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To whom correspondence may be addressed: Dept. of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, BMC A13, SE-221 84 Lund, Sweden. Tel.: 46-46-222-8573; Fax: 46-46-222-0615.
To whom correspondence may be addressed: Dept. of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, BMC A13, SE-221 84 Lund, Sweden. Tel.: 46-46-222-4077; Fax: 46-46-222-0615.
* This work was supported by grants from the Swedish Science Council (VR-M); the Bergvall, Crafoord, Hedborg, Kock, Segerfalk, Zoega, and Österlund Foundations; and the Medical Faculty of Lund University (to L.-Å. F. and K. M.) and in part by the National Health and Medical Research Council of Australia (to R. C.) and the Deutsche Forschungsgemeinschaft (to G. M.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3 and Table 1.
Amyloid β (Aβ) is generated from the copper- and heparan sulfate (HS)-binding amyloid precursor protein (APP) by proteolytic processing. APP supports S-nitrosylation of the HS proteoglycan glypican-1 (Gpc-1). In the presence of ascorbate, there is NO-catalyzed release of anhydromannose (anMan)-containing oligosaccharides from Gpc-1-nitrosothiol. We investigated whether these oligosaccharides interact with Aβ during APP processing and plaque formation. anMan immunoreactivity was detected in amyloid plaques of Alzheimer (AD) and APP transgenic (Tg2576) mouse brains by immunofluorescence microscopy. APP/APP degradation products detected by antibodies to the C terminus of APP, but not Aβ oligomers detected by the anti-Aβ A11 antibody, colocalized with anMan immunoreactivity in Tg2576 fibroblasts. A 50–55-kDa anionic, sodium dodecyl sulfate-stable, anMan- and Aβ-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C terminus). anMan-containing HS oligo- and disaccharide preparations modulated or suppressed A11 immunoreactivity and oligomerization of Aβ42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 3-β[2(diethylamino)ethoxy]androst-5-en-17-one (U18666A) and decreased when Gpc-1 autoprocessing was stimulated by ascorbate. Neither overexpression of Gpc-1 in Tg2576 fibroblasts nor addition of copper ion and NO donor to hippocampal slices from 3xTg-AD mice affected A11 immunoreactivity levels. However, A11 immunoreactivity was greatly suppressed by the subsequent addition of ascorbate. We speculate that temporary interaction between the Aβ domain and small, anMan-containing oligosaccharides may preclude formation of toxic Aβ oligomers. A portion of the oligosaccharides are co-secreted with the Aβ peptides and deposited in plaques. These results support the notion that an inadequate supply of vitamin C could contribute to late onset AD in humans.
peptides that have a central role in Alzheimer disease (AD) are derived from the amyloid precursor protein (APP). APP is a copper- and heparan sulfate (HS)-binding membrane protein, which is expressed in many cell types, including neural cells and fibroblasts (
). Processing of APP involves several proteases and regulatory proteins, collectively designated α-, β-, and γ-secretases (supplemental Fig. S1, a–c). Single α- or β-cleavages result in the release of the large ectodomain, whereas the C-terminal fragments (APP-CTF-α and APP-CTF-β, respectively) remain tethered to the membrane. Combined β- and γ-cleavages are amyloidogenic and lead to release of the C-terminal cytoplasmic domain of APP and the generation of Aβ peptides, mostly Aβ40 and Aβ42. Aβ peptides first form soluble oligomers and then insoluble aggregates that accumulate as amyloid fibrils and senile plaques in the brain of AD patients. Soluble Aβ oligomers formed at early stages of AD are believed to be particularly toxic and responsible for early memory failure (
The role of glycosaminoglycans, such as HS, in APP processing and amyloid formation is not fully understood. Although intact HS chains can change protein conformations into amyloidogenic forms, small HS oligosaccharides are without effect (
). The origin of amyloid-associated HS is unclear, but some of the HS co-deposited with extracellular Aβ in both sporadic and familial AD as well as in Tg2576 mice can be derived from the HS proteoglycans glypican-1 (Gpc-1) and syndecan-3 produced by glial cells (
). Cleavage of the HS chains occurs at the relatively rare N-unsubstituted glucosamine (GlcNH3+) sites. During cleavage, GlcNH3+ is converted to anhydromannose (anMan), which becomes the reducing terminal sugar of the released HS degradation products. These products accumulate in Rab7-positive late endosomes (
The reducing terminal anMan residue in the released HS degradation products contains a free aldehyde that can form an unstable aldimine bond to amino groups in proteins, i.e. a Schiff base. A stable covalent bond could then be formed by reduction or rearrangement (supplemental Fig. S1e and Refs.
), we decided to investigate whether anMan-containing HS degradation products generated by Gpc-1 autoprocessing interact with APP degradation products and whether such HS is ultimately deposited in amyloid plaques. For this purpose, we examined normal human and AD brains as well as brains and/or fibroblasts from wild-type, Tg2576, and 3xTg-AD mice for anMan- and Aβ-immunoreactive components. We show here that anMan immunoreactivity is present in amyloid plaques from human AD and Tg2576 mouse brains. In extracts of fibroblasts from Tg2576 mice, we found that anMan immunoreactivity co-precipitated with APP-CTF-β, yielding a 50–55-kDa, Aβ(4G8)-immunoreactive, sodium dodecyl sulfate (SDS)-stable species. After radiolabeling with 35SO4, an anionic pool comprising both [35S]HS and 70–75-kDa Aβ(4G8)-immunoreactive species was obtained. The addition of anMan-containing HS oligo- or disaccharides to Aβ42 peptide monomers modulated or suppressed the transient appearance of A11 immunoreactivity and inhibited Aβ42 oligomerization. Aβ A11 immunoreactivity in Tg2576 fibroblasts increased when NO-dependent cleavage of HS in Gpc-1 was suppressed. Conversely, when such cleavage was initiated by ascorbate in copper- and NO-supplemented Tg2576 fibroblasts or hippocampal slices from 3xTg-AD mice, A11 immunoreactivity was nearly eliminated.
APP is a precursor to the amyloidogenic Aβ peptides in AD, but the normal function of APP and its degradation products remains poorly understood. The N-terminal ectodomain is growth factor-like and has a neurotrophic role, whereas the cytoplasmic C terminus participates in cell adhesion and gene regulation (for recent reviews, see Refs.
). Moreover, Aβ can efficiently generate reactive oxygen species in the presence of transition metals, such as copper, and form e.g. carbonyl groups in amino acid side chains or stable dityrosine-cross-linked dimers (
). If the generated APP-CTF-β fragments remain oligomeric, this may facilitate Aβ peptide oligomerization upon subsequent cleavage by γ-secretase (Fig. 9a).
Tg2576 fibroblasts express a mutant APP that is especially sensitive to β-secretase cleavage, resulting in increased production of APP-CTF-β compared with wild-type cells. Here, we show that this is accompanied by increased formation of anMan-containing HS degradation products that colocalize with APP-CTF-β. We also show that anMan-containing HS oligosaccharides and especially disaccharides modulate/suppress Aβ A11 immunoreactivity and Aβ oligomerization in vitro. As the NO-sensitive GlcNH3+ moieties are often clustered in Gpc-1 HS (
), anMan-containing di- and tetrasaccharides should be prominent products when NO-catalyzed HS degradation is stimulated by ascorbate in vivo. Moreover, as Gpc-1 colocalizes with APP, a high local concentration of anMan-containing HS oligosaccharides may be generated.
The reaction Aβ + HS-Di ↔ Aβ·HS-Di (where Di represents disaccharide), which probably involves aldimine formation, should have an equilibrium far to the left. Therefore, a large excess of HS disaccharide may be required to generate sufficient complex formation. In cultured Tg2576 fibroblasts, some of the HS·CTF-β complexes appear to be converted to 50–55-kDa, negatively charged, SDS-stable, oligomeric HS-CTF-β conjugates. anMan-Aβ aldimines may be stabilized by reduction in late endosomes. Alternatively, prolonged exposure of Aβ to an excess of anMan-containing saccharides can result in the rearrangement of the aldimines, generating a form of protein glycation.
A portion of newly synthesized HS in Tg2576 cells co-chromatographed with a 70–75-kDa Aβ-immunoreactive species, suggesting that resident 50–55-kDa HS-CTF-β conjugates were derived from a larger precursor possibly with longer N-acetylated glucosamine-containing HS oligosaccharide adducts. Endoheparanase and exoglycosidases may then degrade these oligosaccharides to the shorter, possibly highly sulfated stubs that were resistant to degradation by the bacterial HS lyase.
An APP-CTF-β tetramer should have an molecular mass of approximately 40 kDa. If the 50–55-kDa component is a tetrameric HS-APP-CTF-β conjugate, the HS adduct should amount to approximately 2.5–3.8 kDa in each Aβ domain. As there are at least three amino groups in Aβ that could be used as binding sites for anMan-containing HS oligosaccharides, each adduct may be very small (supplemental Fig. S1e).
Tg2576 fibroblasts displayed A11 immunoreactivity, indicating the presence of potentially toxic Aβ oligomers. However, the A11-positive material appeared to be mainly located separately from the anMan immunoreactivity. In general, accumulation of misfolded proteins is accompanied by increased formation of anMan-containing HS oligosaccharides as observed previously in tumor cells (
) and now in hippocampal slices from 3xTg-AD mice (Fig. 8). In scrapie-infected neural cells, the anMan-positive HS degradation products co-immunoprecipitated and co-migrated with the scrapie prions, suggesting that anMan-containing HS oligosaccharides were covalently bound.
U18666A treatment markedly increased A11 immunoreactivity in Tg2576 fibroblasts. U18666A impedes transport from early to late endosomes and suppresses formation of anMan-containing HS oligosaccharides from Gpc-1 in normal fibroblasts and in T24 carcinoma and N2a neuroblastoma cell lines (
). However, in Tg2576 fibroblasts, suppression of HS degradation was less pronounced. It is possible that Gpc-1 autoprocessing is maximally stimulated in Tg2576 cells and that HS-depleted HS-CTF-β conjugates accumulate in early endosomes of drug-treated cells. Excessive APP processing in combination with inadequate generation of HS degradation products may result in accumulation of oligomers selectively recognized by antibody A11 (Fig. 9, a and b).
Ascorbate releases NO from Gpc-1-SNO, which results in deaminative degradation of HS, generating more anMan-containing HS oligosaccharides. As shown here, treatment with ascorbate suppresses A11 immunoreactivity. This was demonstrated in vitro with untreated as well as U18666A-treated Tg2576 fibroblasts, with GFP-Gpc-1-transfected fibroblasts, and by ex vivo incubation of slices of hippocampus from 3xTg-AD mice. The effect of ascorbate was greatly potentiated by supplementation with Cu(II) ion and NO donor. A11 immunoreactivity that was almost completely eliminated in Tg2576 fibroblasts reappeared when ascorbate was withdrawn. This indicates that the A11-positive conformation is reversible. There may also be a rapid turnover of A11-positive higher oligomers, and fewer A11-positive smaller oligomers should be formed when the generation of anMan-containing HS degradation products is stimulated by ascorbate.
We speculate that reversible and temporary interaction of APP-CTF-β with anMan-containing HS oligosaccharides modulates the conformation of the Aβ portion of APP-CTF-β. This may serve to maintain the Aβ domain in a non-toxic conformation after cleavage by γ-secretase (Fig. 9c). HS·Aβ complexes (temporary or permanent) are negatively supercharged, which may also limit oligomer/aggregate formation and confer resistance to proteolytic degradation. In AD, there may be insufficient formation of HS oligosaccharides to meet the needs, resulting in secretion of partially conjugated Aβ oligomers as indicated by the presence of anMan-containing HS in AD plaques.
Other studies indirectly support a role for Gpc-1 in APP processing and Aβ clearance. It was recently reported that removal of NO synthase 2 in APP transgenic mice resulted in a greater spectrum of Aβ-like pathologies (
). As NO is required for S-nitrosylation of Gpc-1, NO-catalyzed deaminative cleavage of the HS chains in Gpc-1 and subsequent formation of HS oligosaccharides would be diminished. Moreover, formation of anMan-containing HS oligosaccharides is markedly reduced when pre-endosomal cholesterol traffic is slow or blocked as in Niemann-Pick type C disease (
) are examples of proteins that can affect early steps in the trafficking of APP. Thus, Gpc-1 is yet another protein involved in regulating APP processing but that is downstream of β-cleavage.
Finally, it is remarkable that it takes three different mutations to generate AD-like pathology in mice, whereas humans without mutations in any of these genes can spontaneously develop AD. There are indeed many differences between mice and men, but one is that mice can synthesize ascorbate, whereas humans cannot. It is well documented that ascorbate is important for neuroprotection, and certain neurons may contain up to 10 mm ascorbate (for reviews, see Refs.
). The present study supports the possibility that intracellular copper dysregulation, failing NO production, and/or an inadequate supply of vitamin C could contribute to late onset AD in humans.
We thank Prof. Gunnar Pejler, Uppsala University, and Prof. Peter Påhlsson, Linköping University, for generous gifts of monoclonal antibodies. We also thank Dr. Håkan Toresson, Lund University, for help with transfer of 3xTg-AD mice and Sol Da Rocha Baez for excellent technical assistance.