©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Proteoglycan-mediated Inhibition of A Proteolysis
A POTENTIAL CAUSE OF SENILE PLAQUE ACCUMULATION (*)

(Received for publication, April 10, 1995)

Rekha Gupta-Bansal (§) Robert C. A. Frederickson Kurt R. Brunden

From the Discovery Research Group, Gliatech Inc., Cleveland, Ohio 44122

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Senile plaques of Alzheimer's disease brain contain, in addition to beta amyloid peptide (Abeta), multiple proteoglycans. Systemic amyloidotic deposits also routinely contain proteoglycan, suggesting that these glycoconjugates are generally involved in amyloid plaque formation and/or persistence. We demonstrate that heparan sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG) inhibit the proteolytic degradation of fibrillar, but not non-fibrillar, Abeta at physiological pH. In accordance with the proteolysis studies, high affinity binding of proteoglycans to fibrillar Abeta(1-40) and Abeta(1-42) is observed from pH 4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar peptide is only seen at pH < 6. This differing pH dependence of binding suggests that a lysine residue is involved in proteoglycan association with fibrillar Abeta, whereas a protonated histidine appears to be needed for binding of the glycoconjugates to non-fibrillar peptide. Scatchard analysis of fibrillar Abeta association with proteoglycans indicates a single affinity interaction, and the binding of both HSPG and CSPG to fibrillar Abeta is completely inhibited by free glycosaminoglycan chains. This implies that these sulfated carbohydrate moieties are primarily responsible for proteoglycanbulletAbeta interaction. The ability of proteoglycans to bind fibrillar Abeta and inhibit its proteolytic degradation suggests a possible mechanism of senile plaque accumulation and persistence in Alzheimer's disease.


INTRODUCTION

Senile plaques are one of the classical neuropathological features of Alzheimer's disease (AD) (^1)brain(1) , and a prevailing hypothesis is that these structures lead to the dystrophic neurites and dying neurons that cause the dementia associated with this disease. The major component of senile plaques is polymeric fibrils of Abeta(2) , a 39-42-amino-acid peptide which is formed after proteolytic processing of the amyloid precursor protein (APP)(3, 4, 5, 6) . Other macromolecules(7, 8, 9) , including a variety of proteoglycans(10, 11, 12) , co-localize with senile plaques. Interestingly, proteoglycans are also invariably associated with plaques of peripheral systemic amyloidoses(13, 14, 15) , suggesting they may play an important role in plaque accumulation and/or persistence.

Abeta (16, 47) and APP (16, 17) have been shown to bind with relatively high affinity to certain heparan sulfate proteoglycans (HSPG), and these interactions were suggested to be mediated in part via the core protein of the proteoglycans. Work from this laboratory (18) has demonstrated that Abeta is also capable of association with a variety of glycosaminoglycan chains. The binding of Abeta with proteoglycan core protein and/or glycosaminoglycan chains provides an explanation for the localization of CSPG(10) , DSPG(11) , and HSPG (12) with senile plaques.

It has been suggested that the association of proteoglycans with Abeta might result in an enhanced rate of fibrillation of the amyloid peptide (13) or that proteoglycans may inhibit the degradation of Abeta(13, 19) . Either of these actions could lead to increased numbers of senile plaques, with a concomitant increase of neuropathology. There is precedent for the glycosaminoglycan moieties of proteoglycans affecting protein breakdown, as heparan sulfate has been demonstrated to slow plasmin-mediated digestion of basic fibroblast growth factor(20) . In this study, we have examined whether proteoglycans of the classes found associated with senile plaques are capable of decreasing the susceptibility of Abeta to proteolytic degradation. The results reveal that whereas both non-fibrillar and fibrillar Abeta can be readily cleaved by proteases, complexes of proteoglycan and fibrillar amyloid peptide are relatively resistant to breakdown. The polymeric form of Abeta binds proteoglycans with significantly greater affinity than does non-fibrillar peptide at physiological pH, and the association of CSPG and HSPG with fibrillar Abeta appears to be mediated primarily via glycosaminoglycan chains. These data thus supply the first indication that senile plaques in AD brain may accumulate and persist due to the inability of naturally occurring proteases to act on Abetabulletproteoglycan complexes.


MATERIALS AND METHODS

Proteoglycan Preparations

EHS mouse HSPG was obtained commercially (Collaborative Biomedical Products). CSPG were purified from bovine nasal septum cartilage essentially as described(21) .

Characterization of Abeta

Fibrillar Abeta(1-40) (Bachem Inc.) and Abeta(1-42) (synthesized at Gliatech Inc.) were prepared by resuspending lyophilized peptide in H(2)O (2 mM stock) and allowing the solutions to age for 1-3 weeks. The extent of fibril formation was quantified using a Thioflavin T fluorescence assay as described(26) . Non-fibrillar Abeta preparations were made by resuspending lyophilized peptide in hexafluoroisopropanol, with subsequent relyophilization. The hexafluoroisopropanol-treated lyophilized peptides were suspended in H(2)O (2 mM stock) and used within 3 days of solubilization. The non-fibrillar Abeta solutions did not bind Thioflavin T as determined by the absence of specific fluorescence.

Proteolysis of AbetabulletProteoglycan/Glycosaminoglycan Complexes

In a typical assay, non-fibrillar or fibrillar Abeta(1-40) (4.3 µg) and proteoglycans (30 µg) were incubated for 1-2 h at room temperature in 10-15 µl final volume of Tris (0.1 M) or phosphate-buffered saline (PBS) at pH 7.4. Papain (2 µg) was subsequently added and the reaction mixture incubated at 37 °C for 18 h. In some experiments, papain was replaced with cathepsin B (Sigma). In these studies, fibrillar Abeta(1-40) was incubated either in the presence or absence of CSPG as above, except in 20 mM sodium acetate, pH 5.2, containing 120 mM NaCl, 0.5 mM EDTA, and 1 µM dithiothreitol. Cathepsin B (0.1 unit) was subsequently added and incubated as above. Digested products were subjected to 16.5% Tris-Tricine SDS-PAGE (27) and the gels were stained with Coomassie Blue to visualize undegraded 4-kDa Abeta, which was quantitated using a densitometer (PDI; model DNA 35).

Additional studies were performed in which 30 µg each of chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, heparin, (all from Sigma) or heparan sulfate (Seikagaku America, Inc.) were added as above instead of proteoglycan.

AbetabulletProteoglycan Binding Assay

Polyvinyl microtiter plates (Costar high binding) were coated with proteoglycans (2 µg/50 µl) in PBS buffer overnight at 4 °C. After aspirating the proteoglycan solution, wells were blocked with PBS containing 2% bovine serum albumin for 2 h at room temperature. Control wells were prepared as above, except proteoglycan was omitted from the initial overnight incubation. Aliquots (100 µl) of either fibrillar or non-fibrillar Abeta(1-40) and Abeta(1-42) at varying concentrations in blocking solution were added to the wells. Following a 2-h incubation at room temperature, the wells were extensively rinsed with PBS. Proteoglycan-bound Abeta was detected by the addition of mouse Abeta antibody (4G8 at 1:2000 dilution in blocking solution; antibody provided by Drs. K. S. Kim and H. M. Wisniewski, Institute for Basic Research in Developmental Diabetics, Staten Island, NY), which was allowed to incubate for 1 h at room temperature. After washing the plate with PBS, a peroxidase-conjugated goat anti-mouse antibody (1:2000 dilution in blocking solution; Sigma) was added and allowed to incubate for 1 h. The plate was again rinsed thoroughly with PBS, and peroxidase-catalyzed color development was initiated by adding 100 µl of 3,3`,5,5`-tetramethyl benzidine substrate (Kirkegard & Perry, Inc.). The reaction was quenched by adding 100 µl of 1 M phosphoric acid, and the plates were read at 450 nm in a microplate reader (Dynatech). The ability of proteoglycans or glycosaminoglycans to inhibit Abeta binding to immobilized proteoglycans was evaluated by adding varying concentrations of these agents to a constant concentration of Abeta (typically 1-2 µM). The amount of Abeta bound in the wells was determined by subtracting control wells from the experimental wells. Data were analyzed as described below.

Studies examining the pH-dependent binding of fibrillar and non-fibrillar Abeta(1-40) were performed as above in buffer systems containing 120 mM NaCl, 2.7 mM KCl, and 10 mM of each of the following buffers: sodium acetate, pH 4 and 5; sodium phosphate, pH 6, 7, and 8; sodium bicarbonate/carbonate, pH 9 and 10.

Data Analysis

The estimated K of fibrillar Abeta-proteoglycan interaction was based on the concentration of added Abeta ([Abeta]) at 50% maximal binding (Microcal Origin program). Scatchard analyses of binding data were performed by plotting bound/free (OD/[Abeta]) versus bound (OD). The concentration of Abeta fibrils could not be accurately determined due to the uncertainty of fibril size (i.e. [Abeta] = [Abeta]/c, where c = number of Abeta monomers comprising an average fibril). The K values obtained based on monomeric Abeta concentration (K) are thus greater than the true K values for fibrillar peptide (K = K/c,).

To more accurately determine binding affinities, Kvalues were obtained using proteoglycans and glycosaminoglycans as competitive inhibitors of Abeta binding to immobilized proteoglycan. The K values were derived from , where L = the concentration of Abeta ([Abeta]) used in the inhibition studies.

can be utilized to obtain accurate K values with fibrillar Abeta, even with the uncertainty of fibril concentration. In this case, L would represent [Abeta], which can be replaced by the term ([Abeta]/c). The K term in would be K, as described above. Substituting these terms into results in

[Abeta] and K are readily determined parameters.


RESULTS

Proteoglycans Protect Abeta from Proteolysis

To determine whether proteoglycans can protect Abeta from proteolytic degradation, either non-fibrillar or fibrillar Abeta(1-40) were incubated for 1-2 h at pH 7.4 with or without HSPG and CSPG. The nonspecific protease papain was subsequently added to the mixtures, and after overnight incubation the extent of Abeta proteolysis was evaluated by SDS-PAGE analysis. Both fibrillar Abeta(1-40) ( Fig. 1and 2) and non-fibrillar Abeta(1-40) (Fig. 2) were degraded by the protease in the absence of proteoglycan. Addition of CSPG or HSPG to the non-fibrillar peptide solution did not appreciably affect Abeta degradation (Fig. 2). In contrast, a significant amount of fibrillar Abeta(1-40) persisted following preincubation with both of the proteoglycans prior to papain treatment ( Fig. 1and Fig. 2). Essentially identical results were obtained when Abeta(1-42) was used instead of Abeta(1-40) or when papain was replaced with the nonspecific enzyme mixture Pronase (data not shown).


Figure 1: SDS-PAGE showing proteoglycan-mediated inhibition of Abeta(1-40) proteolysis. Fibrillar Abeta(1-40) (4.3 µg) was treated with papain (2 µg) either in the absence (lanes B) or presence of 30 µg of bovine CSPG (lanes C) or HSPG (lanes D). Lane A shows Abeta (4.3 µg) that was not subjected to papain digestion. The samples were analyzed on 16.5% Tris-Tricine SDS gels as described under ``Materials and Methods.''




Figure 2: Proteoglycan-mediated protection of Abeta(1-40) from proteolytic degradation. Non-fibrillar or fibrillar Abeta(1-40) and HSPG (lane C), bovine CSPG (lane D), or chondroitin sulfate glycosaminoglycan (lane E) were incubated at pH 7.4 and subsequently subjected to proteolysis with papain as described under ``Materials and Methods.'' Lane A shows Abeta(1-40) in the absence of papain, while lane B reveals Abeta treated with papain in the absence of proteoglycan. The data are normalized to the amount of Abeta remaining after treatment relative to lane A conditions. The inset shows a similar study in which papain was substituted with cathepsin B. Lane A contains fibrillar Abeta(1-40) in the absence of cathepsin, lane B shows fibrillar Abeta(1-40) treated with cathepsin in the absence of CSPG, and lane C contains the amyloid peptide treated with cathepsin in the presence of CSPG.



To confirm that the results obtained with papain and Pronase extended to more specific and physiologically relevant proteases, we examined whether cathepsin B degrades Abeta in the absence or presence of CSPG. This enzyme has been shown to be released by microglia (25) and thus might represent one of the proteases found in the milieu of the brain. As with papain, cathepsin B cleaved fibrillar Abeta in the absence of proteoglycan, while CSPG inhibited cathepsin proteolysis of the amyloid peptides (Fig. 2, inset).

The proteoglycan-mediated inhibition of Abeta degradation is most likely explained by steric shielding of the amyloid peptide by glycosaminoglycan chains, which should be naturally resistant to proteases. This would suggest that free glycosaminoglycans might also reduce the papain-catalyzed degradation of Abeta. However, neither chondroitin sulfate (Fig. 2, lane E), nor a variety of other glycosaminoglycans (data not shown) inhibited the proteolysis of either fibrillar or non-fibrillar Abeta. This indicates that intact proteoglycan is required for effective protection of amyloid peptide.

Binding of Proteoglycans to Abeta

Inhibition of fibrillar Abeta proteolysis by proteoglycans implied that these glycoconjugates were binding tightly to this form of the amyloid peptide. The reduction of Abeta degradation appeared to be specific to proteoglycans, since other proteins that are associated with senile plaques (C1q and laminin) had no effect on papain-mediated cleavage of Abeta (data not shown). To directly analyze proteoglycan association with Abeta, a solid-phase binding assay was developed in which proteoglycans were immobilized to the wells of microtiter dishes. Abeta was subsequently added, and bound peptide was quantitated via an enzyme-linked immunoabsorbant methodology. As demonstrated in Fig. 3, neither HSPG or CSPG showed appreciable binding to non-fibrillar Abeta(1-40) or Abeta(1-42) at physiological pH. While virtually no binding of non-fibrillar amyloid peptides could be demonstrated with CSPG, the HSPG preparation consistently bound a small amount of both Abeta peptides (compare Fig. 3, A and C to B and D). However, the amounts of non-fibrillar peptides that associated with HSPG were very low compared to the amount of fibrillar Abeta(1-40) or Abeta(1-42) that bound to either CSPG or HSPG (Fig. 3). The ability of proteoglycans to bind substantial amounts of fibrillar, but not non-fibrillar, Abeta at pH 7.4 coincides with the proteoglycan-mediated inhibition of degradation of only the fibrillar form of amyloid peptide. Scatchard transformations of the binding data (Fig. 3, insets) reveal that the association of fibrillar Abeta(1-40) and Abeta(1-42) with both HSPG and CSPG is governed by a single affinity interaction. The actual affinities of fibrillar Abetabulletproteoglycan binding were not calculated from these data, since the Abeta concentrations are expressed as monomeric peptide and thus overestimate the concentration of Abeta fibrils. The average amyloid fibril is likely to be composed of hundreds of monomers(29) .


Figure 3: Binding of proteoglycans to non-fibrillar and fibrillar Abeta(1-40) or Abeta(1-42) at pH 7.4. HSPG (panels A and C) and CSPG (panels B and D) were immobilized onto microtiter wells, and the binding of non-fibrillar Abeta () and fibrillar Abeta (bullet) were analyzed as described under ``Materials and Methods.'' Panels A and B show data obtained with Abeta(1-40), whereas panels C and D demonstrate binding with Abeta(1-42). Representative binding profiles are shown. When fibrillar Abeta binding data were analyzed by Scatchard transformation (inset), a linear relationship was obtained for all combinations of proteoglycan and amyloid peptide, indicating binding governed by a single affinity.



To more completely characterize the nature of proteoglycan binding to Abeta, both fibrillar and non-fibrillar peptide preparations were examined for their ability to bind CSPG over a broad pH range. As seen in Fig. 4, the binding of proteoglycan to Abeta fibrils (closed circles) is relatively constant from pH 5-7, whereas at higher pH there is a decrease in CSPG-amyloid peptide interaction. Half-maximal binding is seen at pH 9, suggesting that the protonation of 1 or more lysine residues is necessary for proteoglycan binding to Abeta fibrils. In contrast, non-fibrillar Abeta did not associate with CSPG until pH values were 6 or lower (Fig. 4, open circles). This implies that binding of proteoglycan to the non-fibrillar peptide was governed, at least in part, by protonation of histidine(s). These data show that the nature of proteoglycan binding to the two forms of Abeta is significantly different, and only the fibrillar amyloid peptide would appear to play a role in proteoglycan binding at physiological pH.


Figure 4: Binding of fibrillar and non-fibrillar Abeta(1-40) to CSPG as a function of pH. Fibrillar Abeta(1-40) (bullet) and non-fibrillar Abeta(1-40) () were examined for their ability to bind immobolized CSPG at different pH values, as described under ``Materials and Methods.'' The optical density values obtained in the binding assay are shown for each pH value.



Since the saturation binding studies presented in Fig. 3could not be used to accurately determine the affinities of proteoglycanbulletAbeta interaction, analyses were done in which HSPG and CSPG were added as competitive inhibitors of fibrillar Abeta binding to the immobilized proteoglycans (Fig. 5). The K values obtained for solution-phase proteoglycan inhibition of Abeta binding to substrate-attached HSPG and CSPG at pH 7.4 reveal high-affinity interactions of these proteoglycans with both Abeta(1-40) and Abeta(1-42) (see Table 1). Examination of the inhibition data by log-logit transformation suggested one class of non-interacting binding sites for both proteoglycans with either amyloid peptide (Fig. 5, insets). Therefore, the inhibition data are in agreement with the single affinity binding observed in the Abeta saturation analyses.


Figure 5: Inhibition of fibrillar Abeta(1-40) and Abeta(1-42) binding to substrate-bound proteoglycans by soluble proteoglycans. HSPG (panels A and C) and bovine CSPG (panels B and D) were immobilized onto microtiter wells. Fixed amounts (1.5 µM) of fibrillar Abeta(1-40) (panels A and B) or Abeta(1-42) (panels C and D) were added with varying concentrations of HSPG (panels A and C) or CSPG (panels B and D). The amount of bound Abeta was assayed as described under ``Materials and Methods.'' Plotting logit(percent bound) versus log(inhibitor) for each curve (insets) revealed a linear relationship with a slope of -2.3 (Hill coefficient of 1), suggestive of a single affinity interaction.





Although the binding of both CSPG and HSPG to fibrillar Abeta is governed by a single affinity interaction, it was not known whether the proteoglycan association was mediated via the core-protein, glycosaminoglycan chains, or a combination of both. To examine this question, free heparan sulfate and chondroitin sulfate glycosaminoglycans were utilized as competitive inhibitors in the Abeta binding assay. Both of these anionic polysaccharides were effective inhibitors of amyloid peptide binding to HSPG and CSPG (Fig. 6), with their K values listed in Table 1. Again, log-logit transformation of these data (Fig. 6, insets) suggested single affinity, non-interacting binding sites. The ability of free glycosaminoglycans to completely inhibit amyloid peptide binding to proteoglycan would seem to indicate that the intact glycoconjugates complex with fibrillar Abeta via these sulfated sugar chains. The larger K values obtained with the glycosaminoglycans relative to the proteoglycans is likely to reflect the fact that there are many of these sugar moieties per proteoglycan molecule. For example, the proteoglycans bind Abeta(1-42) with affinities that are approximately 25-75-fold greater than those determined for the free glycosaminoglycans. However, the sugar content (by weight) per mole of the proteoglycans is 25-100 times that of the glycosaminoglycan chain preparations.


Figure 6: Inhibition of fibrillar Abeta(1-40) and Abeta(1-42) binding to substrate-bound proteoglycans by glycosaminoglycans. HSPG (panels A and C) and bovine CSPG (panels B and D) were immobilized onto microtiter wells. Fixed amounts (1.5 µM) of fibrillar Abeta(1-40) (panels A and B) or Abeta(1-42) (panels C and D) were added with varying concentrations of heparan sulfate (panels A and C) or chondroitin sulfate (panels B and D). The amount of bound Abeta was assayed as described under ``Materials and Methods.'' Plotting logit(percent bound) versus log(inhibitor) for each curve (insets) revealed a linear relationship with a slope of -2.3 (Hill coefficient of 1), suggestive of a single affinity interaction.



In addition to proteoglycan association with Abeta being prevented by glycosaminoglycans of the same class as those of the immobilized proteoglycan, we found that free CS and HS polysaccharides could inhibit amyloid peptide binding to HSPG and CSPG, respectively (data not shown). The ability of dissimilar glycosaminoglycan chains to inhibit the binding of the same proteoglycan indicates that the binding of these polysaccharides to Abeta is relatively nonspecific with regard to sugar composition.


DISCUSSION

A body of evidence is accumulating implicating Abeta and APP in the etiology of AD(30, 31, 32, 33, 34) . In AD, Abeta is assembled into polymeric crossed beta-fibrils within senile plaques, and studies (35, 36) have suggested that fibrillar Abeta is directly neurotoxic. It should be noted, however, that the in vitro models used to demonstrate Abeta-induced neurotoxicity are unlikely to mimic the conditions of the AD brain, and there are alternative explanations of the role of Abeta in causing AD neuropathology(19, 37, 38, 39) .

Senile plaques contain proteoglycans in addition to amyloid peptide (10, 11, 12) . Systemic amyloid deposits resemble senile plaques in that polymeric beta-fibrils are found associated with proteoglycans/glycosaminoglycans(13, 14, 15) . In one study of peripheral amyloidosis(40) , the temporal deposition of proteoglycans appeared to occur concurrently with the formation of amyloid fibrils, suggesting that proteoglycans played an active role in plaque formation.

The data presented here support the hypothesis that proteoglycans play an important role in the accumulation and persistence of plaques. We have found that mouse HSPG and bovine CSPG have the ability to bind fibrillar Abeta(1-40) and impart protection against proteolytic attack. An identical protective effect is elicited by CSPG purified from rat astrocytes (data not shown), indicating that this activity is not species-specific. Enzymes with relatively nonspecific substrate specificity (i.e. papain and Pronase) were employed in these studies since the stability of proteoglycanbulletAbeta complexes to such exhaustive proteolytic conditions would suggest similar resistance to proteases found in the brain.

The inability of proteoglycans to affect the degradation of non-fibrillar Abeta reflects a relative lack of interaction with this form of peptide at physiological pH. Previous work from this laboratory (18) indicated that glycosaminoglycans only associated with the truncated Abeta(1-28) peptide at pH values below 7. This pH profile suggested that 1 or more histidine residues of Abeta(1-28) must be protonated for glycosaminoglycan binding. The pH dependence of proteoglycan binding to non-fibrillar Abeta shows a qualitatively similar profile, implicating histidine in the binding of glycosaminoglycans and proteoglycans to non-fibrillar Abeta.

The binding of fibrillar Abeta to proteoglycans at pH 7.4 would imply, however, an interaction that is not mediated through glycosaminoglycan association with histidine(s). A significant reduction in proteoglycan binding to fibrillar Abeta is observed as the pH increases above 9, indicating 1 or more lysine residues are involved in the association. The critical lysine(s) is likely to reside in the Abeta sequence and not in the proteoglycan core protein since our data indicate that binding of the glycoconjugates is via glycosaminoglycans.

The binding of proteoglycans to lysine residues within fibrillar Abeta would suggest that anionic groups of the former ion-pair with the amyloid fibril. It would seem likely that the sulfated sugars of glycosaminoglycans align with a positive-charge array formed from 1 or more lysine residues from each monomeric subunit of the assembled amyloid fibril. Both Abeta(1-40) and Abeta(1-42) fibrils showed comparable affinities for proteoglycans and glycosaminoglycans, suggesting that there were not significant differences in the arrangement of the residues critical to binding between these two forms of amyloid peptide. The binding of proteoglycan to the longer amyloid peptide may be of the most physiological relevance, as recent data suggest that Abeta(1-42) is found in larger amounts than Abeta(1-40) within the senile plaques of AD brain(41) .

Buee et al.(16) demonstrated that intact HSPG and chemically deglycosylated HSPG could bind to an Abeta(1-28) affinity column, leading these investigators to suggest that the core protein was involved in binding of intact proteoglycan. These authors also showed glycosaminoglycan-mediated binding of HSPG to Abeta. Since saturation binding analyses were not performed, it is not known whether they observed a single affinity interaction between intact HSPG and the truncated amyloid peptide. Because we have investigated binding of proteoglycans to full-length Abeta peptides, it is difficult to make direct comparisons between this prior study and the data presented here. It should be noted, however, that the deglycosylation of proteoglycans could expose regions of the core protein that would normally be sterically shielded by glycosaminoglycan groups, thereby revealing novel Abeta-binding sites.

While our data indicate that Abeta fibrils bind proteoglycans through interaction with glycosaminoglycans, we cannot exclude the possibility that the small amount of non-fibrillar Abeta binding to HSPG observed at pH 7.4 (see Fig. 3, A and C) is mediated, in part, by core protein. No attempts were made to examine the nature of non-fibrillar Abeta-HSPG association through the use of glycosaminoglycan competition studies because of the very low signal obtained in the binding assay.

Although proteoglycans bind fibrillar Abeta via glycosaminoglycan chains, these polysaccharide groups alone are not sufficient to inhibit proteolytic accessibility to the amyloid fibrils. The binding of one or a few glycosaminoglycan groups of a proteoglycan may allow the other sulfated polysaccharide moieties of that molecule to form an effective barrier around the Abeta fibril. Such a shield may not be formed when free glycosaminoglycan chains bind the amyloid peptide.

The ability of proteoglycans to inhibit proteolysis of Abeta has important implications with regard to AD pathology. The association of HSPG with Abeta may be one of the precipitating events in the formation of stable plaque structures. HSPG has been shown to be present in cortical diffuse plaques(11) , and there is belief that these structures are predecessors of senile plaques. Interestingly, diffuse plaques of the cerebellum lack HSPG(42) , and cerebellar dense plaques with associated neuropathology are rare. Thus, HSPG may bind the initial few Abeta fibrils that form in diffuse deposits, and in doing so begin the transformation to senile deposits. The localization of CSPG and DSPG with senile plaques, and not diffuse deposits, suggests that these proteoglycans may associate with fibrillar Abeta later in the maturation process. Another notable difference between diffuse and senile neuritic plaques is the clear presence of activated glia within and around the latter(43, 44, 45) . It is possible that glia are responsible for the release of all or some of the proteoglycans that associate with senile plaques. CSPG are known to be inhibitory to axon growth(46) , and their association with plaques may cause some of the neuritic dystrophy characteristic of AD(37) .

The demonstrated ability of proteoglycans to alter the proteolytic processing of Abeta suggests that drugs developed to inhibit the binding of these glycoconjugates to amyloid peptide might increase the turnover of Abeta by naturally occurring proteases, thereby providing therapeutic benefit to those suffering the consequences of this devastating disease.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed: Gliatech Inc., 23420 Commerce Park Rd., Cleveland, OH 44122. Tel.: 216-831-3200; Fax: 216-831-4220.

^1
The abbreviations used are: AD, Alzheimer's disease; Abeta, beta amyloid peptide; APP, amyloid precursor protein; CSPG, chondroitin sulfate proteoglycan; DSPG, dermatan sulfate proteoglycan; HSPG, heparan sulfate proteoglycan; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Drs. Stephen Yates and Clark Tedford for their helpful discussions.


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