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Mediates Neuronal Abeta42 Uptake and Lysosomal Trafficking Rodrigo A Washington University School of Medicine Digital Commons@Becker Open Access Publications 2010 Low-density lipoprotein receptor-related protein 1 (LRP1) mediates neuronal Abeta42 uptake and lysosomal trafficking Rodrigo A. Fuentealba Washington University School of Medicine in St. Louis Qiang Liu Washington University School of Medicine in St. Louis Juan Zhang Washington University School of Medicine in St. Louis Takahisa Kanekiyo Washington University School of Medicine in St. Louis Xiaoyan Hu Washington University School of Medicine in St. Louis See next page for additional authors Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs Part of the Medicine and Health Sciences Commons Recommended Citation Fuentealba, Rodrigo A.; Liu, Qiang; Zhang, Juan; Kanekiyo, Takahisa; Hu, Xiaoyan; Lee, Jin-Moo; LaDu, Mary Jo; and Bu, Guojun, ,"Low-density lipoprotein receptor-related protein 1 (LRP1) mediates neuronal Abeta42 uptake and lysosomal trafficking." PLoS One.,. e11884. (2010). https://digitalcommons.wustl.edu/open_access_pubs/698 This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker. For more information, please contact [email protected]. Authors Rodrigo A. Fuentealba, Qiang Liu, Juan Zhang, Takahisa Kanekiyo, Xiaoyan Hu, Jin-Moo Lee, Mary Jo LaDu, and Guojun Bu This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/open_access_pubs/698 Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Mediates Neuronal Ab42 Uptake and Lysosomal Trafficking Rodrigo A. Fuentealba1,2., Qiang Liu1., Juan Zhang1, Takahisa Kanekiyo1, Xiaoyan Hu2, Jin-Moo Lee2, Mary Jo LaDu3, Guojun Bu1,4* 1 Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2 Department of Neurology, Washington University School of Medicine, St. Louis, Missouri, United States of America, 3 Department of Anatomy and Cell Biology, University of Illinois at Chicago, Chicago, Illinois, United States of America, 4 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, United States of America Abstract Background: Alzheimer’s disease (AD) is characterized by the presence of early intraneuronal deposits of amyloid-b 42 (Ab42) that precede extracellular amyloid deposition in vulnerable brain regions. It has been hypothesized that endosomal/ lysosomal dysfunction might be associated with the pathological accumulation of intracellular Ab42 in the brain. Our previous findings suggest that the LDL receptor-related protein 1 (LRP1), a major receptor for apolipoprotein E, facilitates intraneuronal Ab42 accumulation in mouse brain. However, direct evidence of neuronal endocytosis of Ab42 through LRP1 is lacking. Methodology/Principal Findings: Here we show that LRP1 endocytic function is required for neuronal Ab42 uptake. Overexpression of a functional LRP1 minireceptor, mLRP4, increases Ab42 uptake and accumulation in neuronal lysosomes. Conversely, knockdown of LRP1 expression significantly decreases neuronal Ab42 uptake. Disruptions of LRP1 endocytic function by either clathrin knockdown or by removal of its cytoplasmic tail decreased both uptake and accumulation of Ab42 in neurons. Finally, we show that LRP1-mediated neuronal accumulation of Ab42 is associated with increased cellular toxicity. Conclusions/Significance: These results demonstrate that LRP1 endocytic function plays an important role in the uptake and accumulation of Ab42 in neuronal lysosomes. These findings emphasize the central function of LRP1 in neuronal Ab metabolism. Citation: Fuentealba RA, Liu Q, Zhang J, Kanekiyo T, Hu X, et al. (2010) Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Mediates Neuronal Ab42 Uptake and Lysosomal Trafficking. PLoS ONE 5(7): e11884. doi:10.1371/journal.pone.0011884 Editor: Cheng-Xin Gong, New York State Institute for Basic Research, United States of America Received March 19, 2010; Accepted July 6, 2010; Published July 29, 2010 Copyright: ß 2010 Fuentealba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Institutes of Health (NIH) grants R01AG027924 (to G.B.), R01AG19121 (to M.J.L.) and P01AG021184 (to G.B. and M.J.L.), Zenith Fellows Awards from the Alzheimer’s Association (to G.B. and M.J.L.), and the NIH Neuroscience Blueprint Interdisciplinary Center Core Grant P30NS057105 (to Washington University). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction synaptotoxicity in Ab42-containing neurons [4,5,6]. Since the endosomal/lysosomal pathway plays an important role in Alzheimer’s disease (AD) is a neurodegenerative disorder of the mediating intracellular degradation of Ab, an impairment of this central nervous system characterized by a progressive decline in pathway has been suggested as a possible mechanism for increased cognitive functions and neuronal loss. AD was originally attributed neuronal Ab42 load within multivesicular bodies, especially in the to the accumulation of extracellular amyloid and intraneuronal tau postsynaptic compartments [3,7,8]. The molecular mechanisms deposition [1]; however, mounting evidence indicates that that lead to Ab42 accumulation during AD are not clear. Recent intracellular accumulation of amyloid-b 42 (Ab42) is an early evidence indicates that autophagy, an upstream branch of the pathological marker that precedes neuronal cell death and endosomal/lysosomal cascade, is impaired in AD brains and correlates with cognitive decline. Significant intraneuronal Ab42 might be responsible for intraneuronal Ab42 accumulation [9]. In is found in neuronal cell bodies of both AD patients and patients addition to intraneuronal Ab production, Ab42 internalized from with mild cognitive impairment, a preclinical condition that extracellular space might represent a possible mechanism that precedes AD [2,3]. Animal models for AD corroborate the contributes to intracellular Ab42 accumulation [10,11,12,13,14]. presence of intraneuronal Ab42 prior to the appearance of Therefore, understanding the mechanisms of Ab42 internalization amyloid plaques. Functionally, intraneuronal Ab42 load is is crucial for defining the neuropathological process of AD and for associated with decreased long-term potentiation and increased designing novel methods of AD therapy. PLoS ONE | www.plosone.org 1 July 2010 | Volume 5 | Issue 7 | e11884 Ab42 Uptake through LRP1 Apolipoprotein E (apoE) secreted by glia in the brain is required synthesis, we analyzed cell surface levels of mLRP4 by FACS in for the internalization of lipoproteins by neuronal apoE receptors non-permeabilized cells. Approximately 80% of the cells showed and plays important roles in brain lipid transport and neuronal expression of cell surface mLRP4 (Fig. 1B). To assess whether cell repair [15]. It has been suggested that apoE regulates Ab42 surface mLRP4 could interact and internalize ligands, we accumulation in neurons during AD. Strong co-immunostaining of performed uptake experiments with fluorescently labeled RAP. intracellular apoE and Ab42 was evident in APP transgenic mice RAP is an LRP1 chaperone of 39-kDa that binds LRP1 with high [16]. The e4 allele of the APOE gene represents the most prevalent affinity and is commonly used as a model extracellular ligand genetic risk factor for late-onset AD [17,18,19], and apoE4 causes for LRP1 [23,36]. Cell-associated RAP was primarily detected lysosomal leakage when applied exogenously to cultured cells intracellularly, as demonstrated by confocal microscopy [20,21]. Furthermore, apoE4 targeted-replacement mice induced (Figure 1C) and increased in N2a-mLRP4 cells compared to to accumulate endogenous Ab42 show increased neurodegener- N2a-pcDNA3 cells. To quantify the amount of internalized RAP, ation compared to apoE3 mice [22]. Notably, metabolism of both we performed FACS analysis of similarly treated cultures. Cells apoE and Ab42 are regulated by the low-density lipoprotein (LDL) were detached and additionally treated with pronase to remove receptor-related protein 1 (LRP1), a large endocytic receptor any cell-surface labeled RAP. N2a-mLRP4 cells have an 8-fold belonging to the LDL receptor family [15]. increase in the uptake of fluorescently labeled RAP compared to LRP1 is synthesized as a 600-kDa precursor protein that interacts N2a-pcDNA3 cells (Fig. 1D), indicating that mLRP4 expressed in with the ER chaperone receptor-associated protein (RAP) [23,24] N2a cells is fully functional in mediating ligand internalization. and matures in the biosynthetic pathway into an extracellular, Based on these results, we analyzed the uptake of Ab42 in N2a- ligand-binding subunit of 515-kDa (LRP1-515) and a transmem- pcDNA3 cells and N2a-mLRP4 cells by performing binding/ brane 85-kDa subunit (LRP1-85) that binds several adaptor proteins endocytosis assays and fluorescence microcopy analysis. We for efficient endocytic trafficking and signaling
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