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Glypican-1 as an Aß binding HSPG in the human brain: its localization in DIG domains and possible roles in the pathogenesis of Alzheimer’s disease ¹

NORIFUMI WATANABE², WATARU ARAKI^*, DE-HUA CHUI, TAKAO MAKIFUCHI, YASUO IHARA and TAKESHI TABIRA

National Institute for Longevity Sciences, Morioka, Obu, Aichi;
^* Department of Demyelinating Disease and Aging, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo;
Laboratory for Alzheimer’s Disease, RIKEN Brain Science Institute, Wako-shi, Saitama;
Department of Clinical Research, National Saigata Hospital, Ogata, Nakakubiki, Niigata; and
Department of Neuropathology, Faculty of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan

² Correspondence: National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan. E-mail: watanabn{at}nils.go.jp

SPECIFIC AIMS

The amyloid deposition whose major component is the 39-43 amino acid peptide, termed ß-amyloid protein (Aß), is considered to be important in Alzheimer’s disease (AD) pathology, but the precise mechanism of its accumulation in AD brain is unclear. Although there are studies of the association between Aß and heparan sulfate proteoglycans (HSPGs), they demonstrated colocalization in plaques or binding ability of HSPGs derived from tissues other than human brain. It is not fully understood whether HSPGs are involved in neuronal cell death in AD brain. Our aims are to identify Aß binding HSPG(s) from human brain and to investigate their roles in Aß accumulation and neuronal cell death.

PRINCIPAL FINDINGS

1. Identification of glypican-1 as an Aß binding HSPG from human brain
To identify HSPGs derived from the human brain with the capacity to bind Aß, control human brain lysates were separated using anion exchange DEAE-Sepharose chromatography and fractions containing possible Aß binding protein(s) were determined by Aß binding assay. We found that fractions containing HSPGs exclusively showed the binding activity to fibrillar Aß in an HS chain-dependent manner. Earlier studies reported that Aß binding to HS chains prevents heparanase-catalyzed degradation of HS chains. Thus, we examined whether preincubation of the HSPG containing DEAE fractions with Aß alters the sensitivity of HSPGs to heparitinase treatment. Without preincubation with Aß, several bands (200, 100, 60, 40 kDa) were detected with 3G10 mAb in the DEAE fractions, indicating they contained plural HSPGs. Preincubation with Aß resulted in the disappearance of the 60 kDa band; the intensity of the other bands was relatively unchanged, suggesting that an HSPG with the 60 kDa core protein bound to Aß preferentially and prevented heparitinase-catalyzed degradation of HS chains. Glypicans are known as HSPGs with a 60 kDa core protein; six glypicans (glypican-1 to -6) have been cloned. Glypican-1 is the major HSPG expressed in the adult brain. We performed the same incubation experiments to clarify the identity of the 60 kDa band using anti-glypican-1 mAb. A 60 kDa band of glypican-1 core protein was detected in the lysates; preincubation of the lysates with Aß resulted in a marked decrease of this 60 kDa band and the appearance of a smear band (>100 kDa) probably representing intact glypican-1. Pretreatment of lysates with heparitinase before incubation with Aß recovered the 60 kDa band, suggesting that Aß was unable to bind to heparitinase-treated glypican-1. These results suggest that glypican-1 derived from the human brain can bind to Aß in an HS chain-dependent manner.

2. Aß binding to glypican-1 depends on its aggregation state
It was reported that heparin or mouse EHS HSPG binds fibrillar Aß (fAß) but not non-fibrillar Aß (non-fAß) with high affinity. In the present in vitro analysis of Aß binding to glypican-1, the core protein of glypican-1 could be detected after incubation with non-fibrillar Aß but not after with fibrillar Aß at levels similar to those observed in the untreated sample, suggesting that non-fibrillar Aß had little or no binding ability to glypican-1. Binding of non-fibrillar Aß to HSPGs was not observed on dot blot membranes.

3. Glypican-1 is a major HSPG in DIG domains from human brains
Glypican-1 is a GPI-anchored HSPG; most, if not all, GPI-anchored proteins are localized in special membrane domains called detergent-insoluble, glycosphingolipid-enriched (DIG) domains. We next examined whether glypican-1 is present in the DIG fraction from brain tissues. DIG was recovered in fractions 4–6, into which flotillin (a marker protein of DIG) was exclusively fractionated. Western blot with anti-glypican-1 showed that glypican-1 was mainly present in fraction 5, indicating localization of glypican-1 to the DIG domains. Western blot with 3G10 mAb indicated that HSPGs other than glypican-1 were almost undetectable in the DIG fraction. An insoluble pellet from the DIG fraction was solubilized with 6 M guanidine-HCl and the solubilized sample was used for dot blot Aß binding assay. Aß bound to guanidine soluble samples from the DIG fraction; this binding was significantly inhibited by heparitinase pretreatment, indicating that Aß could bind to DIG-resided glypican-1 in an HS chain-dependent manner.

4. Co-localization of glypican-1 and Aß in DIG fractions from AD brain
Recently, DIG domains or "rafts" have received attention with regard to the pathogenesis of AD, because accumulation of Aß in these domains was demonstrated and appeared to correlate with the extent of Aß deposition in the brain. Thus, it is possible that glypican-1 participates in the process of Aß accumulation by interaction with Aß in such specific microdomains. To clarify this, we examined whether glypican-1 and Aß are co-fractionated in DIG fractions from AD brains. The fractions were analyzed by Western blots with antibodies to human glypican-1, Aß, Aß40, Aß42, or APP. Glypican-1 was recovered mainly in DIG fractions. Full-length APP was fractionated predominantly in the high density fractions, and to a smaller extent in DIG fractions. We observed that significant amounts of Aß40 and Aß42 were present in the DIG fractions as monomers and SDS stable dimers. BAN50, whose epitope is located in Aß1-10, labeled Aß monomers more strongly than Aß dimers, suggesting these SDS stable dimers were formed in a way that the amino-terminal portion of Aß was masked, modified, or deleted.

5. Preferential role of glypican-1 in Aß42 accumulation in DIG domains
Given that Aß binds to glypican-1 and these two proteins were accumulated in DIG domains, there may be a correlation between them. To examine this, we quantified levels of Aß and glypican-1 in the DIG fraction using ELISA. There was a strong correlation between Aß42 and glypican-1 (ctrl; r=0.9517, AD; r=0.8756) whereas no correlation between Aß40 and glypican-1 (ctrl; r=0.3559, AD; r=0.0854) was observed. These results suggest that glypican-1 plays a preferential role in the accumulation of Aß42 in DIG domains.

6. Effect of glypican-1 overexpression on cell viability
To explore the function of glypican-1 other than plaque formation, we generated transfectants that overexpressed glypican-1 in a tetracycline-inducible manner. Western blot analysis showed that the trasnsfectants were induced to express a large amount of glypican-1 protein when cultured with tetracyclin (Fig. 1 A). Time course experiments demonstrated that induction of glypican-1 expression was begun 5 min after addition of tetracyclin and reached maximal levels after 12 h. Cell viability of these cells with or without the induction of glypican-1 was analyzed using WST assay. As shown in Fig. 1 , overexpression of glypican-1 decreased viability of cells coexpressing APP-carrying Swedish mutation of familial Alzheimer’s disease (Fig. 1B c). Neither glypican-1 nor Swedish APP overexpression alone affected cell viability (Fig. 1B a, b). Since the production of Aß in Swedish APP-expressing cells was increased, the observed effect of glypican-1 on cell viability may be due to enhanced Aß toxicity by binding to glypican-1. To examine this, transfectants were cultured with exogenously added Aß for 4 days, then cell viability was measured. The viability of all cells examined was not affected by adding non-fAß40, fAß40, or Aß40-1 even though glypican-1 expression was induced (Fig. 1C a–f, j–l). In contrast, Aß42 significantly reduced cell viability only when glypican-1 was overexpressed (Fig. 1C g, i). These results indicate that cells that overexpress glypican-1 become more susceptible to Aß42 toxicity, resulting in enhanced cell death.

View larger version (42K): [in this window] [in a new window]   Figure 1. Time course of induction of glypican-1 expression in sweAPP-SH cells and effect of overexpression of glypican-1 on cell viability. A) SweAPP-GPC1-SH cells were cultured with tetracyclin and subjected to Western blot. The same membrane was reprobed by anti-APP Aß as an internal protein control. B) a–c: Transfectans were cultured with () or without (•) tetracyclin. C) After incubation in medium with () or without (•) tetracyclin overnight, transfectants were cultured with non-fAß40 (a–c), fAß40 (d–f), Aß42 (g–i), or Aß40-1 (j–l) in the presence or absence of tetracyclin for 4 days. D) After overnight incubation in medium with () or without (•) tetracycline, transfectants were treated with thapsigargin (a–c), tunicamycin (d–f), or brefeldin A (g–i). Cell viability was monitored by WST assay. Assays were performed in triplicate and mean values ± SE at 450 nm were measured. Quantitative values are expressed as % of cell viability in untreated cells.

7. Effect of glypican-1 overexpression on ER stress
It has been reported that ER stress is an important factor in the neuropathology of a wide variety of neurological disorders, including AD. Studies have shown that neurotoxicity elicited by Aß is at least partially mediated by ER, raising the possibility that the ER stress response is influenced by overexpression of glypican-1, which may result in enhanced susceptibility of cells to Aß42 toxicity. We examined the effect of glypican-1 expression on the stress response. Cell death induced by thapsigargin was accelerated when glypican-1 was overexpressed in cells coexpressing Swedish APP (Fig. 1D c). Such an acceleration was not observed in cells that expressed glypican-1 or Swedish APP alone (Fig. 1D a, b). The stress response by tunicamycin and brefeldin A did not alter the cell survival even though cells were coexpressing Swedish APP and glypican-1 (Fig. 1D d–i). These results suggest that glypican-1, together with Aß, makes cells more vulnerable to some but not all stresses.

CONCLUSIONS AND SIGNIFICANCE

Although HSPGs are co-localized in senile plaques and may promote amyloid formation, deposition, and/or persistence by binding to Aß, it remains uncertain how HSPGs are involved in AD pathogenesis and which HSPG has a pathogenic role in AD. The findings here suggest that glypican-1 binds to Aß through HS chains and may be involved in accumulation of Aß in DIG domains and/or the formation of plaques at an initial stage. Glypican-1 may act as a negative factor to neuronal cell survival, probably by binding with Aß. Individuals whose expression levels of glypican-1 are relatively high might have a higher risk of AD. It is necessary to define more precisely the exact role of glypican-1 in these pathological events. A better understanding of normal and pathological functions of glypican-1 may lead to the development of new therapeutic approaches for AD.

View larger version (23K): [in this window] [in a new window]   Figure 2. Proposed roles of glypican-1 in AD. Glypican-1 is involved in Aß accumulation in DIG or raft domains; hence 1) fibril and plaque formations are accelerated by binding to HS chains on glypican-1, and 2) neuronal cell death induced by Aß42 or certain stresses is enhanced when glypican-1 levels are increased.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1040fje;

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