The Low Density Lipoprotein Receptor-related Protein 1 Mediates Uptake of Amyloid β Peptides in an in Vitro Model of the Blood-Brain Barrier Cells

Kaoru Yamada,Tadafumi Hashimoto,Chiori Yabuki,Yusuke Nagae,Masanori Tachikawa,Dudley K Strickland,Qiang Liu,Guojun Bu,Jacob M Basak,David M Holtzman,Sumio Ohtsuki,Tetsuya Terasaki,Takeshi Iwatsubo

doi:10.1074/jbc.m801487200

Abstract

The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.

Highlights

Aggregation and deposition of amyloid ␤-peptide (A␤)2 in the brain are crucial events in the pathogenesis of Alzheimer disease (AD) [1]
A␤ Is Internalized by TR-blood-brain barrier (BBB) Cells in a Specific Manner— We reasoned that if A␤ is transported across the BBB, A␤ should be internalized by brain microvascular endothelial cells (BMECs)
We BMECs that delineate BBB rapidly internalize chaperone-free conducted A␤ internalization assays using a range of cell lines A␤ peptides in the brain parenchyma and efflux them into

Summary

The abbreviations used are

A␤, amyloid ␤ peptide; AD, Alzheimer disease; BBB, blood-brain barrier; BMEC, brain microvascular endothelial cell; P-gp, P-glycoprotein; tPA, tissue-type plasminogen activator; RAP, receptor-associated protein; PICUP, photo-induced cross-linking of unmodified proteins; MEF, mouse embryo fibroblast; siRNA, small interference RNA; RNAi, RNA interference; RAGE, receptor for advanced glycation end products; HUVEC, human umbilical vein endothelial cell; ANOVA, analysis of variance. LRP1-mediated A␤ Clearance in TR-BBB Cells recapitulate the transport of A␤ and other macromolecules across the BBB and with which a precise molecular mechanism of A␤ transport across the BBB could be elucidated. We adopted the TR-BBB cells, a conditionally immortalized cell line derived from brain capillary endothelial cells of transgenic rats expressing temperature-sensitive large T antigen [19], whose inactivation upon incubation at 37 °C renders the cells into a nonimmortalized state similar to primary BMECs. TR-BBB cells have been shown to express a number of receptors and transporters expressed in endothelial cells comprising the BBB (e.g. GLUT-1, P-glycoprotein (P-gp), and other influx or efflux transporters, allowing for the characterization of their functions in vitro [20]. Using TR-BBB cells, we established an in vitro model of A␤ uptake and found that LRP1 is involved in the rapid and robust A␤ internalization in TR-BBB cells. Our observations provide a new clue to the molecular mechanism of A␤ uptake and transport across the BBB

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION