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.

A consistent rat model for the study of the consequences of congophilic and fibrillar A beta-amyloid in brain has been developed. One hundred percent of animals receiving infusions of synthetic beta-amyloid protein (A beta 1-40) plus a specific heparan sulfate proteoglycan (HSPG) for 1 week or 7 weeks (following 2 week infusions) demonstrated Congo red and thioflavin S-positive deposits adjacent to the infusion site. Extracellular amyloid fibrils were identified by electron microscopy and were immunogold decorated with A beta antibody. Significant increases in Congo red staining were observed in animals infused with A beta plus HSPG versus those infused with only A beta. Infusion of A beta alone was variable with respect to congophilic amyloid persistence, which occurred in 50% of animals and only when endogenous HSPGs accumulated at A beta deposition sites. By 7 weeks, only animals infused with A beta plus HSPG demonstrated compaction of the Congo red material from amorphous, wispy deposits (at 1 week) to stellate deposits resembling a Maltese cross. These spherical amyloid deposits were very similar to Congo red-stained amyloid plaques in human Alzheimer's disease brain, and in vitro data suggest that they were probably formed in vivo following interactions with endogenous brain components.

Effects of 1,2-dimethylhydrazine on the number of epithelial cells present in the villi, crypts, and mitotic pool along the rat small intestine.

Young adult male rats received 1,2-dimethylhydrazine (25 mg/kg) twice weekly for 2 months and once a week thereafter for up to 6 months. Histological samples of duodenum, jejunum, and upper, mid-, and terminal ileum were prepared from groups killed at each month. Using cell counts, the average number of epithelial cells was determined per representative section of villi and crypts and was used as an index of villus size and crypt size, respectively. The average number of mitotic figures in representative crypt sections was also determined. All three parameters increased during treatment, and the increments showed a specific pattern in relation to time and intestinal region. Mitotic number showed a consistent change all along the small intestine: close to 20% rise by 3 months; decrease to near control level by the fourth month; and a rise thereafter. Probably, a systemic stimulation of mitotic activity by 1,2-dimethylhydrazine took place. The crypt size index changed similarly, showing highly significant correlation with mitotic number. This correlation indicated that average mitotic time and cell cycle time remained unchanged and the number of divisions increased in progenitor cells. Calculations showed that only a fraction of the progenitor cells was involved. These were probably "initiated" cells. There was a net increase of initiated cell numbers with time, but a sharp drop at 4 months indicated that there is a mechanism inducing a regression of the initiated cell population. Villus size increased linearly in the duodenum and jejunum. In the ileum, there was also a net increase but with some initial fluctuation. In general, villus size seemed to increase so as to maintain a fairly stable turnover time. This would mean that the increased mitotic activity was balanced by increasing differentiation.

Morphometric study of the rat duodenal epithelium during the initial six months of 1,2-dimethylhydrazine carcinogenesis.

Male Wistar rats weighing 100 g received 1,2-dimethylhydrazine (25 mg/kg) s.c.) twice a week for 2 months and once a week thereafter for an additional 4 months. Groups of four to six rats were sacrificed monthly. Paraffin sections of duodenum were prepared and stained with periodic acid-Schiff and hematoxylin for cell counts and with toluidine blue for measuring nucleolar area. As an index of villus size and crypt size, the mean number of epithelial cells per representative sections of villi and crypts were used. Mitotic activity was assessed by counting the mean number of mitotic figures per representative crypt section. Nucleolar area was assessed from the analysis of drawn (camera lucida) images of nucleoli of columnar cells at six levels of the epithelium: lower, mid, and upper parts of the crypts and villi. Villus size increased progressively during the 6-month treatment, from 272 +/- 2 (S.E.) to 349 +/- 8. Crypt size increased from 118 +/- 2 in a wavy fashion, showing maximum (139 +/- 5, 143 +/- 2) at 3 and 6 months and minimum (123 +/- 3, 127 +/- 1) at 1 and 4 months. Mitotic number displayed a similar pattern of increase so that the percentage of mitotic figures in the crypts (mitotic index) remained constant (about 5%) in control and experimental animals. Nucleolar area in the controls decreased with age from 4.2 +/- 0.08 sq micron in lower crypt at 1 month to 2.8 +/- 0.04 sq micron at 6 months. During 1,2-dimethylhydrazine treatment, lower crypt nucleoli increased to 4.5 +/- 0.12 sq micron after 3 months and decreased slightly thereafter, reaching 4.0 +/- 0.14 sq micron by the sixth month. The nucleoli furthermore decreased gradually along epithelium (nucleolar compaction) by an average of 0.23% per cell position in control as well as treated animals. It appeared, then, that the main effect of 1,2-dimethylhydrazine was the enlarging of the four parameters measured. This effect seemed to relate in some manner to tumor formation as all the enlargements were attenuated in intestinal tissue adjacent to tumors.

Sulfate content and specific glycosaminoglycan backbone of perlecan are critical for perlecan's enhancement of islet amyloid polypeptide (amylin) fibril formation.

Islet amyloidosis is characterized by the deposition and accumulation of amylin in pancreatic beta-cells and is observed in 90% of patients with type 2 diabetes. Previous studies have also revealed the presence of the specific heparan sulfate proteoglycan, perlecan, colocalized to islet amyloid deposits, similar to perlecan's known involvement with other amyloid proteins. In the present study, perlecan purified from the Engelbreth-Holm-Swarm (EHS) tumor was used to define perlecan's interactions with amylin (i.e., islet amyloid polypeptide) and its effects on amylin fibril formation. Using a solid phase-binding immunoassay, human amylin, but not rat amylin, bound immobilized EHS perlecan with a single dissociation constant (Kd) = 2.75 x 10(-6) mol/l. The binding of human amylin to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans (GAGs), and was completely abolished by 10 micromol/l heparin. Using thioflavin T fluorometry, Congo red staining, and electron microscopy methodology, intact perlecan was found to enhance amylin fibril formation in a dosage-dependent manner, with the majority of these effects attributed to the heparan sulfate GAG chains of perlecan. Other sulfated GAGs and related macromolecules were also effective in the enhancement of amylin fibril formation in the order of heparin > heparan sulfate > chondroitin-4-sulfate = dermatan sulfate = dextran sulfate > pentosan polysulfate, implicating the importance of the specific GAG/carbohydrate backbone. The sulfate content of heparin/heparan sulfate was also important for the enhancement of amylin fibril formation in the order of heparin > N-desulfated N-acetylated heparin > completely desulfated N-sulfated heparin > completely desulfated N-acetylated heparin. These studies suggest that the enhancement effects of perlecan on amylin fibril formation are mediated primarily by both specific GAG chain backbone and GAG sulfate content, and implicate perlecan as an important macromolecule that is likely involved in the pathogenesis of islet amyloidosis.

Conformation and fibrillogenesis of Alzheimer A beta peptides with selected substitution of charged residues.

A key pathological feature of Alzheimer's disease (AD) is the formation and accumulation of amyloid fibers within the neurophil as senile plaques and in the walls of cerebral and meningeal blood vessels. The major component is the 39 to 42 residue amyloid beta protein (A beta), which is an internal proteolytic fragment of the membrane-associated amyloid precursor protein. Aggregation of A beta into amyloid fibers that could be cytotoxic may be a factor in the AD-related neuronal loss. To understand the steps and molecular interactions involved in the transition from a soluble to fibrous form of A beta, and to test molecular models that postulate ion pairing between beta-strands, we have synthetized four peptides having substitutions in specific, charged residues. These included an A beta fragment, residues 11 to 25, and having histidine-to-aspartate replacements at positions 13 (H13D) and 14 (H14D), an aspartate-to-lysine at position 23 (D23K) and a 28-mer full-length extracellular domain where the positive charge cluster at His13-His14-Gln15-Lys16 was replaced by an uncharged Gly13-Gly14-Gln15-Gly16 (GGQG). Fourier-transform infrared spectroscopy and fiber X-ray diffraction determined that the H13D and H14D substitutions had negligible effect on beta-sheet formation, suggesting that these residues are not critical for the intramolecular interactions necessary for folding in the beta-conformation. However, negative-stain electron microscopy revealed that the loss of the His13 or His14 resulted in only protofilament formation, suggesting that these residues are involved in amyloid fibril assembly. By contrast, the D23K substitution virtually eliminated folding into a beta-sheet conformation, with appreciable secondary structure being detected only following extended incubation times. The complete absence of the centrally charged region GGQG arrested amyloid assembly at the protofilament stage and also reduced the stability of the beta-conformation, suggesting a contribution of Lys16 in maintaining secondary structure. While it has been conclusively demonstrated by previous investigations that amyloid formation is dependent to a large extent on hydrophobically driven interactions, our results indicate that charge-charge interactions function in concert with non-ionic interactions to stabilize the beta-sheet conformation and assembly of AD amyloid fibers.

Proteoglycans in the pathogenesis of Alzheimer's disease and other amyloidoses.

Proteoglycans and the amyloid P component are two constituents of amyloid that appear to be present regardless of the type of amyloid protein deposited, the extent of amyloid deposition and the tissue or organ involved. This article reviews the literature concerning proteoglycans and/or glycosaminoglycans in amyloidosis and describes recent studies which demonstrate their localization to the characteristic lesions of Alzheimer's disease and the amyloid plaques containing PrP protein in the prion diseases. Additionally, the possible interaction of proteoglycans with various amyloidogenic proteins, including the beta-amyloid protein in Alzheimer's disease is discussed. It is postulated that proteoglycans localized to a number of different amyloids play a common role in the pathogenesis of amyloidosis. Some of these hypothesized roles include 1) inducing amyloidogenic precursor proteins to form amyloid fibrils containing a predominant beta-pleated sheet structure, 2) influencing amyloid deposition to occur at specific anatomical sites within tissues and/or 3) aiding in prevention of amyloid degradation once amyloid has formed.

Familial dementia with PrP-positive amyloid plaques: a variant of Gerstmann-Sträussler syndrome.

We present a 22-year follow-up of a large and unusual kindred previously reported as familial Alzheimer's disease (FAD). However, detailed clinical and neuropathologic evaluation of family members and brain autopsy on another affected individual now make the diagnosis of FAD unlikely. Our patient, as well as members of this family, had numerous amyloid plaques and rare neurofibrillary tangles. These plaques were quite atypical for Alzheimer's disease (AD); many were quite large (up to 500 microns in diameter) and contained several amyloid cores, some with neuritic components. The plaques were present throughout the cerebral cortex and striatum, but not in the cerebellum. By electron microscopy, they had radiating star-shaped amyloid cores containing 8- to 10-nm fibrils, and a few dystrophic neurites. They were strongly immunoreactive with antiserum to prion protein but did not react with the antiserum to the amyloid A4 protein of AD. Although the cerebellum was uninvolved, this family appears to represent another clinical and neuropathologic variant of Gerstmann-Sträussler syndrome.

Age-related deposition of glia-associated fibrillar material in brains of C57BL/6 mice.

With advancing age, clusters of unusual granules appear in the brains of C57BL/6 (B6) mice. At the light, confocal laser and electron microscopic levels, the granules represent aggregations of fibrillar material often associated with astrocytes. The fibrillar material is largely free of normal organelles and has been located within astrocytic somata and processes, although in many cases the material is found in the neuropil and is surrounded by a discontinuous membrane. The deposits occur predominantly in hippocampus, but also in piriform cortex, cerebellum and less frequently in some other brain regions. They become evident about six months of age and increase markedly in both number and size thereafter. Incidence of the deposits varies greatly among inbred mouse strains. At six to 12 months of age, granules are abundant in male and female B6, and are absent in BALB/c, CBA, DBA/2 and A mice. In hybrid strains with a B6 background the deposits are also present and thus appear to manifest dominant genetic heritability. Similar granular structures have been described in adult brains of the senescence accelerated mouse and have been noted, albeit very rarely, in aged mice from other strains. While immunostaining of the granules with several polyclonal antisera was found by preabsorption with antigens to be non-specific, immunolabeling with monoclonal antibodies to heparan sulfate proteoglycan core protein and to laminin suggest these or related molecules as components of the fibrillar material. The presence of glycosaminoglycans is supported by staining with periodic acid-Schiff and Gomori's methenamine silver methods. The functional significance of the murine deposits is not yet clear. The deposits do not represent senile plaques with beta-amyloid deposition, but they might mimic the deposition of extracellular matrix molecules that is hypothesized to be a precursor condition for plaque formation and cerebral amyloidosis. Furthermore, the genetic differences in the incidence of the fibrillar deposits has potential to model aspects of familial neurodegenerative diseases.

Peripheral distribution of dermatan sulfate proteoglycans (decorin) in amyloid-containing plaques and their presence in neurofibrillary tangles of Alzheimer's disease.

We used a polyclonal antibody and a mixture of three monoclonal antibodies (MAb), all recognizing the protein core of the small dermatan sulfate proteoglycan (DSPG) (known as PG-II or decorin) derived from human skin fibroblasts, to immunolocalize this molecule in the characteristic lesions in Alzheimer's brain. All antibodies demonstrated positive decorin immunostaining in both the amyloid deposits of neuritic plaques (NPs) and the filamentous structures within neurofibrillary tangles (NFTs). Unlike heparan sulfate proteoglycans (HSPGs), which tend to be evenly distributed throughout NPs containing amyloid fibrils, decorin was primarily localized to the periphery of the spherically shaped amyloid plaques and to the edges of amyloid fibril bundles within the plaque periphery. Decorin was also immunolocalized to the paired helical and straight filaments within NFTs 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.

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis.

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.

Sulfated glycosaminoglycans in Alzheimer's disease.

Using the sulfated alcian blue and alcian blue-MgCl2 techniques for demonstrating sulfated glycosaminoglycans (GAGs), we have shown sulfated GAGs to be associated with the amyloidotic lesions of Alzheimer's disease, the neuritic plaques, the neurofibrillary tangle, and the congophilic angiopathy. To determine how specific these findings are to Alzheimer's disease, other neurologic disorders with neurofibrillary tangles and filamentous inclusions were examined. These included progressive supranuclear palsy, Pick's disease, subacute sclerosing panencephalitis, and postencephalitic parkinsonism. Sulfated GAGs were not demonstrated in the neurofibrillary tangles or filamentous structures in any of these disorders. The relationship of GAGs to the pathology of Alzheimer's disease is discussed as is their possible importance in determining the characteristic morphology of the amyloidotic lesion.

Novel purification and detailed characterization of perlecan isolated from the Engelbreth-Holm-Swarm tumor for use in an animal model of fibrillar A beta amyloid persistence in brain.

Co-infusion of the specific heparan sulfate proteoglycan (HSPG), perlecan, and beta-amyloid protein (A beta) into rodent hippocampus leads to a consistent animal model to study the effects of fibrillar A beta amyloid in brain [Snow, A.D. et al. (1994) Neuron 12, 219-234]. In the present study, we describe our rapid novel method of perlecan isolation. The isolation method does not require cesium chloride centrifugation and exploits a newly discovered aggregating property of a approximately 220 kDa PG observed during gel filtration chromatography, which allowed it to be affectively separated from non-aggregating perlecan. Fifty or 100 g of EHS tumor were routinely extracted using 4 M guanidine-HCl, followed by anion-exchange and gel filtration chromatography. SDS-PAGE (before and after digestion with heparitinase/heparinase or nitrous acid) followed by staining with silver demonstrated no other contaminating proteins in the perlecan preparations. Western blots using a specific perlecan core protein antibody (HK-102) following heparitinase digestion showed a characteristic doublet at 400 and 360 kDa indicative of intact perlecan core protein. Absence of contamination by other basement membrane components produced by the EHS tumor was confirmed by absence of immunoreactive bands on Western blots using antibodies against laminin, fibronectin, or type IV collagen. One week continuous co-infusion of perlecan obtained from this methodology, with A beta (1-40) into rodent hippocampus, led to deposition of fibrillar A beta amyloid in 100% (10 of 10) of animals. The detailed protocol for isolation and characterization of perlecan from EHS tumor ensures perlecan of the highest quality, and maximizes the potential effects of A beta amyloid deposition/persistence in brain using the animal model. High quality perlecan obtained from this novel isolation method will also allow future studies utilizing in vitro assays to determine the potential interactions of this specific HSPG with other macromolecules.

Temporal relationship between glycosaminoglycan accumulation and amyloid deposition during experimental amyloidosis. A histochemical study.

The temporal relationship between glycosaminoglycan (GAG) accumulation and amyloid deposition was determined in two models of amyloid induction. In the first model amyloid was induced rapidly using amyloid-enhancing factor and AgNO3 as an inflammatory stimulus. In the second model amyloid was induced by daily injections of azocasein. Congo red staining was used to demonstrate amyloid, and both the sulfated Alcian blue and Alcian blue (pH 5.7)-MgCl2 methods were used to demonstrate GAGs. In the rapid model, amyloid was first detected in the splenic perifollicular areas 36 hours after induction. The initial presence of GAGs was also seen at 36 hours and in the exact locale where the amyloid was found. Amyloid was first seen in the liver about the central veins at 48 hours. GAGs in the liver appeared coincidentally with and in the same location as the amyloid. In the traditional model (azocasein injections) amyloid did not appear in the spleen until day 6 to 7, again with coincidental GAG deposition. The present study shows that amyloid-associated GAGs appear in the tissues together with the AA protein and are not a function of the tissue type. The appearance of the GAGs is also not a function of the nature of the inflammatory agent or its length of action but appears to be part of the process involved in the deposition of the AA protein.

Characterization of tissue and plasma glycosaminoglycans during experimental AA amyloidosis and acute inflammation. Qualitative and quantitative analysis.

Qualitative and quantitative methods were used to determine changes in glycosaminoglycans (GAGs) in the spleen and plasma during initial stages of experimental amyloidosis and acute inflammation. GAG deposition in the spleen during the early stages of amyloidosis consists of a 16-fold heparin and heparan sulfate increase. Though splenic weights do increase during protracted inflammation only minor changes arise in splenic GAGs in the absence of amyloid deposition. An overall increase in plasma GAGs, consisting of a 4.5-fold chondroitin-4-sulphate increase, occurred at the time of GAG deposition in the tissues (spleen, liver) and probably accounts for the minor GAG changes seen in the spleen during inflammation. The time course of splenic heparin/heparan sulfate increase during amyloid deposition coincides with the histochemical changes previously described. Plasma GAG changes follow a pattern similar to that of acute phase protein reactants. The results suggest that GAG metabolism, in particular heparin/heparan sulfate, are intimately involved in the process of AA amyloidogenesis.

Electrophoresis of glycosaminoglycans isolated from normal human plasma. Direct evidence for the presence of a heparin-like molecule.

It has frequently been suggested that there may normally be small quantities of heparin in human blood, but amounts so small that their direct demonstration is not possible by available methods [1-4]. In this communication, we provide direct electrophoretic visualization that heparin or a closely related substance, is present in normal human plasma. After isolating glycosaminoglycans (GAGs) from 2-3 mls of plasma, a modification of the discontinuous high voltage electrophoretic method developed by Cappelletti et al. was used to successfully separate and identify the GAGs present in six normal adults (4 men and 2 women). In each case, in addition to chondroitin-4-SO4, chondroitin-6-SO4, and heparan-SO4, we were able to show the presence of a band which corresponds to that of heparin.

A close ultrastructural relationship between sulfated proteoglycans and AA amyloid fibrils.

Two cationic reagents, Ruthenium red (RR) and Cuprolinic blue (CB), were used to assess the morphologic and structural relationship between sulfated proteoglycans and AA amyloid fibrils in amyloidotic spleen and liver, and in isolated fibril preparations. Amyloidotic tissue fixed in the presence of RR showed RR granules, measuring 15 to 25 nm in diameter, over areas of electrondense fibrils. In isolated fibril preparations, RR granules were specifically localized on amyloid fibrils. Amyloidotic tissue fixed in the presence of CB at 0.1 M and 0.7 M MgCl2 showed both granule and filamentous (50 to 90 nm in length) staining only over areas of amyloid fibrils. This same staining localization was also seen in isolated fibril preparations. The RR and CB granules and filaments, are believed to represent proteoglycan monomers with the glycosaminoglycan chains collapsed onto the protein core. The persistent CB staining at 0.7 M magnesium chloride suggested that highly sulfated proteoglycans were present. The glycosaminoglycan moiety has previously been identified as heparin/heparan sulphate. The intimate structural relationship between sulfated proteoglycans and AA amyloid fibrils, both in situ and in isolated fibril preparations, further suggests that these highly negatively charged molecules may have an important role in the pathogenesis of amyloidosis. Several pathogenetic scenarios are suggested.

Sulfated glycosaminoglycans: a common constituent of all amyloids?

In the present investigation, we analyzed whether sulfated glycosaminoglycans are a common constituent in many different types of amyloid. Serial sections of amyloidotic tissue were stained for the presence of: (a) amyloid by using Congo Red, and (b) glycosaminoglycans by using both the sodium sulfate Alcian blue method and Alcian blue, pH 5.7, with varying concentrations of magnesium chloride. Our results show that sulfated glycosaminoglycans are always associated anatomically with amyloid deposits regardless of the nature of the protein deposited. Sulfated glycosaminoglycans were found in tissues containing AA, AL, inherited cutaneous amyloid, and senile cardiac amyloid (prealbumin). Additionally, we provide evidence that sulfated glycosaminoglycans are closely associated with the amyloid of medullary carcinoma of the thyroid (prothyrocalcitonin), and neuritic plaques, neurofibrillary tangles, and congophilic angiopathy in Alzheimer's disease. It is postulated that these sulfated glycosaminoglycans can influence the folding of diverse proteins such that all forms of amyloid show a significant beta-pleated sheet component.

Heparin modulates the composition of the extracellular matrix domain surrounding arterial smooth muscle cells.

Heparin and related molecules influence vascular wall structure by their ability to inhibit smooth muscle cell (smc) proliferation and migration. However, little is known as to whether heparin has an effect on the extracellular matrix. In the present study, the effect of heparin on the content and regional distribution of elastin, collagen, and proteoglycans (PGs) in blood vessels following experimental injury was determined. Two groups of rats were subjected to left common carotid balloon injury and were infused with either 0.9% saline or heparin in a saline solution, for 2 weeks. Using a new morphometric method of analysis, the authors determined changes in volumes of elastin, collagen, and PGs contained within an 'extracellular matrix domain (ECM domain),' the average envelope of connective tissue surrounding each smc. Heparin treatment inhibited intimal thickening and decreased the elastin content in the ECM domain in the upper and lower arterial intima. Collagen also was found to be significantly decreased 5.0-fold and 7.6-fold in the ECM domains of upper and lower intima, respectively, of heparin-treated animals. The decrease in both elastin and collagen was balanced by a significant increase in amorphous and filamentous electron-dense material. Heparin also caused a significant 1.8-fold and 1.9-fold increase in the PG content in the ECM domain in the upper and lower intima, respectively. Immunohistochemical analysis, using antibodies to elastin and PG subclasses, supported the morphometric observations. This study has shown that heparin administered in vivo can alter the accumulation and distribution of each of the major vascular ECM components in a specific and differential manner.

Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.

Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to beta-amyloid protein (A beta)-containing amyloid deposits in brains of patients with Alzheimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuse A beta plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuse A beta plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A beta plaques of cerebellum. Tissues from cases of AD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A beta in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain; 2) a polyclonal antibody directed against synthetic A beta (1-40); 3) Congo red; 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. All cases (7 of 7) of AD contained diffuse A beta plaques in the cerebellum as identified by positive Bielschowsky staining and A beta immunoreactivity. None of these cases demonstrated positive Alcian blue staining (at 0.3 and 0.7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellar plaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebellum, all plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (by Alcian blue staining at 0.7 mol/L MgCl2) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebellar diffuse plaques, all cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A beta plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in A beta diffuse plaques in cerebellum. Since previous studies indicate that the cerebellum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component needed for amyloid formation and/or persistence in brain as observed in cortical areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Perlecan binds to the beta-amyloid proteins (A beta) of Alzheimer's disease, accelerates A beta fibril formation, and maintains A beta fibril stability.

Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar beta-amyloid (A beta) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with A beta and its effects on A beta fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length A beta peptides bound immobilized perlecan at two sites, representing both high-affinity [K(D) = approximately 5.8 x 10(-11) M for A beta (1-40); K(D) = approximately 6.5 x 10(-12) M for A beta (1-42)] and lower-affinity [K(D) = 3.5 x 10(-8) M for A beta (1-40); K(D) = 4.3 x 10(-8) M for A beta (1-42)] interactions. An increase in the binding capacity of A beta (1-40) to perlecan correlated with an increase in A beta amyloid fibril formation during a 1-week incubation period. The high-capacity binding of A beta (1-40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of A beta (1-40) amyloid fibril formation, causing a significant increase in A beta fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with A beta (1-40) alone. Perlecan also initially accelerated the formation of A beta (1-42) fibrils within 1 h and maintained significantly higher levels of A beta (1-42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with A beta (1-42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on A beta (1-40) fibril formation and maintenance of A beta (1-42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of A beta fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, alpha1-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of A beta, but also accelerates A beta fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of A beta amyloidosis in Alzheimer's disease.