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Anhydromannose (anMan)-containing heparan sulfate (HS) derived from the proteoglycan glypican-1 (Gpc-1) is generated in endosomes by an endogenously or ascorbate induced SNO-catalyzed reaction. Processing of the amyloid precursor protein (APP) and APP-like protein 2 (APLP2) by β- and γ-secretases into amyloid beta (Aβ) and Aβ-like peptides also takes place in these compartments. Moreover, anMan-containing HS suppresses the formation of toxic Aβ assemblies in vitro. We show by using deconvolution immunofluorescence microscopy with an anMan-specific monoclonal antibody as well as 35S-labeling experiment that expression of APP/APLP2 is required for ascorbate-induced transport of HS from endosomes to the nucleus. Nuclear translocation was observed in wild-type mouse embryonic fibroblasts (Wt-MEF), Tg2576 MEF and N2a neuroblastoma cells but not in APP-/- and APLP2-/- MEF. Transfection of APP-/- cells with a vector encoding APP restored nuclear import of anMan-containing HS. In Wt-MEF and N2a neuroblastoma cells exposed to β- or γ-secretase inhibitors, nuclear translocation was greatly impeded, suggesting involvement of APP/APLP2 degradation products. In Tg2576 MEF, the β-inhibitor blocked transport but the γ- inhibitor did not. During chase in ascorbate-free medium, anMan-containing HS disappeared from the nuclei of Wt-MEF. Confocal immunofluorescence microscopy showed that they appeared in acidic, LC3-positive vesicles in keeping with an autophagosomal location. There was increased accumulation of anMan-containing HS in nuclei and cytosolic vesicles upon treatment with chloroquine indicating that HS was degraded in lysosomes. Manipulations of APP expression and processing may have deleterious effects upon HS function in the nucleus.

Amyloid beta 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-SNO. We have investigated whether these oligosaccharides interact with amyolid beta 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 amyolid beta oligomers detected by the anti-amyloid beta A11 antibody, colocalized with anMan-immunoreactivity in Tg2576 fibroblasts. A 50-55-kDa anionic, SDS-stable, anMan- and amyloid beta-immunoreactive species was obtained from Tg2576 fibroblasts using immunoprecipitation with anti-APP (C-terminal). anMan-Containing HS oligo- and disaccharide preparations modulated or suppressed A11-immunoreactivity and oligomerization of amyloid beta 42 peptide in an in vitro assay. A11 immunoreactivity increased in Tg2576 fibroblasts when Gpc-1 autoprocessing was inhibited by 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 amyolid beta domain and small, anMan-containing oligosaccharides may preclude formation of toxic amyloid beta oligomers. A portion of the oligosaccharides co-secrete with the amyloid beta peptides and are deposited in plaques. These results support the notion that inadequate supply of vitamin C could contribute to late onset AD in humans.