3B and 3C), correlatingwith decorin immunostaining of in 1-pm deplasti-
cized sections. Little to no immunogold labeling for decorin was observed in "Eoutside of the areas containing filaments. In addition, little to no immunogold labeling of filaments of "Ewas found when the adjacent serial sections from Figure 3A were immunolabeled with a polyclonal antibody against the AA amyloid protein (not shown), when TBS was used instead of the decorin primacy antibody (not shown), or when the decorin MAb were used in the presence of excess decorin antigen (Figure 3D).
Discussion The present study has demonstrated that the small DSPG core protein (decorin) is localized within the amyloid plaques and NETS in AD brain. These results therefore indicate that more than one class of PG is associated with amyloid plaques. Unlike HSPG core protein, previously shown to be evenly distributed throughout NPs containing amyloid fibrils (Snow et al., 1988), decorin was primarily localized to the periphery ofthe amyloid plaque and to the edges of amyloid fibril bundles within the plaque periphery. The lack of decorin immunostaining in the center of amyloid plaques indicates that this PG is restricted in its distribution, although the possibility does exist that masking of decorin antigenicity in the center of the plaque may be due to other protein components. However, this seems unlikely because no particular protein or macromolecule has been found preferentially localized to the center of amyloid plaques. At present it is not known why amyloid cores are usually spherically shaped and are confined to a certain size (usually 6-22 pm in diameter for the amyloid core itself) (Selkoeet al., 1986)within the brains of Alzheimer's disease, Down's syndrome, and normal aging patients. The unique and specific localization of decorin to the periphery of the amyloid plaque suggests that these molecules may contribute to regulating the size of amyloid accumulation within the cores of NPs and/or in maintaining their sphericalshape. For example, decorin has been implicated in influencing collagen fibril formation and the ultimate diameter of collagen fibrils formed (Scott, 1988; Flint et al., 1984; Vogel et al., 1984; Obrink, 1973). The DSPGs may be interacting directly with the BAP constituent of amyloid or with other components present, such as HSPGs. The DSPGs may also serve as a barrier in preventing the interaction of trophic factors (such as basic FGF) within the plaque region with nearby accumulatingneurons and/or astrocytes and their processes, thereby containing the plaque to a certain size. Decorin not only was localized to amyloid deposits in plaques but was also associated with collagen fibrils surrounding blood vessels, both within the parenchyma and outside the brain (i.e., meningeal vessels). The specific localization of decorin to collagen fibrils in brain agrees with previous studies in other tissues (Uldbjerg and Danielsen, 1988; Scott, 1980) which demonstrated this association
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Figure 3. Ultrastructural immunolocalization of dermatan sulfate core protein (decorin) in neurofibrillary tangles. All tissue sections shown are from the hippocampus of either a 72-year-old male or a 74-year-old male with Alzheimer's disease. lmmunogold labeling was accomplished according to the method of Mar and Wight (1988) on deplasticized ultra-thin sections. (A) Low magnification of bundles of filaments (n) in an extraneuronal NFT lmmunogold labeling of decorin (polyclonal, 1:lO dilution). (B) Higher magnification from A demonstrates positive decorin immunogold labeling (polyclonal, 1:lO dilution) primarily associated with bundles of filaments in the NFT. (C) Positive immunogold labeling of decorin (mixture of three MAb, 1:5 dilution) primarily localized to the filaments in an intraneuronal NFT (arrowheads). (0)Only a few gold particles are associated with bundles of filaments when the adjacent serial section from A is immunolabeled with a mixture of three MAb in the presence of excess decorin antigen. Bars: A = 1.2 km; B = 0.3 pm; C = 0.4 pm; D = 0.5 pm.
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to occur specifically at the “d” band of the collagen fibril in ten-
don (Scott and Orford, 1981) and bone (Scott and Haigh, 1985). Previous studies have demonstrated patients with Alzheimer’s disease (in comparison to controls) to show alterations in blood vessel walls, including increased collagen deposition (Bohl et al., 1986). Since decorin is known to be associated with collagen, one could speculate that in AD brain an increase in collagen accumulation would also involve an increased accumulation of decorin in the walls of blood vessels. The overall abundance of decorin in the walls of blood vessels may then become associated with amyloid deposits in nearby plaques and/or with NFTs, since previous studies have suggested a close association between blood vessels and amyloid plaques in AD brain (Miyakawa et al., 1982). The identification and immunolocalization of decorin to paired helical and straight filaments in extraneuronal and intraneuronal NFTs correlates with observations previously reported, including the positive staining of NFTs with the cationic dyes Alcian blue (Snow et al., 1987) and cuprolinic blue (Snow et al., 1988). In these latter studies, positive NFT staining was observed at 0.3 M magnesium chloride, which was virtually abolished at 0.7 M magnesium chloride, suggesting the presence of dermatan sulfate and/or chondroitin sulfate proteoglycans. In addition, the lack of NFT immunostaining using antibodies to the protein core of the basement membrane-derived HSPG further suggested that DSPGs and/or CSPGs were primarily present. A recent study demonstrated that an MAb against heparan sulfate GAG chains immunostained approximately 10-20% of extraneuronal and intraneuronal NFTs (Snow et al., 1990). Therefore, in conjunction with the results of the present study, a combination of both heparan sulfate and DS (decorin) may be present in some NFTs, as well as in the amyloid deposits of NPs. Preliminary studies (Snow and Wight, unpublished data) suggest that amyloid cores isolated from the brains of AD patients also contain immunoreactivity for both decorin and HSPG core protein. The significance of the presence of both HSPG and decorin in BAP-containing amyloid deposits is not clear, but a binding interaction between these particular PGs and components within the amyloid core (such as BAP) may occur. Preliminary data with [3~S]-sulfate-labeledPGs isolated from endothelial cells indicate that both HSPGs and DSPGs bind to the BAP (residues 1-28) of AD with differences in their binding affinities (Snow et al., 1989b). The differences in the binding affinity between these specific PGs for the BAP may ultimately explain not only the observed differences in their distribution in the AD brain but also their potential importance in the pathogenesis of this disease.
Acknowledgments We wish to than&Dr Dennis SelRoe and Marcia Podlisny for the ‘Angela” antiserum ana’ for their suggestions in critical review ofthe manuscript, Dr Robert Kisilevs&yfor the A A amyloia’protein antibody, ana’ Dr Colin Masters for the BAPIA4 antibody.
Literature Cited Abraham R, Selkoe DJ. Potter H (1988): Immunochemical identification of the serine protease inhibitor alphal-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52:487
Bohl J, Storkel S, Schulze E, Tichank G, Haas S, Krausbeck E, Gather W, Katzman K, Brocks D (1986): Alzheimer’s disease: a cerebral amyloidosis, a cooperative study to correlate clinical, biochemical and pathologicanatomical findings. Pharmacopsychology 19:196 Coria F, Castano E, Prelli F, Larrondo-Lillo M, Van Duinen S, Shelanski ML, Frangione B (1988): Isolation and characterization of amyloid P component from Alzheimer’s disease and other types of cerebral amyloidoses. Lab Invest 58:454 Day AA, McQuillan CI, Termine JD, Young MR (1987): Molecular cloning and sequence analysis of the [DNA for small proteoglycan I1 of bovine bone. Biochem J 248:801 Fisher LW, TermineJD, Dejter SW Jr, Whitson SW, Yanagishita M, Kumura JH, Hascall VC. Kleinman HK, HassellJR, Nilsson B (1983): Proteoglycans of developing bone. J Biol Chem 258:6588 Flint MH, Craig AS, Reilly HC, Gillard GC, Parry DAD (1984): Collagen fibril diameters and glycosaminoglycan content of skins - indices of tissue maturity and function. Connect Tissue Res 13:69 Gatenby J. Cowdry EV (1929): Microtomist’s vade-mecum. Philadelphia, P Blakiston’s Son & Co. 53 Glenner GG, Wong CW (1984): Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 120:885 Gloss1J, Beck M, Kresse H (1984): Biosynthesis of proteodermatan sulfate in cultured human fibroblasts. J Biol Chem 259:14144 Grundke-Iqbal I, Iqbal K, Tung Y, Quinlan M, Wisniewski HM, Binder LI (1986): Abnormal phosphorylation of the microtubule-associated protein tau in Alzheimer cytoskeletal pathology. Proc Natl Acad Sci USA 83:4913 Haugh MC, Probst A, Ulrich J, Kahn J, Anderton BH (1986): Alzheimer neurofibrillary tangles contain phosphorylated and hidden neurofilament epitopes. J Neurol Neurosurg Psychiatry 49:1213 Hausser H. Hoppe W, Rauch U, Kresse H (1989): Endocytosis of a small dermatan sulphate proteoglycan. Identification of binding proteins. Biochem J 263:137 Kitamoto T, Ogomori K, TateishiJ , Prusiner SB (1987): Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57:230 Kosik KS, Joachim CL, Selkoe DJ (1986): Microtubule-associated protein tau is a major antigenic component of paired helical filaments in Alzheimer’s disease. Proc Natl Acad Sci USA 83:4044 Lee VMY, Balin BJ, Otvos L Jr, TrojanowskiJ Q (1991): A68: a major subunit of paired helical filaments and derivatized forms of normal tau. Science 251:675 Mallory FB (1961): Pathological technique. New York, Hafner Publishing Mar H, Wight TN (1989): Correlative light and electron microscopic immunocytochemistry on reembedded resin sections with colloidal gold. In Hayat MA, ed. Colloidal gold, principles, methods and applications. New York, Academic Press, 358 Mar H, Wight TN (1988): Colloidal gold immunostaining on deplasticized ultra-thin sections. J Histochem Cytochem 36:1387 Mar H, Tsukada T, Gown AM, Wight TN, Baskin DG (1987): Correlative light and electron microscopic immunocytochemistry on the same section with colloidal gold. J Histochem Cytochem 35:419 Masters CL, Simms G, Weinman NA, Multhaup G. McDonald BL, Beyreuther K (1985): Amyloid plaque core protein in Alzheimer’s disease and Down syndrome. Proc Natl Acad Sci USA 82:4245 Miyakawa T, Shimoji A, Kuramoto R, Higuchi Y (1982): The relationship between senile plaques and cerebral blood vessels in Alzheimer’s disease and senile dementia: morphological mechanism of senile plaque production. Virchows Arch [Cell Pathol] 40:121 Mori H, Kondo J , Ihara Y (1987): Ubiquitin is a component of paired helical filaments in Alzheimer’s disease. Science 235:1641
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Obrink B (1973): The influence of glycosaminoglycans on the formation of fibers from monomeric tropocollagen in vitro. Eur J Biochem 31:129 Perry G, Freidman R, Shaw G, Chau V (1987): Ubiquitin is detected in neurofibrillary tangles and senile plaque neurites in Alzheimer’s disease brains. Proc Natl Acad Sci USA 84:3033 Puchtler H, Sweat F, Levine M (1962): On the binding of congo red by amyloid. J Histochem Cytochem 10:355
TN (1988): The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer’s disease. Am J Pathol 1333456 Snow AD, Mar H, Nochlin D, Sekiguchi R, Kimata K, Koike Y, Wight TN (1990): Early accumulation of heparan sulfate in neurons and in the beta-amyloidprotein containing lesions of Alzheimer’s disease and Down’s syndrome. Am J Pathol 137:1253
Rosenberg LC, Choi HU, Tang L, Johnson TL, Pal S, Webber C, Reiner A, Poole AR (1985): Isolation of dermatan sulfate proteoglycansfrom mature bovine articular cartilages. J. Biol Chem 260:6304
Snow AD, Mar H, Nochlin D, Wight TN (1989~):Congo red staining on 1 micron de-plasticizedsections for the detection of lesions in Alzheimer’s disease and related disorders. In Iqbal K, Wisniewski H. Winblad B, eds. Alzheimer’s disease and related disorders. New York, Alan R Liss, 383
Schmidt G, Robenek H, Harrach B, GlosslJ, Nolte V, Hormann H, Richter H, Kresse H (1987): Interaction of small dermatan sulfateproteoglycan from fibroblasts with fibronectin. J Cell Biol 104:1683
Snow AD, Wight TN (1989): Proteoglycansin the pathogenesis of Alzheimer’s disease and other amyloidoses. Neurobiol Aging 10:481
Scott JE (1988): Proteoglycan-fibrillar collagen interactions. Biochem J 252:313 Scott JE (1980): Collagen-proteoglycan interactions. Localization of proteoglycans in tendon by electron microscopy. Biochem J 187:887 ScottJE, Haigh M (1985): Proteoglycan-type-I collagen fibril interactions in bone and non-calcifying connective tissues. Biosci Rep 1:71 Scott JE, Orford CR (1981): Dermatan sulphate-rich proteoglycan associates with rat tail-tendon collagen at the d band in the gap region. Biochem J 197:213 Selkoe DJ, Abraham CR, Podlisny MB, Duffy LK (1986): Isolation of lowmolecular-weight proteins from amyloid plaque fibers in Alzheimer’sdisease. J Neurochem 46:1820 Siedlak SL, Cras P, Kawai M, Richey P, Perry G (1991): Basic fibroblast growth factor binding is a marker for extracellular neurofibrillary tangles in Alzheimer’s disease. J Histochem Cytochem 39:899 Snow AD, Kinsella MG, Prather PB, Nochlin D, Podlisny MB, Kisilevsky R, HassellJR, Wight TN (1989b):A characteristicbinding affinity between heparan sulfate proteoglycans and the A4 amyloid protein of Alzheimer’s disease. J Neuropathol Exp Neurol 48:352 Snow AD, Lara S. Nochlin D, Wight TN (1989a):Cationic dyes reveal proteoglycans structurally integrated within the characteristic lesions of Alzheimer’s disease. Acta Neuropathol 78:113
Snow AD, Willmer J, Kisilevsky R (1987): Sulfated glycosaminoglycans in Alzheimer’s disease. Hum Pathol 18:506 Stopa EG, Gonzalez A, Chorsky R, Corona RJ, Alvarez J, Bird ED, Baird A (1990): Basic fibroblast growth factor in Alzheimer’s disease. Biochem Biophys Res Commun 171:690 Uldbjerg N, Danielsen CC (1988): A study of the interaction in vitro between type I collagen and a small dermatan sulphate proteoglycan. Biochem J 251:643 Vogel KG, Fisher LW (1986): Comparisons of antibody reactivity and enzyme sensitivity between small proteoglycans from bovine tendon, bone, and cartilage. J Biol Chem 261:11334 Vogel KG, Paulsson M, Heinegard D (1984): Specific inhibition of type I and type I1 collagen fibrillogenesisby the small proteoglycan of tendon. Biochem J 223:587 Voss B, GlosslJ, Cully Z, Kresse H (1986): Immunocytochemicalinvestigation on the distribution of small chondroitin sulfate-dermatan sulfate proteoglycan in the human. J Histochem Cytochem 341013 Wong CW, Quaranta V, Glenner GG (1985): Neuritic plaques and cerebrovascular amyloid in Alzheimer’sdisease are antigenetically related. Proc Natl Acad Sci USA 8223729 Young RD (1985): The ultrastructural organization of proteoglycans and collagen in human and rabbit scleral matrix. J Cell Sci 7495
Snow AD, Mar H, Nochlin D, Kimata K, Kat0 M, Suzuki S, HassellJ, Wight
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Vol. 40, NO, 1, pp. 101-113, 1992 Printedin l.%S.A.
Original Artide
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Peripheral Distribution of Dermatan Sulfate Proteoglycans (Decorin) in Amyloid-containing Plaques and Their Presence in Neurofibrillary Tangles of Alzheimer’s Disease’ ALAN D. SNOW,2 HENDERSON MAR, DAVID NOCHLIN, HANS KRESSE, and THOMAS N. WIGHT Departments of Pathology (HM,,nVW)and Neuropathology (ADS,DN), University of Wasbington, Seattle, Wasbington, and Inshtute of Physiological Chemistry and Patbobiochemistry, University of Munster; Munster; Germuny (HK). Received for publication April 30, 1991 and in revised form August 14, 1991; accepted August 27, 1991 (lA2315).
We used a polydonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the s m a l l dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristiclesions in Alzheimer’s brain. All antibodies demonstrated positive decorin immunoStainingin both the amyloid depositsof neuritic plaques (NPs) and the filamentous structures within neurofbrillatytangles (NFTs).Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout N P s containing amyloid fibrils, decorin was pri-
Introduction Current studies investigating the pathogenesis of Alzheimer’sdisease (AD) have primarily focused on the identification and characterization of components within the characteristic brain lesions (i.e., the neuritic plaque, the neurofibrillary tangle, and cerebrovascular amyloid deposits). Understanding the nature of these components and determining their precise location within the enveloping lesions should lead to further clarification of initiating and processing events critical to the pathogenesis of this disease. Much research effort has focused on a major protein component present in the amyloid deposits of neuritic plaques (NPs) and in the walls of blood vessels (i.e., congophilic angiopathy), known as the betaamyloid protein (BAP) (Wong et al., 1985; Glenner and Wong, 1984) or A4 (Masters et al., 1985). In addition to the BAP, other protein components present within the NP and/or cerebrovascular amyloid deposits include the serine protease inhibitor, alphalanti-chymotrypsin (Abraham et al., 1988) and amyloid P component (Coria et al., 1988). In associationwith the paired helical and
Supported by the Alzheimer’sDisease Research Program ofthe American Health Assistance Foundation, the French Foundation for Alzheimer’s Research, and NIH grant AGO5136. Correspondence to: Dr. Alan Snow,University of Washington, Dept. of Pathology, Neuropathology Labs RJ-05, Seattle, WA 98195.
marily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorinwas also immunolocalized to the paired helical and straight filaments within NF% and to collagen fibrils surrounding blood vessels. The unusual distribution of decorin confined to the periphery of amyloid plaques in AD brain suggests that this particular PG may play an important role in the development of the amyloid plaque. (JIlistochem Cytochem 40:105-1U,195’2) KEY WORDS:Decorin; Dermatan sulfate; Proteoglycans; Amyloid;
Plaques; Alzheimer’s disease; Neurofibrillary tangles.
straight filaments of NFTs, major components identified include microtubule-associatedphosphoproteins, including the tau protein (Lee et al., 1991; Grundke-Iqbal et al., 1986; Kosik et al., 1986), ubiquitin (Mori et al., 1987; Perry et al., 1987). and neurofilaments (Haugh et al., 1986). Previous studies have also identified a specific group of complex anionic macromolecules known as proteoglycans (PGs), specifically localized to the amyloid deposits in both NPs and in the wall of blood vessels (Snow and Wight, 1989; Snow et al., 1987,1988,1989a). The particular class of PG present in the amyloid deposits of these lesions has been identified by immunocytochemistry as a basement membrane-derived heparan sulfate proteoglycan (HSPG) (Snow et al., 1988). In this latter study, antibodies to the protein core of the basement membrane-derived HSPG identified and localized HSPG core protein to the amyloid deposits in NPs and congophilic angiopathy, to “primitive”plaques (those containing BAP immunoreactivity but lacking positive staining with congo red or thioflavin S), and in subsets of some adjacent neurons and astrocytes,whereas HSPG core protein was not immunolocalized to NFTs. However, a recent study (Snow et al., 1990)utilizing a monoclonal antibody (MAb) that recognizes a specific epitope (glucosamine sulfate alpha144 glucuronic acid determinant) in heparan sulfate chains of a basement membrane-derived HSPG identified and immunolocalized heparan sulfate to 10-20% of NFTs. Since a number of histochemical and immunocytochemical 105
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studies (Siedlak et al., 1991; Stopa et al., 1990; Snow et al., 1987,1989a;1990)suggest that PGs and/or GAGs are associated with NFTs, and since previous studies imply that heparan sulfate is associated only with some NFTs (Siedlak et al., 1991; Snow et al., 1990), it was important to confirm whether other PGs are localized to NFTs, as well as to other characteristic lesions (i.e., amyloid plaques and cerebrovascular amyloid deposits) in AD. In the present investigation, a polyclonal antibody and a mixture of three mAb primarily recognizing the small dermatan sulfate proteoglycan (DSPG) known as “decorin” or “PG-11”(Schmidt et al., 1987; Voss et al.. 1986; Glossl et al., 1984), were employed to determine whether this particular PG is associated with either NPs, NETS, or congophilic angiopathy in Alzheimer’s brain. Decorin, a small PG that decorates collagen fibrils (Scott and Haigh, 1985; Scott and Orford, 1981; Scott, 1980), contains one GAGchain and has a core protein size of 36,500 M W based on deduced amino acid sequence from full-length cDNA clones (Day et al., 1987; Vogel and Fisher, 1986; Rosenberg et al., 1985; Glossl et al., 1984; Fisher et al., 1983). In the present study, decorin was identified as a new component associated with the amyloid deposits of NPs and the filamentous structure within Unlike HSPGs. which tend to be evenly distributed throughout plaques containing amyloid fibrils, decorin was primarily localized to the periphery of the amyloid plaque as a whole and to the edge of amyloid bundles within this periphery. In addition, decorin was associated with the paired helical and straight filaments within
m.
m.
Materials and Methods Autopsy Material and Tissue Processing. Brain tissue, including hippocampus and amygdala from eight patients with AD (confirmed at autopsy) were used in the present study. Four tissue specimens (an 84-yearold female, a 76-year-old female, a 72-year-old male, a 64-year-old male) were obtained within 2-5 hr after death and were fuced in either 10% formalin for 24 hr or Carnoy’s solution for 4 hr (Gatenby and Cowdry, 1929) before routine paraffin embedding. The brain tissue from the other four patients (an 82-year-old male, a 79-year-old female, a 72-year-old male, and a 63-year-old female) had previously been fixed in 10% formalin and were obtained from the University of Washington Alzheimer’s Disease Research Center. From each block of paraffin-embedded material, 6-pm serial sections were cut and placed on gelatin-coated slides. For electron microscopy, small pieces of hippocampus and amygdala were fixed in a solution of 3% paraformaldehydeand 0.25% glutaraldehyde in 0.05 M phosphate buffer and processed as described previously (Snow et al., 1988). Proteoglycan and Amyloid Protein Antibodies. For the immunohistochemical studies a number of different PG and amyloid protein anti-
bodies were used. These included: (a) an affinity-purified polyclonal antibody (used at a dilution of 1:lO)against the decorin core protein of human skin fibroblasts (Voss et al., 1986; Glossl et al., 1984); (b) a mixture of three MAb (used at dilutions of 1:5 or 1:lO) against the decorin core protein of human skin fibroblasts (Schmidt et al., 1987). The titer of this antibody mixture was 1/20 ofthe polyclonal antibody sera according to ELISA data (not shown); (c) a polyclonal antibody against residues 1-42 of the BAP or A4 protein of AD (used at dilution of 1:100and 1:400) (gift of Dr. Colin Masters, Perth, Australia); (d) a polyclonal antibody (known as antiserum A or ‘Angela”)raised to the native HPLC-purifiedBAP from Alzheimer’s cortex (gift of Dr. Dennis Selkoe, Boston, MA) (Selkoe et al., 1986); and (e) a polyclonal antibody against the AA amyloid protein (used at a dilution of 1:lO) (gift of Dr. Robert Kisilevsky, Kingston, Canada). Histochemical and Immunocytochemical Detection of Amyloid and Decorin in Alzheimer‘s Brain Tissue. Congo red staining (Puchder et al., 1962) was used on paraffin and 1-pm deplasticized sections (Snow et al., 198%) to detect NPs, NFIS, and congophilic angiopathy in cach ofthe cases. Additionally,antibodies raised to the major low molecular weight amyloid protein (Selkoe et al., 1986)or against residues 1-42 of the BAP identified amyloid deposits in NPs and congophilic angiopathy. Adjacent serial sections were immunostained with either the affhity-purified polyclonal antibody against decorin (1:lO dilution) or the mixture of three MAb against decorin (1:5 or 1:lO dilution), enabling us to determine whether decorin was localized to fibrillar amyloid and/or NFIS. Some paraffh sections were also stained for collagen using the Van Gieson stain to determine whether there was an association between DSPGs and collagen (Mallory, 1961). Decorin was also immunolocalized in the hippocampus in six cases of AD, using ultra-thin (1-pm)sectionsafter r e m d of epon (Mar and Wight, 1988,1989). Briefly, before immunostainingslides with 1-2-pm plastic sections were submerged in a solution of saturated sodium hydroxide in absolute alcohol (sodium ethoxide), diluted 1:1with fresh absolute alcohol, for 15-20 min at room temperature, and were processed for immunocytochemical staining as previously described (Snow et al., 1988; Mar et al., 1987). Immunostainingof tissue sections was accomplished using the avidinbiotin-immunoperoxidase method, employing the appropriate biotinlabeled secondary antibodies, followed by incubation with avidin-conjugated horseradish peroxidase complex (Vector Labs; Burlingame, CA). Peroxidase activity was produced by treatment with 3.3-diaminobenzidine as previously described (Snow et al., 1988). For immunocytochemical staining the primary antibody was used initially through a series of dilutions to obtain the best specificity with the least background staining. Only the optimal dilutions of primary antibody are reported. Some tissue sections were also pre-treated for 5 min with 88% formic acid before immunostaining (Kitamoto et al., 1987). In addition, there was the possibility of masking of decorin core protein by dermatan sulfate GAG chains. Therefore, digestion of dermatan sulfate GAG chains was accomplished by incubating some tissue sections with 0.50 units of chondroitinax ABC (Miles Laboratories; Elkhart, IN) in 0.1 M Tris-acetate buffer (pH 7.3) at 37’C for 4 hr. After the incubation period tissue sections were rinsed in buffcr and immunostained using the decorin antibody. ~
Figure 1. Dermatan sulfate proteoglycans(decorin) in neuritic plaques and neurofibrillarytangles in Alzheimer‘s brain tissue. All tissue sections shown are from the hippocampus of either a 72-year-old male or a 74-year-old male with Alzheimer‘s disease. lmmunostaining was accomplished according to the avidin-biotin-peroxidase method on 1-pm deplasticized sections. (A) Positive decorin immunostaining(polyclonal, 1:lO dilution, chondroitinaseABC pre-treatment) of collagen fibrils (arrows) in and surrounding a blood vessel. (6)Positive decorin immunostaining(polyclonal, 1:lO dilution) of two amyloid plaques (arrows). Note that intense immunostaining is primarily localized to the periphery of the plaques. No positive decorin immunostaining is observed in the cell body of the adjacent neuron. Tissue was also treated with 88% formic acid for 5 min before immunostaining. (C) Positive decorin immunostaining(polyclonal, 1:lO dilution) of amyloid plaque (arrows). Strong immunostaining of plaque periphery is obsetved, whereas the center of the amyloid plaque contains little to no positive decorin immunoreactivity. Formic acid pre-treatment. (D) Positive decorin immunostaining (monoclonal, 1 5 dilution) of an NFT (arrowheads). Adjacent serial section demonstrates positive cungo red staining (Snow et al., 198%) of same NFT as viewed under polarized light (not shown). (E) Intense positivedecorin immunostaining(polyclonal, 1:lO dilution)of an NFT (arrowhead). Formicacid pre-treatment. (F) Lack of positivedecorin immunostaining(polyclonal, 1:lO dilution) in nontangle-bearingneuron. Note lack of positive decorin immunostainingof lipofuscin granules (arrowheads) in the cell body of the neuron. Formic acid pre-treatment. Bars: A,D = 11 pm; B,F = 13 pm; C,E = 14 pm.
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To rule out nonspecific binding of the decorin polyclonal antibody, sections were treated with another amyloid protein antibody of the same IgG species known not to be present in AD brain (i.e., the AA amyloid protein) (gift of Dr. Kisilevsky) (used at 1:lO dilution), or with Tris-bufferedsaline (TBS) instead of the primary antibody. In addition, sectionswere immunostained with the decorin antibodies in the presence of excess decorin antigen. With an ELISA assay, the polyclonal decorin antibody demonstrated a loss of binding after pre-absorption with dermatan sulfate antigen. The ELISA procedure for antibody testing was as follows. Decorin was purified (Hausser et al., 1989) and used for coating Nunc immunoplates I1 96F at a concentration of 10 nmol hexuronic acidlml. Fifty p1(in 0.1 M Tris-HC1, pH 7.4. 50 mM NaCI) were added per well. After 90 min at 37T, antigen was removed and the wells were washed three times with 400 ~10.1%Tween 20 in PBS (pH 7.4). The wells were post-absorbed with 400 PI, 1% gelatin/O.l% SDS in PBS for 90 min at 37’C. A series of decorin antibody dilutions in PBS/1% Tween 20 (50 PI) were then allowed to react for 90 min at 37°C. Three washings followed as described above, as well as another blocking step with 400 p1 of 1% gelatin10.1% SDS in PBS for 15 min at 37°C. The secondary antibody goat anti-rabbit IgG-peroxidase conjugate (Sigma; St Louis, MO) was diluted 500-fold with PBS/O.1%Tween 20/3% bovine serum albumin. Color development occurred using 200 p1of 1 mM ABTS/740 FM hydrogen peroxide in 50 mM sodium citrate (pH 4.0). For pre-absorption, 1 ml of antiserum was incubated with purified decorin (0.9 pmol of hexuronic acid) in 0.1 M %is-HC1 (pH 7.4), 1 M NaCl for 48 hr at 4’C under constant mixing. Only 11140 ofthe original titer against decorin was still present after pre-absorption (not shown). Immunodetectionof Decorin at the Ultrastructural Level in Alzheimer‘s Brain. Decorin was also immunolocalized in the hippocampus in two cases of AD at the ultrastructural level. To achieve optimal results, we found it necessary to remove the epon from ultra-thin sections before colloidal gold immunostaining (Mar and Wight, 1988,1989). This technique avoided interference with immunostaining for decorin believed to be due to the resin (Mar and Wight, 1989).For thin-section plastic removal, thin sectionswere etched by submersion in a solution of saturated sodium hydroxidein absolute alcohol, diluted 1:1 with fresh absolute alcohol, for 1.5 min at room temperature, followed by several rinses in absolute alcohol. Grids were then hydrated to water through a graded alcohol series (95%, two times; 7Ooh, 50%, 30%. distilled water, 1 min each). For immunostaining, grids were blocked with 10% normal goat serum in 0.05 M TBS (pH 7.6) for 10 min, followed by running in TBS four times for a total period of 10 min. Primary antisera (i.e., decorin or the AA amyloid antibodies) were diluted 1:10 in filtered TBS just before use. Grids were incubated overnight on droplets of antisera at 4’C. Each grid was then rinsed on 6 droplets of TBS for a total of 15 min before incubation with colloidal gold. Goat anti-rabbit IgG (for the polyclonal antibodies) or goat anti-mouse IgG (for the monoclonal antibodies) conjugated to 10-nm colloidal gold in TBS was used. Grids were incubated on gold solutions for 1 hr before rinsing in 7-10 drops
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of fresh TBS, followed by rinsing in filtered PBS four times for 10 min. This was followed by fixation in 2% glutaraldehyde in PBS for an additional 10 min. Grids were then rinsed three times in distilled water and floated on drops of 2% neutral uranyl acetate for 30 min. For the reembedding of the thin sections, grids were then rehydrated in an alcohol series of30%,50%, 70%. 90%, and 100% (three exchanges for 1 min each). Grids were then immersed in 2% epon in 100% ethanol (2 min) and blotted immediately between a folded piece of No. 50 Whatman filter paper. The grids were set up by their edges on a piece of silicon rubber [back side of an embedding mold from Pelco (Redding, CA) with small shallow slits cut into it]. Polymerizationwas carried out overnight in a covered petri dish at 60°C before removal from slits and counterstaining with 4% aqueous uranyl acetate and lead citrate. Samples were viewed using a JEOLlOOB electron microscope at 60 kV.
Results Amyloid deposits in NPs and in the walls of blood vessels, as well as NFTs in AD brain, were identified either by positive immunostaining with BAP antibodies or by positive congo red staining (as viewed under polarized light) (Snow et al., 1989~).To determine whether decorin was localized to sites of amyloid deposition and/or NFT accumulation, an affinity-purified polyclonal antibody and a mixture of three MAb against decorin were used on serial sections. In both paraffin-embedded and 1-pm deplasticized sections, decorin appeared to be primarily immunolocalized to the collagenous region surrounding small and medium-sized arterioles in AD brain parenchyma (Figure 1A). A similar association of decorin with collagen was also found in normal human brain parenchyma (not shown). These sites were identified as containing collagen because they stained positively with the Van Gieson stain in paraffinembedded sections (not shown). Meningeal vessels that contained amyloid deposits in their walls, as shown by positive congo red staining under polarized light or by positive BAP immunoreactivity, demonstrated decorin primarily localized to collagen in the adventitia of these blood vessels (not shown). In paraffin-embedded sections, “primitive” or “diffuse” plaques, which were negative with congo red on adjacentsections, were found to be negative for decorin immunoreactivity (not shown). No positive immunostainingin these tissue sites described above was observed by use of a polyclonal antibody to the AA amyloid protein or when Tris-bufferedsaline was used instead of the primary antibody. In addition, pre-absorption experiments using the MAb in the presence of excess dermatan sulfate antigen demonstrated no positive immunostaining in these areas.
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Figure 2. Ultrastructuralimmunolocalizationof dermatan sulfate core protein (decorin) in amyloid plaques and congophilic angiopathy. All tissue sections shown are from the hippocampus of a 72-year-old male (same section as in Figure 1) with Alzheimer‘s disease. lmmunogold labeling was accomplished according to the method of Mar and Wight (1988) on deplasticized ultra-thin sections. (A) Unlabeled star-shaped amyloid plaque with radiating bundles of amyloid fibrils. The center (c) and the periphery(p) of the amyloid plaque are shown. (E)When immunolabeled with the decorin polyclonal antibody (1:lO dilution) or MAb (not shown), the periphery (p) of the amyloid plaque shown in A demonstrates decorin primarily localized to the periphery of amyloid bundles (arrowheads). This is in contrast to heparan sulfate proteoglycan core protein which has been previously shown (Snow et al., 1988) to be evenly distributed over amyloid fibrils in plaques. (C) Higher magnification of the center (c) of the amyloid plaque shown in A. Less immunogold labeling of decorin (arrowheads) (polyclonal, 1:lO dilution) in center of plaque in comparisonwith periphery of plaque (E) is observed. This correlates with the lack of positive decorin immunostainingin the center of amyloid plaques demonstrated in 1-pm deplasticized tissue sections (See Figure 1). (0)As a negative control, the adjacent serial section (from A) was immunostainedwith either a polyclonal antibody (1:lO dilution) to the AA amyloid protein or with a mixture of three MAb to decorin in the presence of excess decorin antigen (not shown). Only a few gold particles immunolabel the same plaque as in A. (E) Collagen (c) fibrils (in cross-section) in the adventitia of an arteriole in the brain parenchyma are shown. (F) Higher magnificationof collagen area from E demonstratespositivedecorin immunogoldlabeling(polyclonal, 1:lO dilution) associatedwith collagen fibrils. Dermatan sulfate proteoglycans are known to be associated with collagen fibrils and serve as an internal positive control. Ears: A,E = 1 pm; E = 0.4 pm; C,D = 0.3 pm; F = 0.15 wm.
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Positive decorin immunostaining was also observed in amyloid plaques in 1-pm sections after plastic removal (Figures 1B and IC). Positive immunostaining with the decorin antibodies tended to be primarily localized to the periphery of amyloid plaques (Figure IC), with little to no immunoreactivity in the center of these plaques. Of the 12 amyloid plaques studied, 10 of these demonstrated positive immunostaining primarily localized to the periphery of the plaque, whereas the other two plaques were not immunostained. Amyloid plaques containing dystrophic neurites demonstrated decorin immunoreactivityconfined to the periphery of the amyloid core as described above. No apparent decorin immunolocalization to neurites was observed (not shown). NF?s (Figures 1D and 1E) (identified by positive congo red staining in adjacent serial sections) were also immunostained positively with the decorin antibodies in AD brain. Neurons that did not contain NFTs were not immunostained with the decorin antibody (Fig. IF). Ultrastructural immunolocalization of the decorin antigen was evident only after removal of epon from ultra-thin sections before colloidal gold immunostaining (Mar and Wight, 1988,1989).Nonneuritic star-shaped amyloid plaques contained both a central core region (marked c in Figure 2A) and a periphery (marked p in Figure 2A). Decorin was primarily localized to the periphery of the amyloid plaque, and to the edges of amyloid bundles in the peripheral region (Figure 2B). Little to no immunogold labeling for decorin was found in the center of amyloid plaques (Figure 2C) in comparison with the periphery (Figure 2B). This correlated well with the lack of decorin immunostaining observed in the center of 10 of 12 amyloid plaques demonstrated in 1-pm deplasticized tissue sections (see Figure IC).As negative controls, the adjacent serial thin section from Figure 2A was immunostained with a polyclonal antibody to the AA amyloid protein (Figure 2D), or using TBS instead of the primary antibody (not shown). In addition, no positive immunostaining was observed using the decorin MAb preabsorbed with excess dermatan sulfate antigen. Little immunogold labeling was observed in both the periphery and the center of the identical amyloid plaque. Collagen fibrils present in the adventitia of arterioles in the brain parenchyma (Figure 2E) also demonstrated positive immunogold labeling of decorin (Figure 2F). The association of decorin with collagen indicated that the antibody was active and effectively localized to collagen, as others have demonstrated (Mar and Wight, 1988; Scott and Haigh, 1985;Young, 1985; Scott and Orford, 1981). Decoriri was also immunolocalizedto most of the extraneuronal and intraneuronal NFTs in AD brain at the ultrastructural level (Figures 3A-3C). Immunogold labeling for decorin was primarily associated with bundles of filaments (most likely a mixture of both paired helical and straight filaments) in NF?s (Figures 3B and 3C), correlatingwith decorin immunostaining of in 1-pm deplasti-
cized sections. Little to no immunogold labeling for decorin was observed in "Eoutside of the areas containing filaments. In addition, little to no immunogold labeling of filaments of "Ewas found when the adjacent serial sections from Figure 3A were immunolabeled with a polyclonal antibody against the AA amyloid protein (not shown), when TBS was used instead of the decorin primacy antibody (not shown), or when the decorin MAb were used in the presence of excess decorin antigen (Figure 3D).
Discussion The present study has demonstrated that the small DSPG core protein (decorin) is localized within the amyloid plaques and NETS in AD brain. These results therefore indicate that more than one class of PG is associated with amyloid plaques. Unlike HSPG core protein, previously shown to be evenly distributed throughout NPs containing amyloid fibrils (Snow et al., 1988), decorin was primarily localized to the periphery ofthe amyloid plaque and to the edges of amyloid fibril bundles within the plaque periphery. The lack of decorin immunostaining in the center of amyloid plaques indicates that this PG is restricted in its distribution, although the possibility does exist that masking of decorin antigenicity in the center of the plaque may be due to other protein components. However, this seems unlikely because no particular protein or macromolecule has been found preferentially localized to the center of amyloid plaques. At present it is not known why amyloid cores are usually spherically shaped and are confined to a certain size (usually 6-22 pm in diameter for the amyloid core itself) (Selkoeet al., 1986)within the brains of Alzheimer's disease, Down's syndrome, and normal aging patients. The unique and specific localization of decorin to the periphery of the amyloid plaque suggests that these molecules may contribute to regulating the size of amyloid accumulation within the cores of NPs and/or in maintaining their sphericalshape. For example, decorin has been implicated in influencing collagen fibril formation and the ultimate diameter of collagen fibrils formed (Scott, 1988; Flint et al., 1984; Vogel et al., 1984; Obrink, 1973). The DSPGs may be interacting directly with the BAP constituent of amyloid or with other components present, such as HSPGs. The DSPGs may also serve as a barrier in preventing the interaction of trophic factors (such as basic FGF) within the plaque region with nearby accumulatingneurons and/or astrocytes and their processes, thereby containing the plaque to a certain size. Decorin not only was localized to amyloid deposits in plaques but was also associated with collagen fibrils surrounding blood vessels, both within the parenchyma and outside the brain (i.e., meningeal vessels). The specific localization of decorin to collagen fibrils in brain agrees with previous studies in other tissues (Uldbjerg and Danielsen, 1988; Scott, 1980) which demonstrated this association
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Figure 3. Ultrastructural immunolocalization of dermatan sulfate core protein (decorin) in neurofibrillary tangles. All tissue sections shown are from the hippocampus of either a 72-year-old male or a 74-year-old male with Alzheimer's disease. lmmunogold labeling was accomplished according to the method of Mar and Wight (1988) on deplasticized ultra-thin sections. (A) Low magnification of bundles of filaments (n) in an extraneuronal NFT lmmunogold labeling of decorin (polyclonal, 1:lO dilution). (B) Higher magnification from A demonstrates positive decorin immunogold labeling (polyclonal, 1:lO dilution) primarily associated with bundles of filaments in the NFT. (C) Positive immunogold labeling of decorin (mixture of three MAb, 1:5 dilution) primarily localized to the filaments in an intraneuronal NFT (arrowheads). (0)Only a few gold particles are associated with bundles of filaments when the adjacent serial section from A is immunolabeled with a mixture of three MAb in the presence of excess decorin antigen. Bars: A = 1.2 km; B = 0.3 pm; C = 0.4 pm; D = 0.5 pm.
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to occur specifically at the “d” band of the collagen fibril in ten-
don (Scott and Orford, 1981) and bone (Scott and Haigh, 1985). Previous studies have demonstrated patients with Alzheimer’s disease (in comparison to controls) to show alterations in blood vessel walls, including increased collagen deposition (Bohl et al., 1986). Since decorin is known to be associated with collagen, one could speculate that in AD brain an increase in collagen accumulation would also involve an increased accumulation of decorin in the walls of blood vessels. The overall abundance of decorin in the walls of blood vessels may then become associated with amyloid deposits in nearby plaques and/or with NFTs, since previous studies have suggested a close association between blood vessels and amyloid plaques in AD brain (Miyakawa et al., 1982). The identification and immunolocalization of decorin to paired helical and straight filaments in extraneuronal and intraneuronal NFTs correlates with observations previously reported, including the positive staining of NFTs with the cationic dyes Alcian blue (Snow et al., 1987) and cuprolinic blue (Snow et al., 1988). In these latter studies, positive NFT staining was observed at 0.3 M magnesium chloride, which was virtually abolished at 0.7 M magnesium chloride, suggesting the presence of dermatan sulfate and/or chondroitin sulfate proteoglycans. In addition, the lack of NFT immunostaining using antibodies to the protein core of the basement membrane-derived HSPG further suggested that DSPGs and/or CSPGs were primarily present. A recent study demonstrated that an MAb against heparan sulfate GAG chains immunostained approximately 10-20% of extraneuronal and intraneuronal NFTs (Snow et al., 1990). Therefore, in conjunction with the results of the present study, a combination of both heparan sulfate and DS (decorin) may be present in some NFTs, as well as in the amyloid deposits of NPs. Preliminary studies (Snow and Wight, unpublished data) suggest that amyloid cores isolated from the brains of AD patients also contain immunoreactivity for both decorin and HSPG core protein. The significance of the presence of both HSPG and decorin in BAP-containing amyloid deposits is not clear, but a binding interaction between these particular PGs and components within the amyloid core (such as BAP) may occur. Preliminary data with [3~S]-sulfate-labeledPGs isolated from endothelial cells indicate that both HSPGs and DSPGs bind to the BAP (residues 1-28) of AD with differences in their binding affinities (Snow et al., 1989b). The differences in the binding affinity between these specific PGs for the BAP may ultimately explain not only the observed differences in their distribution in the AD brain but also their potential importance in the pathogenesis of this disease.
Acknowledgments We wish to than&Dr Dennis SelRoe and Marcia Podlisny for the ‘Angela” antiserum ana’ for their suggestions in critical review ofthe manuscript, Dr Robert Kisilevs&yfor the A A amyloia’protein antibody, ana’ Dr Colin Masters for the BAPIA4 antibody.
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