Megalin interacts with APP and the intracellular adapter protein FE65 in neurons

Abstract

Increasing evidence has implicated megalin, a low-density lipoprotein receptor-related protein, in the pathogenesis of Alzheimer's disease (AD). In the brain, megalin is expressed in brain capillaries, ependymal cells and choroid plexus, where it participates in the clearance of brain amyloid β-peptide (Aβ) complex. Recently, megalin has also been detected in oligodendrocytes and astrocytes. In this study we demonstrate that megalin is widely distributed in neurons throughout the brain. Additionally, given that FE65 mediates the interaction between the low density lipoprotein receptor-related protein-1 and the amyloid precursor protein (APP) to modulate the rate of APP internalization from the cell surface, we hypothesize that megalin could also interact with APP in neurons. Our results confirm that megalin interacts with APP and FE65, suggesting that these three proteins form a tripartite complex. Moreover, our findings imply that megalin may participate in neurite branching. Taken together, these results indicate that megalin has an important role in Aβ-mediated neurotoxicity, and therefore may be involved in the neurodegenerative processes that occur in AD.

Introduction

Megalin, also known as low density lipoprotein-related protein-2 (LRP2) and glycoprotein 330, is the largest member of the low-density lipoprotein receptor (LDLR) family. Megalin expression was first detected in various absorptive epithelial cells throughout the body, such as the kidney and the intestinal brush border. In the central nervous system, megalin was initially described in brain capillaries, ependymal lining of the ventricular walls and choroid plexus (Chun et al., 1999, Kounnas et al., 1994, Zheng et al., 1994), and later in neural progenitors in embryonic mouse spinal cord (Wicher et al., 2005), and postnatal mouse spinal cord oligodendrocytes (Wicher et al., 2006). More recently, megalin has been described in cultured astrocytes (Bento-Abreu et al., 2008) and neurons (Ambjørn et al., 2008, Chung et al., 2008, Fleming et al., 2009).

Megalin binds and internalizes a number of ligands that represent a wide variety of molecules, including lipoproteins, hormones, vitamin-binding proteins, drugs, and immunorelated proteins (Hjälm et al., 1996, Moestrup & Verroust, 2001). Since many are involved in neuroprotection, they are considered potential new targets in AD research (Birn, 2006, Carro et al., 2005, Christensen & Birn, 2002, Dietrich et al., 2008, Moestrup & Verroust, 2001).

In the brain, megalin participates in endocytosis and transport of amyloid β-peptide (Aβ) complex across the blood–cerebrospinal fluid barrier at the choroid plexus (Carro et al., 2005, Zlokovic et al., 1996), and the blood-brain barrier (Deane et al., 2004, Hammad et al., 1997, Zlokovic et al., 1996). This process involves the interaction of megalin's cytoplasmic tail domain with several adaptor and scaffold proteins (Biemesderfer, 2006, May et al., 2003), including Dab2, MAGI-I, GIPC, ANKRA, MegBP, and ARH (Bonifacino & Traub, 2003, Jaeger & Pietrzik, 2008). The adaptor protein FE65 has been shown to mediate the interaction between another member of the LDLR superfamily, the LRP1, and the amyloid precursor protein (APP) (Cam & Bu, 2006, Pietrzik et al., 2004, Yoon et al., 2005).

Due to the growing evidence that megalin expression in the central nervous system is not restricted to tight-junction epithelia, but is also expressed in neurons, the objective of our study was to assess the expression of megalin in different subpopulations of neurons, explore the interaction between megalin, APP and FE65, and discuss its possible functional role in the central nervous system.