Intracellular or extracellular accumulation, aggregation, and deposition of Aβ in the brain are pathogenic events of AD (Selkoe, 2001
). One of the major pathways for Aβ clearance is its cellular uptake and degradation by neurons, glia, and cells along the brain vasculature (Bu, 2009
). In this study, we focused on investigating individual and cooperative roles of LRP1 and HSPG in neuronal Aβ uptake. Using complementary approaches, we showed that neuronal cell surface HSPGs constitute major Aβ binding sites and are critical for Aβ uptake. Although not required for Aβ binding, LRP1 cooperates with HSPG in neuronal Aβ uptake.
HSPG is composed of a core protein with long polysaccharide chains of repeated heparin sulphate. There are two groups of HSPGs: extracellular matrix HSPGs (e.g. perlecan, agrin, and collagen XVIII) and cell surface HSPGs (syndecan and glypican) (van Horssen et al., 2003
). Both extracellular matrix and cell surface HSPGs were detected by immunohistochemistry in senile plaques, cerebral amyloid angiopathy, and neurofibrillary tangles (van Horssen et al., 2003
). These evidences strongly suggest that HSPGs may interact with Aβ and play some roles in AD pathogenesis. Consistent with this notion, we demonstrated that heparin binds to Aβ (supplemental figure 5A
) and significantly inhibits neuronal Aβ binding and uptake. Heparin binds to several classes of extracellular proteins, including growth factors, matrix proteins, and apolipoproteins (Bergamaschini et al., 2009
). Heparan sulphate was shown to bind to the amino acid 13–16 region (HHQK) of Aβ (Brunden et al., 1993
) and to antagonize the binding of Aβ to HSPG in vitro
(Castillo et al., 1997
). To block heparin binding proteins and/or displace HSPG binding proteins at the cell surface, GT1-7 cells were pretreated with heparin and then incubated with Aβ42 after washing. No significant effect of heparin pretreatment on Aβ42 uptake was detected (supplemental figure 5B
), suggesting that heparin antagonizes the interaction of Aβ to HSPG through its binding to Aβ. In addition, HSPG-deficiency in CHO cells significantly decreased Aβ binding and uptake. Therefore, our findings indicate that Aβ binding to neuronal cell surface HSPG may be the first and critical step for its internalization, degradation, accumulation, and/or toxicity. Aβ binding to cell surface and/or extracellular matrix HSPG may also be important for its aggregation and eventual formation of amyloid plaques.
We showed that heparin suppresses Aβ binding and internalization in neuronal cell lines and mouse primary neurons. Similar findings have been demonstrated in brain vascular smooth muscle cell (Kanekiyo and Bu, 2009
) and in microglia (Giulian et al., 1998
). However, heparin and heparinase treatment did not affect Aβ uptake in brain capillary endothelial cells (Yamada et al., 2008
). Because heparan sulphate biosynthesis differs depending upon tissue/cell types (Kreuger et al., 2006
), the discrepancies in cellular Aβ uptake may result from differences in tissue/cell-specific HSPG structure. Several studies have demonstrated that heparin and heparin sulphate suppress Aβ cellular toxicity (Woods et al., 1995
; Bergamaschini et al., 2002
), likely by inhibiting cellular Aβ binding and/or uptake. Cells internalize and degrade Aβ through the lysosomal pathway (Hu et al., 2009
). We demonstrated that LRP1 and HSPG mediate uptake of Aβ to lysosomes and its subsequent cellular clearance in N2a cells. However, when the capacity is overwhelmed through continuous exposure to high concentrations of extracellular Aβ, intracellular accumulation and aggregation of Aβ may be induced, leading to cellular toxicity. We recently showed that enhancement of Aβ uptake through LRP1 leads to eventual lysosomal accumulation and slightly decreased cell viability when cells were exposed to high Aβ concentrations and long periods of incubation (Fuentealba et al., 2010
). Furthermore, the remnants of these aggregates after cell death may induce extracellular amyloid plaque formation through its seeding effect (Hu et al., 2009
). In this view, heparin and heparin-like compounds may be promising candidates for AD therapy because of their competitive function against HSPGs. Bergamaschini et al.
demonstrated that the effect of long-term, peripheral treatment with a low-molecular-weight heparin significantly decreased Aβ concentration and deposition in the brain of APP transgenic mice (Bergamaschini et al., 2004
LRP1 plays critical roles in cellular Aβ uptake in the brain. Harris-White et al.
showed that TGFβ2-mediated intraneuronal accumulation of brain-injected Aβ depends on LRP1 function (Harris-White et al., 2004
). We showed that the internalization of Aβ was also decreased in LRP1-deficient neuronal cells and increased in LRP1-overexpressing neuronal cells. LRP1 expression restored Aβ uptake in LRP1-knocked down GT1-7 cells. We also demonstrated that treatment with a neutralizing antibody to LRP1 decreased Aβ uptake in mouse primary cortical neuron. In addition, LRP1 enhanced the Aβ endocytosis rate in N2a cells. Importantly, LRP1-mediated Aβ uptake was also inhibited by heparin, indicating that the function of LRP1 in cellular Aβ uptake depends on HSPG. In contrast to HSPG, we showed that neither LRP1 knockdown nor overexpression affects Aβ binding. These results can be explained by two possibilities. First, HSPG may function as a co-receptor for LRP1. Previous work has shown that LRP1 forms a complex with HSPG (Wilsie and Orlando, 2003
). Aβ may initially bind to the HSPG sites on the surface of the complex and then may undergo endocytosis via LRP1, in a process analogous to another LRP1 ligand, coagulation factor VIII (Sarafanov et al., 2001
). We have shown that inhibition of LRP1 endocytosis decreases cellular Aβ uptake in N2a cells (Fuentealba et al., 2010
). LRP1 is a large endocytotic receptor that recognizes more than 30 ligands including lipoproteins, proteinases, proteinase inhibitor complexes, extracellular matrix proteins, bacterial toxins, viruses, and various intracellular proteins (Herz and Strickland, 2001
; Bu, 2009
). While the ligand-binding sites on LRP1 localize primarily on domains II and IV (Herz and Strickland, 2001
), some LRP1 ligands seem to require co-receptors for their interaction (Nykjaer et al., 1992
; Sarafanov et al., 2001
). Aβ was shown to bind directly to domains II and IV of LRP1 (Sagare et al., 2007
); however, another study did not support direct Aβ binding to LRP1 (Yamada et al., 2008
). Our results suggest that HSPG may be one of the candidate co-receptors of LRP1 for Aβ binding and uptake.
We showed that Aβ had a slower rate of endocytosis than RAP in N2a-mLRP4 cells, indicating that LRP1 mediates Aβ endocytosis through different mechanisms from its other ligands. Therefore, the other possibility is that HSPG, which is abundantly expressed in neurons, may capture Aβ on the cell surface and subsequently internalize Aβ through an LRP1-regulated signaling pathway. Increasing evidence also shows that LRP1 functions as a signal-transducing receptor (Herz and Strickland, 2001
) and that LRP1 plays a critical role in neuronal viability by regulating key survival signalling pathways (Fuentealba et al., 2009
). In addition, LRP1 regulates cell migration (Song et al., 2009
) and the integrity of the blood-brain barrier (Yepes et al., 2003
). Aβ binding to HSPG may activate LRP1 signaling pathways, resulting in an enhancement of cellular Aβ uptake. Consistent with a LRP1-mediated signaling function, actin polymerization, which is critical for macropinocytosis and Aβ uptake (Mandrekar et al., 2009
), is impaired in LRP1-deficient cells (Zhou et al., 2009
). This evidence suggests that LRP1 may mediate Aβ cellular uptake by regulating macropinocytosis.
In summary, we have demonstrated that HSPG and LRP1 function cooperatively in neuronal Aβ binding and uptake. We show that HSPG mediates Aβ binding on the cell surface, and that both HSPG and LRP1 play important roles in Aβ uptake. Our findings provide novel insights into the molecular mechanisms of AD pathogenesis. Further studies of the HSPG/LRP1-mediated Aβ pathways may lead to novel therapeutic strategies to treat AD.