Elsevier

Experimental Neurology

Volume 225, Issue 1, September 2010, Pages 85-93
Experimental Neurology

The role of low-density receptor-related protein 1 (LRP1) as a competitive substrate of the amyloid precursor protein (APP) for BACE1

https://doi.org/10.1016/j.expneurol.2010.05.017Get rights and content

Abstract

Cleavage of APP by BACE1 is the first proteolytic step in the production of amyloid-beta (Aβ), which accumulates in senile plaques in Alzheimer's disease. Through its interaction with APP, the low-density receptor-related protein 1 (LRP1) enhances APP internalization. Recently, BACE1 has been shown to interact with and cleave the light chain (lc) of LRP1. Since LRP1 is known to compete with APP for cleavage by gamma-secretase, we tested the hypothesis that LRP1 also acts as a competitive substrate for β-secretase. We found that the increase in secreted APP (sAPP) mediated by over-expression of BACE1 in APP-transfected cells could be decreased by simultaneous LRP1 over-expression. Analysis by multi-spot ELISA revealed that this is due to a decrease in sAPPβ, but not sAPPα. Interaction between APP and BACE1, as measured by immunoprecipitation and fluorescence lifetime assays, was impaired by LRP1 over-expression. We also demonstrate that APP over-expression leads to decreased LRP1 association with and cleavage by BACE1. In conclusion, our data suggest that – in addition to its role in APP trafficking – LRP1 affects APP processing by competing for cleavage by BACE1.

Introduction

Amyloid-β (Aβ) is the major component of senile plaques, one of the histopathological hallmarks in Alzheimer's disease. Cleavage of the amyloid-β precursor protein (APP) by the type I transmembrane aspartyl protease β-site APP cleaving enzyme (BACE1) is the first proteolytic step in the production of Aβ (Hussain et al., 1999, Sinha et al., 1999, Vassar et al., 1999, Yan et al., 1999). Besides APP, other BACE1 substrates that have been identified include the APP homologues APLP1 and APLP2 (Li and Sudhof, 2004), a membrane-bound sialyltransferase (Kitazume et al., 2001), P-selectin glycoprotein ligand-1 (Lichtenthaler et al., 2003), neuregulin1 type III isoform (Willem et al., 2006), the β-subunits of voltage gated sodium channels (Wong et al., 2005) and the low-density lipoprotein receptor-related protein 1 (LRP1) (von Arnim et al., 2005).

LRP1 is a roughly 600 kDa type I integral membrane protein and three of its key ligands (apoE, α2-macroglobulin, and APP) are genetically associated with Alzheimer disease and are found in senile plaques. Within cells LRP1 is found on the cell surface and cycles between the cell membrane and endosomes. LRP1 mediates the binding and clearance of Aβ complexes bound to apoE or α2-macroglobulin in cultured cells and in the brain. LRP1 may also play a crucial role in brain efflux of Aβ isoforms at the blood–brain barrier. However, in addition to its role in Aβ clearance, LRP1 interacts directly with APP in two ways. First, KPI-containing isoforms of APP are among the ligands for the extracellular binding sites on LRP1's 515 kDa N-terminal domain (Herz and Strickland, 2001, Kounnas et al., 1995, Li et al., 2001). Second, the short cytoplasmic tail of LRP1's 85 kDa C-terminal domain contains two intracellular NPXY sites which, through the adaptor protein FE65, bind APP. This binding event leads to increased endocytosis of APP from the cell surface, (Kinoshita et al., 2001, Kounnas et al., 1995, Pietrzik et al., 2002, Rebeck et al., 2001, Trommsdorff et al., 1998, Ulery et al., 2000), which is important since BACE1 cleavage of APP occurs more or less exclusively within intracellular compartments (Kinoshita et al., 2003a, Kinoshita et al., 2003b, Koo and Squazzo, 1994).

LRP1 undergoes proteolysis in an interesting pattern that parallels APP in some ways. It undergoes γ-cleavage resulting in the release of the LRP1 intracellular domain (May et al., 2002). In doing so, LRP1 has been shown to compete with APP for this presenilin 1-dependent γ-secretase activity (Lleo et al., 2005). Ectodomain shedding of LRP1 (Quinn et al., 1999) and cleavage of LRP1 by matrix metalloproteases have also been reported (Rozanov et al., 2004, Higashi and Miyazaki, 2003). Furthermore – and as with APP – β-secretase cleavage of LRP1 leads to release of a secreted LRP1 (sLRP) domain (von Arnim et al., 2005). sLRP normally circulates in plasma (Quinn et al., 1997) and can bind Aβ (Sagare et al., 2007) leading to its clearance from the brain.

Given that both APP and LRP1 are β-secretase substrates, we asked whether they compete to be cleaved by this protease. Since BACE1 has been postulated to be the “rate-limiting” step for APP cleavage, raising the possibility of pharmacologic down-regulation of β-secretase as a treatment for AD, the question of how BACE1 activity is modulated endogenously is of particular interest. We have shown previously that BACE1 and LRP1 interact and that LRP1 is a BACE1 substrate. In this study, we analyzed the impact of LRP1 co-expression on APP cleavage by BACE1 and, vice versa, the impact of APP co-expression on LRP1 cleavage by BACE1 and found that LRP1 and APP can compete for BACE1 cleavage.

Section snippets

Antibodies

As displayed by corresponding numbers in Fig. 1, in Western blots LRP1 constructs were detected using (1) anti-c-Myc (9E10) (Mouse/M4439/Sigma). APP was detected by (2) anti-HA (Rabbit/H6908/Sigma), (3) anti-APP-N-terminal (22C11) (Mouse/MAB348/Millipore), (4) anti-APP-C-terminal (Rabbit/A8717/Sigma) and (5) anti-β-amyloid (6E10) (Mouse/SIG39320/Covance). BACE1 was detected by (7) Anti-GFP-N-terminal (Rabbit/G1544/Sigma). In immunocytochemistry, LRP1 constructs were detected by (8) anti-c-myc

LRP1 co-expression decreases sAPPβ secretion

To analyze the effect of LRP1 over-expression on processing of APP by BACE1, we used the secreted alkaline phosphatase assay (SEAP). Production of sAPP was measured after cells were transfected either with β-galactosidase (β-gal) + HA-SEAP-APP695 + pEGFP-N3 + pcDNA3.1-myc (Fig. 2, lane 2), β-gal + HA-SEAP-APP695 + BACE1-GFP + pcDNA3.1-myc (Fig. 2, lane 3) or β-gal + HA-SEAP-APP695 + BACE1-GFP + LC-LRP-myc (Fig. 2, lane 4) and measured against empty N2A cells as control (Fig. 2, lane 1).

Discussion

Since the identification of BACE1 as the APP β-secretase, multiple additional BACE1 substrates have been identified. However, it is not clear whether cleavage of APP and of non-APP BACE1 substrates is performed by the same β-secretase molecules in the same compartments and may therefore be subject to competitive enzyme kinetics. We here investigate these questions with regard to the BACE1 substrate LRP1.

When APP, BACE, and LRP1 light chain (LC) were coexpressed and sAPP was measured in

Acknowledgments

This work was supported by a grant from Hirnliga e.V. and Heidelberger Akademie der Wissenschaften to C.v.A.

References (38)

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