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

Abstract

The metabolism of amyloid ␤ peptide (A␤) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A␤ 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␤ transport across the BBB, we established a new in vitro model of the initial internalization step of A␤ transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A␤ through a receptor-mediated mechanism. We also provide evidence that A␤ internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptorassociated protein, neutralizing antibody, or small interference RNAs all reduced A␤ uptake. Despite the requirement of LRP1-dependent internalization, A␤ 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␤ uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A␤ 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␤ across BBB. Aggregation and deposition of amyloid ␤-peptide (A␤)2 in the brain are crucial events in the pathogenesis of Alzheimer disease (AD) (1). A␤ is produced from ␤-amyloid precursor protein through sequential proteolytic cleavages by ␤-and ␥-secretases. Missense mutations as well as duplication of the ␤-amyloid precursor protein gene have been identified in pedigrees of early onset familial AD (2-4), some of which have been shown to alter the ␤-amyloid precursor protein processing in such a way as to increase the level of A␤, especially that of the more aggregable species A␤42, leading to formation of A␤ fibrils (5-7). Cognitive deficits in transgenic mice overexpressing familial AD mutant form ␤-amyloid precursor protein, as well as alteration in synaptic plasticity and synapse loss induced by A␤ oligomers implicate aggregated species of A␤ in the neuronal dysfunction and death in AD brains (8 -10).A␤, secreted from neurons in the brain, is thought to be catabolized by specific proteases (e.g. NEP and insulin-degrading enzyme) (11, 12); phagocytotic cells in brains (i.e. microglia and astrocytes) (13, 14) take up and clear soluble or aggregated A␤. Notably, in vivo observations that A␤ injected into rodent brains is rapidly effluxed from brains (15, 16) suggest the presence of a novel pathway for A␤ clearance across the blood-brain barrier (BBB). The BBB is considered as a three-cell archetype composed of brain microvascular endothelial cells (BMECs), astrocytes, and supporting pericytes. It does not normally allow a free exchange of macromolecules between brain and blood, ...

Aβ Immunotherapy: Intracerebral Sequestration of Aβ by an Anti-Aβ Monoclonal Antibody 266 with High Affinity to Soluble Aβ

“…The binding of anti-A␤ antibodies to A␤ was determined by immunoprecipitation of 125 I-A␤ 1-40 in vitro. We have confirmed that 125 I-A␤ 1-40 in solution exists in a monomeric state (Yamada et al, 2008).…”

“…125 I]-A␤ 1-40 was purchased from Perkin-Elmer Life Sciences as described previously (Yamada et al, 2008). [ 14 C]-Inulin was obtained from American Radiolabeled Chemicals.…”

Low-density lipoprotein receptor overexpression enhances the rate of brain-to-blood Aβ clearance in a mouse model of β-amyloidosis

“…S2B), which labels microvessel abluminal surfaces at the astrocyte-vessel interface (24). These results suggest the LDLR overexpression in neurons and astrocytes (18) likely increases BBB-mediated Aβ clearance through an indirect mechanism involving other apoE receptors at the BBB, such as LRP1-a receptor shown to directly mediate Aβ clearance across the BBB (14,15,26). To test this hypothesis, we delivered an anti-LRP1 antibody (N20) or vehicle into the brains of TG mice immediately before coinjection of 12 nM [ The rapid degradation of Aβ in the periphery (t 1/2 = 2-3 min) precludes a direct and sensitive measurement of the rate of Aβ appearance from brain into blood (27,28).…”

La barrière hémato-encéphalique

“…La Le récepteur LRP1 et l'endocytose des peptides Ab LRP1 fait partie de la famille des récepteurs aux lipoprotéines de faible densité (famille LDLR pour low density lipoproteins receptor). In vitro, LRP1 est exprimé au niveau des CEC de rat et semble capable de lier les peptides A afin d'engager leur endocytose [9]. Cependant le processus de transcytose doit encore être élucidé d'autant plus que cette internalisation a pour conséquence la dégradation de ces peptides et non leur transcytose.…”

“…L'expression de NEP au niveau des microvaisseaux cérébraux des patients atteints de maladie d'Alzheimer est diminuée lorsqu'elle est comparée aux patients non atteints [22]. D'autres études suggèrent également des mécanismes de dégradations intracellulaires impliquant vraisemblablement les lysosomes des cellules endothéliales [9]. L'ensemble de ces données suggère que les cellules endothéliales joueraient également un rôle dans la dégradation des peptides A cérébraux et que ces mécanismes de dégradation seraient moins efficaces lors de la maladie, contribuant ainsi à l'accumulation cérébrale de ces peptides.…”